Macroscopic biofilms in fracture-dominated sediment that anaerobically oxidize methane.

Methane release from seafloor sediments is moderated, in part, by the anaerobic oxidation of methane (AOM) performed by consortia of archaea and bacteria. These consortia occur as isolated cells and aggregates within the sulfate-methane transition (SMT) of diffusion and seep-dominant environments. Here we report on a new SMT setting where the AOM consortium occurs as macroscopic pink to orange biofilms within subseafloor fractures. Biofilm samples recovered from the Indian and northeast Pacific Oceans had a cellular abundance of 10(7) to 10(8) cells cm(-3). This cell density is 2 to 3 orders of magnitude greater than that in the surrounding sediments. Sequencing of bacterial 16S rRNA genes indicated that the bacterial component is dominated by Deltaproteobacteria, candidate division WS3, and Chloroflexi, representing 46%, 15%, and 10% of clones, respectively. In addition, major archaeal taxa found in the biofilm were related to the ANME-1 clade, Thermoplasmatales, and Desulfurococcales, representing 73%, 11%, and 10% of archaeal clones, respectively. The sequences of all major taxa were similar to sequences previously reported from cold seep environments. PhyloChip microarray analysis detected all bacterial phyla identified by the clone library plus an additional 44 phyla. However, sequencing detected more archaea than the PhyloChip within the phyla of Methanosarcinales and Desulfurococcales. The stable carbon isotope composition of the biofilm from the SMT (-35 to -43‰) suggests that the production of the biofilm is associated with AOM. These biofilms are a novel, but apparently widespread, aggregation of cells represented by the ANME-1 clade that occur in methane-rich marine sediments.

The histocompatibility (HLA) antigen distribution in diabetes in southern African Blacks (Xhosa).

The frequency distributions of HLA antigens in 25 juvenile-onset diabetics (JOD) and 56 maturity-onset diabetics (MOD) belonging to a southern African black tribe (Xhosa) were compared with those of 153 non-diabetic Xhosa blacks. Unlike the findings in white JODs, there was no increase of B8 or B15 nor a reduced frequency of B7 but an apparently, significantly increased frequency of Bw35 and A2 in both Xhosa JODs and Xhosa MODs respectively. This is the first ethnic group in which an HLA antigen marker has been found for MOD. Furthermore, these findings suggest that diabetes, both JOD and MOD, in white people is a different genetic disease from the diabetes among the Xhosa tribe.

Attenuation of cytomegalovirus-induced endothelial intercellular adhesion molecule-1 mRNA/protein expression and T lymphocyte adhesion by a 2'-O-methoxyethyl antisense oligonucleotide.

BACKGROUND: Intercellular adhesion molecule-1 (ICAM-1) is strongly induced under inflammatory conditions associated with allograft rejection, thereby promoting leukocyte recruitment and activation at the site of inflammation. Enhancement of ICAM-1 expression can also be the result of viral infection, in particular human cytomegalovirus (CMV), a frequent source of complications in the transplant recipient. In vitro studies have shown that CMV infection of endothelial cells (EC) results in the direct enhancement of ICAM-1 expression and consequent leukocyte adhesion/activation suggesting mechanisms by which CMV exacerbates graft vascular disease. Although treatment of EC with ICAM-1-specific antisense oligonucleotides has been shown to attenuate ICAM-1 induction under simulated inflammatory conditions (i.e., TNF-alpha), no studies have addressed their effectiveness on virally-induced ICAM-1 expression. RESULTS: In the current investigation, we show that the progressive increase in endothelial ICAM-1 protein expression that follows inoculation with CMV correlates with a progressive accumulation of ICAM-1 mRNA. Furthermore, we demonstrate that treatment of EC with a partially 2'-O-methoxyethyl modified ICAM-1-specific antisense oligonucleotide before viral inoculation significantly reduces CMV-associated induction of ICAM-1 protein and mRNA expression. Finally, we show that antisense-mediated attenuation in ICAM-1 expression results in a significant reduction of T lymphocyte adhesion to CMV-infected EC monolayers, an interaction that has been implicated in allogeneic T lymphocyte activation, in viral transmission to transiently adherent leukocytes and subsequent hematogenous dissemination. CONCLUSIONS: These findings demonstrate for the first time that antisense oligonucleotides can effectively reverse virally-induced host cellular protein expression, specifically ICAM-1, as well as consequent T lymphocytes adhesion, thus broadening the potential clinical utility of antisense oligonucleotides.

