Prevalence, clonality, and pathogenicity of Staphylococcus epidermidis isolates in newborn feces.

Coagulase-negative staphylococci (CoNS) are the most prevalent pathogens causing late-onset sepsis in neonates. The question is whether neonates acquire endemic hospital-adapted clones or incidentally occurring CoNS strains after birth during their hospital stay. Therefore, a prospective study was performed on the prevalence of CoNS in the stool of babies (born vaginally or by cesarean section) during their first days of life. Their clonal relatedness and potential to induce invasive disease were characterized. CoNS were analyzed from the stool samples of newborns with a load of CoNS above 103 colony-forming units (CFU)/mL. The identification of CoNS was performed phenotypically and genotypically. For typing, repetitive polymerase chain reaction (PCR), pulsed-field gel electrophoresis, and multilocus sequence typing were used. Resistance profiles, biofilm production, the presence of icaAD and of IS256 were determined as well. From a total of 207 stool samples (56 newborns), CoNS were detected in 41% of the newborns, mostly on day 3 for the first time (62.5%). Staphylococcus epidermidis was isolated in 85.7% of cases, harbored no IS256 element, and mostly expressed no biofilm. The isolates were separated into four main clusters by repetitive sequence-based PCR. 24% of the strains showed no antimicrobial resistance. 20% were resistant against four antibiotics of two different antibiotic classes. The remaining strains were resistant only against one antimicrobial substance class. Thus, it can be concluded that newborns do not acquire hospital-adapted endemic, multidrug-resistant S. epidermidis isolates during their first days of life. Yet, the results support the thesis that, during hospital stay, environmental parameters may convert sensible/noninvasive S. epidermidis strains into multidrug-resistant strains with characteristics of invasiveness.

Independent associations of vitamin D metabolites with anemia in patients referred to coronary angiography: the LURIC study.

PURPOSE: Anemia and vitamin D deficiency are both frequent in adult patients. Whether low vitamin D metabolite levels are an independent risk factor for different subtypes of anemia remains to be studied in detail. METHODS: In 3299 patients referred for coronary angiography, we investigated the association of 25-hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D [1,25(OH)2D] with anemia [hemoglobin (Hb) <12.5 g/dl] of specific subtypes. RESULTS: Compared with patients with 25OHD levels in the adequate range (50-125 nmol/l), patients with deficient 25OHD concentrations (<30 nmol/l; 33.6 % of patients) had 0.6 g/dl lower Hb levels. Hb values were 1.3 g/dl lower in patients with 1,25(OH)2D levels <40 pmol/l (5.4 % of patients), compared with patients in the highest 1,25(OH)2D category (>70 pmol/l). Of the participants, 16.7 % met the criteria for anemia. In multivariate-adjusted regression analyses, the odds ratios for anemia in the lowest 25OHD and 1,25(OH)2D categories were 1.52 (95 % CI 1.15-2.02) and 3.59 (95 % CI 2.33-5.52), compared with patients with 25OHD levels in the adequate range and patients with 1,25(OH)2D levels >70 pmol/l. The probability of anemia was highest in patients with combined 25OHD and 1,25(OH)2D deficiency [multivariable-adjusted odds ratio 5.11 (95 % CI 2.66-9.81)]. Patients with anemia of chronic kidney disease had the highest prevalence of 25OHD deficiency and 1,25(OH)2D concentrations of <40 pmol/l. CONCLUSIONS: Low 25OHD and 1,25(OH)2D concentrations are independently associated with anemia. Patients with poor kidney function are most affected. Interventional trials are warranted to prove whether administration of plain or activated vitamin D can prevent anemia.

[Spectrum of microbial colonisation and resistance of a surgical ICU in a systematic comparison of the 10-year time period 1996-2005 using routine microbiological testing]. / Erreger- und Resistenzspektrum einer chirurgischen ITS im systematischen 10-Jahres-Vergleich 1996-2005 im Rahmen des mikrobiologischen Routinemonitorings.