Cytomegalovirus induces sialyl Lewis(x) and Lewis(x) on human endothelial cells.

BACKGROUND: Cytomegalovirus (CMV) is the primary viral cause of complications in transplant recipients. We sought to understand the mechanisms of its dissemination and induction of vascular disease, which may lead to transplant complications. Sialyl Lewis(x) (sLe(x)) and Lewis(x) (Le(x)) are known for their roles in mediating cell adhesion and as tumor-associated carbohydrate antigens. Herein we explore whether CMV induces surface expression of these important molecules in endothelial cells (EC). METHODS: Flow cytometry was used to detect surface expression of sLe(x) and Le(x) on CMV-infected human umbilical vein endothelial cells (HUVEC), with or without ultraviolet inactivation of the virus. To elucidate mechanisms of CMV-mediated induction, mRNA coding for predominant HUVEC sialyltransferases (ST) and fucosyltransferases (FT), key enzymes in sLe(x) and Le(x) synthesis, was analyzed by Northern blot. Dual immunohistochemical staining for sLe(x) and Le(x) expression of human colon and placental tissue was performed to investigate in vivo relevance. RESULTS: sLe(x) expression on CMV-infected HUVEC was strongly up-regulated by 8 days after inoculation. Le(x) expression was detectable earlier and increased steadily over time. In contrast, ultraviolet-inactivated CMV did not induce expression of these molecules. Northern blot assays demonstrated higher levels of important EC glycosyltransferases ST-IV, FT-III, and FT-IV in CMV-infected EC. Finally, high levels of sLe(x) and Le(x) were expressed in CMV-infected EC in vivo. CONCLUSIONS: Given the known biologic functions of sLe(x) and Le(x), we suggest that CMV induction of these molecules may have widespread consequences ranging from CMV dissemination to induction of CMV-associated vascular disease, including thrombosis.

Effectiveness of digitalis with or without acebutolol in preventing atrial arrhythmias after coronary artery surgery.

In this study, a beta-adrenergic blocker in combination with digoxin provided marginal protection against atrial fibrillation/flutter after coronary artery surgery. The economic comparison of patients who did and did not develop atrial fibrillation/flutter indicates that prevention of these arrhythmias can have a significant impact on length of hospital stay and cost of this common surgical procedure.

Heparin therapy during extracorporeal circulation. I. Problems inherent in existing heparin protocols.

Five heparin protocols, representative of about 30 presently used throughout the country, were analyzed. The adequacy of anticoagulation during and the precision of protamine neutralization at the conclusion of extracorporeal circulation were studied. In each of 50 patient's age, height, weight, or surface area was of no help in predicting heparin kinetics. The study group consisted of the 2 patients with the longest and the 2 patients with the shortest heparin half lives, as well as the 2 patients who showed the greatest sensitivity to heparin and the 2 who showed the least. By computer simulation, each was managed according to the five protocols and by a monitoring procedure. The protocols failed to provide safe anticoagulation or precise protamine neutralization, whereas the simplified monitoring approach was uniformly successful.

Antimicrobial activity evaluations of two new quinolones, PD127391 (CI-960 and AM-1091) and PD131628.

The in vitro activities of PD127391 and the new fluorinated-4-quinolone, PD131628, were compared with each other and with five similar fluoroquinolones (ciprofloxacin, enoxacin, fleroxacin, norfloxacin, and ofloxacin). A total of 844 isolates mainly from recent clinical bacteremias and additional stock strains with well-characterized resistance mechanisms were tested. PD127391 had slightly more activity than PD131628 (90% minimum inhibitory concentration (MIC90)] 0.008-0.12) against the Enterobacteriaceae, but both were two- to fourfold more potent than ciprofloxacin. PD131628 activity was equal to or greater than PD127391 when tested against Pseudomonas aeruginosa. PD127391 showed greatest activity against Bacteroides fragilis group strains (MIC90, 2 micrograms/ml) when compared with PD131628 (MIC90 greater than 8 micrograms/ml). Both PD127391 (MIC90s, 0.015-1.0 micrograms/ml) and PD131628 (MIC90s, 0.03 - greater than 8 micrograms/ml) were more active than ciprofloxacin against Gram-positive organisms. Altering the medium pH, adding divalent cations (magnesium), and increasing the inoculum concentration to 10(6) colony-forming units per spot adversely effected the activity of both PD127391 and PD131628. Resistance selection and mutational rates to resistance were identical to previously studied drugs in their class.

In vitro activity evaluations of cefdinir (FK482, CI-983, and PD134393). A novel orally administered cephalosporin.

Cefdinir, a so-called third-generation oral cephalosporin was tested in vitro against over 700 pathogens from patients with bacteremia. Cefdinir was very active against the Enterobacteriaceae with a 50% minimum inhibitory concentration (MIC50) value range of less than or equal to 0.03-8 micrograms/ml. The enteric species having the highest MIC90S (greater than or equal to 16 micrograms/ml) were Citrobacter freundii, and the enterobacters, Morganella morganii, Proteus vulgaris, and Serratia marcescens. Cefdinir was generally two- to fourfold less active than cefixime, but markedly more potent with a wider spectrum compared with older oral cephalosporins, cefaclor or cefuroxime. In contrast to cefixime, cefdinir inhibited Staphylococcus aureus (MIC90, 1 micrograms/ml) and other staphylococci. Pneumococci, beta-hemolytic streptococci, Haemophilus influenzae, Moraxella catarrhalis, and pathogenic Neisseria spp. (MIC90S, 0.12-0.5 micrograms/ml) were cefdinir susceptible, but Pseudomonas aeruginosa, oxacillin-resistant staphylococci and Bacteroides fragilis gr. strains were resistant. Cefdinir was generally bactericidal with a minimal inoculum effect at 10(6) colony-forming units per spot. Cefdinir beta-lactamase hydrolysis by some recently described extended broad spectrum beta-lactamases was suspected. Cefdinir exhibited a wide, balanced spectrum for an oral cephalosporin indicating possible clinical use against susceptible pathogens in respiratory tract, urinary tract, genital and cutaneous infections.

In vitro antimicrobial activity of sparfloxacin (AT-4140, CI-978, PD 131501) compared with numerous other quinolone compounds.

Sparfloxacin (AT-4140, CI-978, PD 131501) was tested against over 800 recent bacteremic strains and compared with ciprofloxacin and six other fluoroquinolones. The 90% minimum inhibitory concentration (MIC90) ranges for the Enterobacteriaceae species were (a) sparfloxacin, 0.03-1 microgram/ml and (b) ciprofloxacin, 0.015-0.25 microgram/ml. Moraxella catarrhalis, Haemophilus influenzae, and Neisseria gonorrhoeae were very susceptible to sparfloxacin (MIC90s, 0.004- less than or equal to 0.03 microgram/ml) and the other comparison drugs. Staphylococcus aureas and other staphylococci were generally susceptible to the tested fluoroquinolones but very susceptible to sparfloxacin and WIN 57273. All beta-hemolytic streptococci, enterococci, and pneumococci had sparfloxacin MICs of less than or equal to 1 microgram/ml. Sparfloxacin was quite active against anaerobic bacteria including Bacteroides fragilis gr. and Gram-positive strains (MIC90s, less than or equal to 2 micrograms/ml). The most resistant enteric bacilli were among Serratia marcescens and the Proteae, especially the Providencia spp. (two- to eightfold higher MICs). Pseudomonas aeruginosa strains were also susceptible to sparfloxacin (MIC90, 2 micrograms/ml). Magnesium ions, CO2 incubation, and low pH had some adverse effect on sparfloxacin MICs, and resistance development was documented among current clinical isolates of staphylococci, pseudomonas, and some enteric species.

In vitro activity of RU29246. The metabolite of a new HR916 cephalosporin ester.