INTRODUCTION: Knowledge on potentially pathogenic microbes including characteristics of their antibiotic resistance in septic patients as well as on the ward- and department-specific microbial spectrum can be considered essential for an efficient initiation of an adequate antimicrobial treatment, which turns out to become pivotal for patient outcome. Permanent changes in microbial patterns and antibiotic resistance can only be identified by a continuous investigation of various microbiological specimens. AIM: Based on the retrospective evaluation of prospectively collected data on microbiological investigations of the surgical ICU in 1996, 2002, 2004 and 2005, the short- and long-term changes by trend of microbial spectrum and antibiotic resistance following reorganisation and restructuring of the University Hospital from the more traditional pavillon-based system to a multidisciplinary complex building in 2003 were investigated. MATERIAL AND METHODS: Twice a week, routine microbiological testing of blood and urinary cultures as well as swabs from wound areas and endotracheal swabs were initiated in septic patients (suspect, manifestation) or in case of their clinical impairment. The microbial spectrum was sub-divided according to Gram-staining (Gram-positive/ -negative), various species and fungi with descriptive absolute and relative data values. -Various groups and time periods were statistically compared using χ² test as appropriate. P values < 0.05 were considered statistically significant. RESULTS: In total (n (Total) = 4 899), microbiological testing resulted in the detection of microbes in 699 and 833 blood and urinary cultures (14.3 % and 17 %, respectively) as well as 1 232 wound swabs (25.1 %) together with 2 135 samples from the endotracheal sites (43.6 %). During the short- (2002 vs. 2004) and long-term analyses (1996 vs. 2005), the proportion of Gram-positive microbes increased. Al-though Gram-positive bacteria can be considered the most frequent microbes for bacteriemia, there was a shift onto urinary and wound infections as well as pneumonias through the observation period. Despite the decreasing incidence of Enterococcus and the consistent proportion of MRSA, the increase of resistant Enterococcus strains (0 % vs. 43.2 %; P < 0.05) is critical. However, in the Gram-negative microbial spectrum there was an increase of the bacteraemia rate but a fall of the detection rate in wound and endotracheal swabs. In parallel, an increase of the detection rate of E. coli in blood (6.5 % vs. 45.5 %; P < 0.05) and endotracheal swabs (9.2 % vs. 16.2 %; P < 0.05) is associated with an increase of multiresistant Enterobacteriaceae strains (0 % vs. 30.7 %; P < 0.05). The portion of multiresistant strains of Pseudomonas with 31 % stayed the same through the 10-year time period. While Candida-based colonisation showed a decreased incidence (25 % vs. 15 %; P < 0.05) during the whole investigation period, there was a relative rise in the frequency of candidemia. CONCLUSION: ICU relocation from the pavillon-based system to a new complex clinic building was not associated with any significant alteration of the microbial spectrum on the surgical ICU. Increasing incidences of resistant Enterococcus and Gram-negative problematic microbes may indicate a general spread of multi-resistant microbes under the steady selecting pressure of a not always adequately initiated antibiotic / antimicrobial therapeutic regimen and underline the required but specific and selected microbiological screening.

Complement-dependent B-cell activation by cobra venom factor and other mitogens?

It has been proposed that two distinct signals are required for the triggering of the precursors of antibody-forming bone marrow-derived cells (B cells): (a) the binding of antigen or of a mitogen to the corresponding receptor sites on B-cell membranes and (b) the interaction of activated C3 with the C3 receptor of B lymphocytes. There is growing evidence that B-cell mitogens and T (thymus-derived cell)-independent antigens are capable of activating the alternate pathway of the complement system (bypass). Therefore, the effect of another potent bypass inducer was investigated with regard to B-cell activation and the role of C3. Purified, pyrogen-free cobra venom factor was mitogenic for both T and B lymphocytes (cortisone-resistant mouse thymus cells and lymph node lymphocytes from congenitally athymic mice). Venom factor could substitute for T cells by restoring the potential of antibody formation to sheep red blood cells in mouse B-cell cultures supplemented with macrophages or 2-mercaptoethanol. Venom factor may be capable of conferring activated C3 to the C3 receptor of B lymphocytes: preincubation of lymphoid cells with homologous serum or plasma, 10 mM EDTA, and sepharose-coupled venom factor converted with serum to an enzyme active against C3, inhibited their capacity to subsequently form rosettes with sheep erythrocytes sensitized with amboceptor and C5-deficient mouse complement. In the absence of EDTA, preincubation of freshly prepared B-cell suspensions with C3-sufficient homologous serum also blocked their subsequent interaction with complement-sensitized erythrocytes and at the same time rendered them reactive to an otherwise T-cell-specific mitogen. Moreover, mitogen induced B-cell proliferation in lymph node (but not in spleen) cell cultures, appeared to depend on the availability of exogenous C3: zymosan-absorbed fetal bovine serum (only 8.3% site-forming units remaining) supported T-cell activation by phytohemagglutinin, concanavalin A, and venom factor, but failed to sustain B-cell stimulation by pokeweed mitogen, lipopolysaccharide, and venom factor. T-cell-dependent antibody formation in composite cultures containing T cells or T-cell-substituting B-cell mitogens, B cells, and macrophages, always required the presence of C3-sufficient serum.

Emergence of a community-associated methicillin-resistant Staphylococcus aureus strain with a unique resistance profile in Southwest Nigeria.

Phenotypic, genotypic, and toxin gene analyses have not yet been done all in one for the Nigerian Staphylococcus aureus population. This study provides a comprehensive overview of the molecular epidemiology and genetic diversity of S. aureus strains at the largest university clinic in Ibadan, Nigeria. From 1,300 patients' clinical samples collected at the University Teaching Hospital in Ibadan, Nigeria, during a 1-year-surveillance in 2007, 346 nonduplicate S. aureus isolates were obtained. All isolates underwent antibiotic susceptibility testing, toxin gene analysis, multilocus sequence typing, agr group typing, and spa typing. For methicillin (meticillin)-resistant S. aureus (MRSA), staphylococcal cassette chromosome mec (SCCmec) typing was also performed. Of the 346 isolates, 20.23% were methicillin resistant. Thirty-three patients' isolates (47.15%) fulfilled the definition criteria for community-associated MRSA (CA-MRSA) according to a review of the medical charts. The majority of MRSA strains analyzed were isolated from surgical or pediatric patients. The commonest types of MRSA infection identified were surgical-site infections (>70%), whereas those for CA-MRSA were conjunctivitis and otitis (19 patients [57.6%]) and accidental skin and subcutaneous tissue infections (14 patients [42.4%]). The methicillin-susceptible S. aureus strains (ST1, ST5, ST15, ST7, ST8, ST25, ST30, ST72, ST80, ST121, and ST508) were heterogeneous by phenotypic and genotypic analyses. The first report of a Panton-Valentine leukocidin-positive ST88 strain (agr III, SCCmec IV) in Nigeria, as well as genetic analyses of this strain, is presented in this study. The ST88 strain was resistant to trimethoprim-sulfamethoxazole as well as to penicillin and oxacillin. CA-MRSA infections are increasing rapidly among young patients with ophthalmologic and auricular infections. Urban regions with populations of lower socioeconomic status and evidence of overcrowding appear to be at high risk for the emergence of this clone.

Comparison of the performance of the rapid antigen detection actim Influenza A&B test and RT-PCR in different respiratory specimens.

Nowadays, influenza antigen detection test kits are used most frequently to detect influenza A or B virus to establish the diagnosis of influenza rapidly and initiate appropriate therapy. This study was conducted to evaluate the performance of the actim Influenza A&B test (Medix Biochemica). Overall, 473 respiratory specimens were analysed in the actim Influenza A&B test and the results were compared with those from an RT-PCR assay; 461 of these samples originated from paediatric patients aged 7 weeks to 6.5 years either with influenza-related symptoms or from the intensive care unit, and 12 samples originated from adults with underlying lung or haematological diseases. Diagnosis of influenza A or B virus could be established using the actim Influenza A&B test (9/473 samples for influenza A virus and 6/473 for influenza B virus). RT-PCR revealed 23 patients with influenza virus (13/473 for influenza A virus and 10/473 for influenza B virus). The sensitivity and specificity of the actim Influenza A&B test were 65 and 100 % compared with the RT-PCR assay. However, 32 external quality assessment samples containing seven different strains of influenza A subtypes H1N1 and H3N2 and the avian H5N1 were detected correctly by the actim Influenza A&B test. No cross-reactivity to a range of bacterial, fungal and other viral pathogens was observed. In conclusion, the actim Influenza A&B test is reliable for positive results due to its high specificity. Nevertheless, negative results from this test need to be confirmed by a more sensitive assay because of the low sensitivity observed with diagnostic samples.

Inhalational anesthetics stereochemistry: optical resolution of halothane, enflurane, and isoflurane.