Compound RU29246 (RU) is the active metabolite of an orally absorpted cephalosporin ester HR916. The RU spectrum of activity includes the majority of Enterobacteriaceae species, Haemophilus influenzae, pathogenic Neisseria spp., Moraxella catarrhalis, Acinetobacter antiratus, staphylococci, and Streptococcus spp. Pseudomonas species and enterococci were routinely resistant to RU. The RU spectrum was most similar to cefixime against the Gram-negative bacilli and to cefuroxime against the Gram-positive organisms. RU was bactericidal and its mean inhibitory concentrations (MICs) were not greatly increased by high inoculum concentrations. Many strains producing various beta-lactamases generally remained susceptible to RU by MIC tests. However, isolates with extended broad spectrum beta-lactamases capable of hydrolyzing cefotaxime and ceftazidime were also resistant to RU. Broth and agar RU MICs were comparable. Its activity was increased against enterococci in the presence of blood products.

Antimicrobial activity of E-1040, a novel thiadiazolyl cephalosporin compared with other parenteral cephems.

E-1040, a new parenteral fourth-generation cephalosporin, was tested against greater than 600 bacteremic pathogens and compared with cefotaxime, ceftazidime, and cefpirome. E-1040 activity against Staphylococcus aureus was comparable (MIC90, 8 micrograms/ml) to ceftazidime, but inferior to cefotaxime (MIC90, 2 micrograms/ml) and cefpirome (MIC90, 0.5 microgram/ml). beta-Hemolytic streptococci and most Gram-positive anaerobes were also susceptible to E-1040. Some strains of coagulase-negative staphylococci, all oxacillin-resistant Staphylococcus spp., enterococci, and Bacteroides fragilis group strains were resistant to E-1040 (MIC90, greater than 64 micrograms/ml). Comparative tests for E-1040 and the three other cephalosporins against pseudomonads and nonenteric Gram-negative bacilli showed E-1040 to be generally most active. The E-1040 MIC90 for Pseudomonas aeruginosa was 1 microgram/ml and for ceftazidime it was 4 micrograms/ml. Haemophilus influenzae, Moraxella catarrhalis, and Neisseria spp. has E-1040 MIC90s ranging from 0.12 to 2 micrograms/ml. Neisseria gonorrhoeae, strains resistant to penicillin, did not have markedly elevated E-1040 MICs compared with penicillin-susceptible strains. Enterobacteriaceae species had all MICs of less than or equal to 8 micrograms/ml for E-1040 and cefpirome, indicating activity against strains producing stably derepressed beta-lactamases. E-1040 appeared to be beta-lactamase stable, little influenced by testing systems or media, and was bactericidal. E-1040 seems to have promise as a parenteral beta-lactam for use on strains resistant to "third-generation" cephalosporins and other families of drugs such as aminoglycosides and fluoroquinolones.

Ascorbic acid-dehydroascorbate induces cell cycle arrest at G2/M DNA damage checkpoint during oxidative stress.

Reactive oxygen species induce cellular damage and have been implicated as mediators for cellular signaling pathways. However, a linkage between the cellular redox status and cell cycle progression has not been demonstrated. We previously demonstrated, using the Chinese hamster ovary cell line AS52, that the cytotoxic and mutagenic effects of oxidative stress is prevented by ascorbic acid (AA), but only when cells are treated with AA prior to treatment with the stressor. To elucidate the mechanism(s) responsible for this effect, we determined the effect of AA on cell cycle progression during oxidative stress. Flow cytometric analyses demonstrated that treatment of AS52 cells with AA (50 microM), prior to treatment with a radical generating system (RGS), enhanced cell cycle arrest at the G2/M DNA damage checkpoint when compared to cells treated with RGS. AA had no effect on cell cycle progression in the absence of oxidative stress. Furthermore, under conditions that prevent the reduction of dehydroascorbate (DHA), the oxidized form of AA, cell cycle arrest was also induced at the G2/M DNA damage checkpoint. These observations demonstrate that during periods of oxidative stress, AA functions as an antioxidant and DHA enhances transient arrest at the G2/M checkpoint by delaying the activation of cyclin B-cdc2. These results suggest the presence of a unique redox mechanism for the regulation of cell cycle progression and also demonstrate a novel mechanism by which AA protects cells from damage due to oxidative stress.

Outbreak of gentamicin-resistant Acinetobacter baumanii in an intensive care unit: clinical, epidemiological and microbiological features.