Halothane (I), enflurane (II), and isoflurane (III), which are among the most important inhalation anesthetics, are currently administered as racemic mixtures. The pure enantiomers have not been described, and no analytical method for resolving the commercially available racemic mixtures has been reported. Complete optical resolution of (+/-)-I and (+/-)-III on per-n-pentylated alpha-cyclodextrin (Lipodex A) and of (+/-)-II and (+/-)-III on octakis(6-O-methyl-2,3-di-O-pentyl)-gamma-cyclodextrin capillary columns has been achieved, making rapid and convenient determination of enantiomeric ratios in samples of all three of these anesthetics possible.

[Microbial spectrum in surgical infections based on a microbiological routine monitoring over the 10-year period from 1995 to 2004]. / Erregerspektrum chirurgischer Infektionen im 10-Jahres-Vergleich 1995-2004 eines mikrobiologischen Routinemonitorings.

INTRODUCTION: Infections belong to the most frequent and dangerous complications in surgery. In addition to the medical aspects, these infections may have a significant impact on the costs and the overall economic efficacy of medical treatment under the present circumstances of DRG. AIM: A systematic, prospective collection and retrospective evaluation of all consecutive microbiological analyses in specimens from the 3 medical floors (except the ICU) of the Department of Surgery at the University Hospital, Magdeburg (Germany) was performed in 1995, 2002 and 2004 to characterise i) the 10-year course (1995 vs. 2004) and ii) possible alterations due to changes in the previously existing pavillon system (2002 vs. 2004). PATIENTS AND METHODS: The microbial spectrum was determined in the 3 most frequent specimen types (blood culture, urine sample, wound swab) including number and percentage of the single microbial groups such as gram-positive and gram-negative Enterobacteriae, pseudomonades and fungi. In addition, the antibiotic resistance of selected microbes was analysed. The primary data were registered in a database and evaluated according to the various questions. RESULTS: Overall, 2 979 microbes were identified in 1995 (2002, 1 338; 2004, 915). On comparing 1995 with 2004, the percentage of gram-positive microbes did not change (50.5 vs. 50.3 %), whereas the percentage of gram-negative enterobacteriae increased: 37.4 vs. 29.1 %. The percentage of detected fungi was only half of that in 1995: 6.2 vs. 12.2 %. In blood cultures, the Klebsiella spp. portion in the group of gram-negative enterobacteriae distinctly increased: 29.6 vs. 18.8 %. While in 2004, MRSA was found in 24.4 % of all detected Staphylococcus aureus strains in swab specimens amounting to a considerable increase compared to 2002 (17.6 %), in 1995, MRSA was not isolated at all in this material. In the fungi group, there was a decrease of the Candida albicans portion vs. the non-C. albicans strains, which was associated with an increasing resistance against fluconazol. This requires treatment with caspofungin, resulting in increased costs vs. those necessary for fluconazol treatment. CONCLUSION: A systematic, microbiological, long-term monitoring is indispensable since i) microbial detection plays a growing role to include the various types of infections in the spectrum of diagnosis for DRG, ii) alterations of the microbial spectrum can only be detected through a long-term observation period (MRSA, fungi) and iii) simultaneously developing antibiotic resistances can be determined (MRSA, ESBL strains in Enterobacteriae, fluconazol-resistant fungi). This can have an infectious, biological, hygienic and cost-determining as well as a health policy relevance among others, with considerable additional costs (e. g., isolation of patients, cost-intensive substitutional medication) with necessary reimbursement.

Genetic classification and distinguishing of Staphylococcus species based on different partial gap, 16S rRNA, hsp60, rpoB, sodA, and tuf gene sequences.

The analysis of 16S rRNA gene sequences has been the technique generally used to study the evolution and taxonomy of staphylococci. However, the results of this method do not correspond to the results of polyphasic taxonomy, and the related species cannot always be distinguished from each other. Thus, new phylogenetic markers for Staphylococcus spp. are needed. We partially sequenced the gap gene (approximately 931 bp), which encodes the glyceraldehyde-3-phosphate dehydrogenase, for 27 Staphylococcus species. The partial sequences had 24.3 to 96% interspecies homology and were useful in the identification of staphylococcal species (F. Layer, B. Ghebremedhin, W. König, and B. König, J. Microbiol. Methods 70:542-549, 2007). The DNA sequence similarities of the partial staphylococcal gap sequences were found to be lower than those of 16S rRNA (approximately 97%), rpoB (approximately 86%), hsp60 (approximately 82%), and sodA (approximately 78%). Phylogenetically derived trees revealed four statistically supported groups: S. hyicus/S. intermedius, S. sciuri, S. haemolyticus/S. simulans, and S. aureus/epidermidis. The branching of S. auricularis, S. cohnii subsp. cohnii, and the heterogeneous S. saprophyticus group, comprising S. saprophyticus subsp. saprophyticus and S. equorum subsp. equorum, was not reliable. Thus, the phylogenetic analysis based on the gap gene sequences revealed similarities between the dendrograms based on other gene sequences (e.g., the S. hyicus/S. intermedius and S. sciuri groups) as well as differences, e.g., the grouping of S. arlettae and S. kloosii in the gap-based tree. From our results, we propose the partial sequencing of the gap gene as an alternative molecular tool for the taxonomical analysis of Staphylococcus species and for decreasing the possibility of misidentification.

Extended-spectrum Beta-lactamase detection with different panels for automated susceptibility testing and with a chromogenic medium.

Infections caused by extended-spectrum beta-lactamase (ESBL)- and ampC beta-lactamase-producing gram-negative bacteria complicate therapy and limit treatment options. Several different panels for ESBL detection with automated systems exist. In addition, a chromogenic agar medium is available for ESBL screening. We compared two automated identification and susceptibility testing systems with regard to their effectiveness in detecting ESBL production in Enterobacteriaceae: the BD Phoenix system (BD Diagnostic Systems, Sparks, MD) and the Vitek 2 system (bioMerieux, Marcy l'Etoile, France). We tested 114 strains using the Etest as the standard, various available panels for both automated systems (for BD Phoenix, the NMIC/ID-50 and NMIC/ID-70 GN Combo panels for combined identification and susceptibility testing of gram-negative bacilli, and for Vitek 2, the ID-GNB panel for identification of gram-negative bacilli and the AST-N020, AST-N041, and AST-N062 panels for susceptibility testing), and a chromogenic agar medium (bioMérieux, Marcy l'Etoile, France). PCR for common ESBL gene families (encoding TEM, SHV, OXA, and CTX-M) and for chromosomal or plasmid-mediated ampC beta-lactamase genes was conducted to complete the study design. For the tested specimens overall, the chromID ESBL agar showed the highest sensitivity (95.8%) but the lowest specificity (10.5%) compared to the sensitivity and specificity of the Etest (chosen as reference by the authors) for the detection of ESBL-producing strains. The BD Phoenix system showed sensitivities of 77.1% and 84.2% and specificities of 61.5% and 75.0%, respectively, for the NMIC/ID-50 andNMIC/ID-70 panels. The sensitivity of the Vitek 2 system ranged from 78.8% (AST-N020) to 80.6% (AST-N062) and up to 84.2% (AST-N041). The specificities of the respective panels were 50.0% (AST-N041 and AST-N062) and 55.6% (AST-N020). In conclusion, the sensitivities and specificities of ESBL detection by the different methods differ depending on the microorganisms under study.

Subtyping of ST22-MRSA-IV (Barnim epidemic MRSA strain) at a university clinic in Germany from 2002 to 2005.

Emergence of the meticillin-resistant Staphylococcus aureus (MRSA) Barnim epidemic strain (ST22-MRSA-IV) was demonstrated recently at University Hospital in Magdeburg, Germany. To aid the study of transmission events, it is important to have an epidemiological typing method with the ability to distinguish among MRSA isolates. The aim of this study was to determine the ability of phenotypic and genotypic methods to type ST22-MRSA-IV strains within a hospital for microevolution events. Forty-two ST22-MRSA-IV strains collected from 2002 to 2005 were analysed using antimicrobial testing, toxin gene analysis, PFGE, spa typing, fluorescent amplified fragment length polymorphism (fAFLP) and determination of staphylococcal interspersed repeat units (SIRUs). Four different antimicrobial patterns were observed. The majority of the isolates (n=31) were resistant towards erythromycin, ciprofloxacin and clindamycin, in addition to penicillin and oxacillin. All strains harboured the sec gene and showed a homogeneous profile of toxin genes. One isolate was typed as spa t022, two as spa t474 and the remainder belonged to spa type t032. PFGE yielded eight profiles and SIRU typing resulted in six different patterns. The fAFLP technique subdivided the individual PFGE profiles, but the grouping of isolates differed from that obtained by PFGE or SIRU typing. These results showed a diversity of ST22-MRSA-IV strains within a narrow clinical setting, indicating microevolution of the Barnim MRSA clone. The ability to distinguish among MRSA strains within an endemic setting will lead to a greater understanding of the transmission of MRSA and is necessary to be able to control the spread of various clones.