The clinical, epidemiological and microbiological features of an outbreak of infection and colonisation caused by gentamicin-resistant Acinetobacter baumanii (GRAB) in an 18-bed intensive care unit (ICU) of a 680-bed adult teaching hospital are described. A retrospective review of medical, laboratory and infection control records was followed by prospective surveillance. Typing of isolates was performed by restriction enzyme analysis (REA) of chromosomal DNA. The incidence of GRAB in the ICU increased from 1.26 cases per 1000 occupied bed days (OBDs) for January to June 1993, to 6.62 per 1000 OBDs for July to December 1993 (Chi square = 4.8, P < 0.05), confirming the existence of an outbreak. For the two year period, 1993 and 1994, a total of 45 cases of GRAB infection or colonisation was identified. Males and females were equally represented, with an age range of 16-79 years and a mean age of 51 years. Admitting diagnoses varied, with multiple trauma and head injury predominating (ten cases). For 35 of the 45 cases the initial site of GRAB isolation was sputum or other respiratory tract specimen. Specific treatment for GRAB was initiated in 23 patients, however no deaths were directly attributable to GRAB infection. The period of time between admission to the ICU and first isolation of GRAB ranged from three to 70 days with a median of nine days. Overall, ten (11%) of 91 staff hand samples and one of 37 (3%) environmental samples yielded GRAB. All GRAB isolates produced similar biochemical profiles and antibiotic resistance patterns, except for a group of five which were ciprofloxacin resistant. Thirty patient isolates, all ten staff hand isolates and the environmental isolate produced identical REA patterns. The remaining five patient isolates (all ciprofloxacin resistant) which were available for typing produced a different REA pattern. Our study has documented a moderate-sized outbreak of GRAB in an ICU setting. Typing of isolates using REA was useful in delineating outbreak strains. Carriage of GRAB on staff hands was demonstrated as the most likely source of infection. Despite institution of infection control measures GRAB now appears endemic in the ICU.

Glenohumeral arthrodesis. Operative and long-term functional results.

Seventy-one shoulders of seventy patients were fused for treatment of various conditions, and the results were analyzed after an average follow up of nine years and six months. The operative technique always included the use of internal fixation. The average position of arthrodesis was 45 degrees of abduction and 25 degrees of flexion of the arm, with the flexed forearm rotated 21 degrees above the horizontal plane, measured with the arm abducted and flexed. In sixty-eight shoulders, one operation achieved a solid fusion; in the other three, a second arthrodesis was required. Complications included tenderness over the outer ends of the internal fixation device, which required its removal from seventeen shoulders; a fracture in the fused extremity in ten patients; and a post-operative infection in one. Relief of pain was adequate in three-quarters of the patients. Three-quarters of the patients could perform activities involving reaching the trunk, one-half could do activities requiring reaching the head, and one-quarter were able to do light work with the arm at shoulder level or higher. The position of fusion had little effect on the result. Eighty-two per cent of the entire group believed that they had benefited from the operation, and none of the results deteriorated with time.

The mechanical performance of the standard Hoffmann-Vidal external fixation apparatus.

A standard Hoffmann-Vidal quadrilateral fixation system for tibial fractures was analyzed using different geometric and material variations of the basic configuration. The fixation stiffness properties were quantitated to provide objective comparison. The results have shown that the bone-pin interface is the least-stiff link in the entire structure, particularly under the anterior-posterior bending mode. Rigidity of the device can be substantially improved by increasing the number of pins, using full threaded pins with a larger diameter, decreasing side connecting-rod distances, and increasing pin-separation distances in each pin group. Symmetrical tightening of the compression screws by hand is sufficient to produce compression of bone at the fracture site. The use of titanium pins tends to reduce stiffness, but using a frame made of titanium can significantly decrease the weight of the apparatus without decreasing its stiffness.

Enzymatic deacylation of teicoplanin followed by reductive alkylation: synthesis and antibacterial activity of new glycopeptides.

Novel glycopeptides derived from teicoplanin were prepared and evaluated for activity against antibiotic-resistant gram-positive pathogens. Removal of the fatty acid sidechains of teicoplanin was accomplished by enzymatic deacylation. The resulting deacylated teicoplanin was subjected to reductive alkylation resulting in mono- and di-alkylated compounds at the 2 possible primary amines. Deacylated teicoplanin was less active than teicoplanin against enterococci and staphylococci (MIC > or =32 microg/ml). All mono- and di-alkylated products regained some activity, and some had potent activity against both staphylococci and glycopeptide-resistant enterococci. MICs of the most potent di-alkylated compounds ranged from 0.25 approximately 2 microg/ml against glycopeptide-resistant enterococci.

Preparation and evaluation of 3,4''-ester derivatives of 16-membered macrolide antibiotics related to tylosin.

A large group of ester derivatives of tylosin-related macrolides was prepared in which the hydroxyl groups at C-3 and C-4'' were acylated by either chemical or biochemical methods. Most of the derivatives exhibited excellent in vitro antimicrobial activity. However, only the 3,4''-diacyl derivatives of tylosin and macrocin showed any significant improvements of in vivo efficacy against experimental infections in rodents.

Estimation of lung oedema in humans by transmission-emission scintigraphy with 99Tcm.

UNLABELLED: We have used a combination of transmission and emission gamma camera techniques to scan the thorax in the anteroposterior plane in 21 patients with partially treated cardiogenic pulmonary oedema and have compared their results with those from 20 age-matched normal subjects who were scanned previously. For transmission scanning, an external 99Tcm flood source was used; for emission scanning we labelled sequentially the vascular compartment with 99Tcm autologous erythrocytes and the interstitium with 99Tcm diethylenetriaminepentaacetic acid (99Tcm-DTPA). From the transmission scans we derived the transthoracic tissue thickness (Tt) and from the emission scans, after correction for attenuation, the regional blood and interstitial volumes. In the lower zone of the right lung, mean (S.D.) Tt in normal subjects was 10.9 (S.D. 3.1) cm and in subjects with lung oedema was 12.5 (S.D. 3.1) cm (P = 0.07). There was a weak correlation between Tt and a radiographic numerical score of oedema severity (r = 0.47, P less than 0.05). In eight subjects with lung oedema, lung tissue thickness (T1) was estimated (by subtraction from Tt of radiographically estimated chest wall thickness). The T1 correlated closely with the radiographic score (r = 0.78, P less than 0.01). There was no significant change in blood or interstitial volumes in oedema. IN CONCLUSION: (1) transmission scanning gives an indication of oedema severity if an allowance is made for chest wall thickness; (2) 99Tcm-DTPA fails to equilibrate fully with oedema liquid during an equilibration period of 5 min.

Experimental otitis media with effusion following middle ear inoculation of nonviable H influenzae.

In order to test the hypothesis that nonviable bacteria can induce middle ear inflammation leading to persistent middle ear effusion (MEE), we conducted an animal experiment using formalin-killed Hemophilus influenzae, the bacterium reported to be the most common pathogen isolated from chronic MEEs. Over 70% of the chinchillas injected with formalin-killed H influenzae type b or a nontypeable isolate developed sterile, straw-colored serous MEEs, and exhibited histological evidence of extensive inflammatory changes of the middle ear mucosal connective tissue and epithelium. Control animals injected with pyrogen-free sterile saline did not exhibit any inflammatory changes or effusions in the middle ears. Our data suggest that endotoxin on the surface of H influenzae, a gram-negative bacterium, may be responsible for the induction of the otitis media with effusion. It is suggested that endotoxin (even when the organisms are no longer viable) may be responsible for the production of serous MEE and inflammatory changes in the middle ear.

Treatment of benign positional vertigo using heels-over-head rotation.

Particle repositioning, using either bedside techniques or whole-body manipulation devices, has been used effectively to treat benign positional vertigo (BPV). We assessed the efficacy of particle repositioning using a device designed and built specifically to treat BPV that rotated patients 360 degrees heels-over-head in their sagittal body plane while their heads were turned to align the posterior semicircular canal with the plane of rotation. Eye movements were monitored during the maneuver by an infrared video recording system that allowed subsequent review of the induced nystagmus. Our results indicate that 1) heels-over-head rotation is an extremely efficacious procedure for treating patients with BPV and 2) the pattern of nystagmus during repositioning is consistent with the theory that free-floating debris is highly likely to account for BPV.