Differentiation of Staphylococcus spp. by terminal-restriction fragment length polymorphism analysis of glyceraldehyde-3-phosphate dehydrogenase-encoding gene.

Classical phenotypic and biochemical testing do not lead to correct identification of the distinct Staphylococcus species. Therefore, the aim of our study was to develop a method for the reliable and accurate determination of distinct Staphylococcus species. In the present study, the 931-934-bp partial sequences of the glyceraldehyde-3-phosphate dehydrogenase-encoding (gap) gene of 28 validly described Staphylococcus species were amplified and sequenced. By using the respective sequence information we performed a terminal-restriction fragment length polymorphism (T-RFLP) analysis. For T-RFLP the partial gap gene was amplified with double-fluorescently labelled primers and digested with the restriction enzymes DdeI, BspHI and TaqI. Distinctive T-RFLP patterns were rendered by the use of capillary electrophoresis with laser-induced fluorescence detection. This molecular method allowed us to identify all 28 Staphylococcus species with high specificity. This was validated by analysis of 34 Staphylococcus epidermidis and 28 Staphylococcus haemolyticus isolates. These results demonstrate the feasibility and applicability of the T-RFLP method based on the partial gap gene sequences for rapid and accurate species identification.

Trichotoxin A40. Purification by counter-current distribution and sequencing of isolated fragments.

The isolation of the membrane-modifying polypeptide antibiotics from the mycelium of Trichoderma viride 5242 was optimized via extraction with dichloromethane and chromatography on Sephadex LH-20. The components trichotoxin A40 and A50 were separated from each other and purified by multiplicative counter-current distribution. The sequence of proteinase-resistant trichotoxin A40 was determined by combined gas chromatography and mass spectrometry of three isolated N-acetylated dodecapeptides and two N-prolylhexapeptides obtained after selective trifluoroacetolysis. Including amino acid exchanges due to natural microheterogeneity, the sequence is Ac-Aib-Gly(LAla)-Aib-LLeu-Aib-LGln-Aib-Aib-Aib(LAla )-LAla-Aib-Aib-LPro-LLeu -Aib-DIva(Aib)-LGlu-LValol. In contrast to the eicosapeptide alamethicin, trichotoxin A40 contains only 18 residues, with a higher proportion of alpha-aminoisobutyric acid (Aib), C-terminal L-valinol (Vol), one D-isovaline (Iva) and no proline at the N-terminal part.

Stereochemistry of 2,4-dimethyleicos-2-enoate from the pyruvylated glycolipid of Mycobacterium smegmatis.

The absolute stereochemistry of 2,4-dimethyleicos-2-enoic acid, isolated from the pyruvylated glycolipid of Mycobacterium smegmatis, has been determined. The two enantiomers of methyl 2,4-dimethyleicos-2-enoate were synthesised for the first time but could not be separated by gas chromatography on cyclodextrin phases. (E)-2-Methyloctadec-2-enoate, an intermediate in the synthesis, is a characteristic component of acyl trehalose glycolipids from Mycobacterium fortuitum. Ozonolysis of the fatty acid ester mixture, obtained from the pyruvylated glycolipid produced 2-methyloctadecanoate. It was identified as the (S)-enantiomer by comparison with (2R) and (2S)-2-methyloctadecanoic acid, intermediates in the synthesis of (4R)- and (4S)-2,4,-dimethyleicos-2-enoic acid, using enantioselective gas chromatography of the methyl esters with heptakis(2,6-di-O-methyl-3-O-pentyl)-beta-cyclodextrin as a chiral stationary phase. The natural acid was therefore determined to be 2E-(4S)-2,4-dimethyleicos-2-enoic acid.

The metabolism of leukotrienes in blood plasma studied by high-performance liquid chromatography.

The metabolism of leukotrienes (B4, C4, D4, and E4) within human plasma was studied and a simple sample preparation is presented. It was demonstrated that leukotriene E4 and leukotriene B4 were stable during incubation at 37 degrees C using the in vitro system. In contrast, leukotriene C4 was metabolized by gamma-glutamyl transpeptidase activities into leukotriene D4 which was further metabolized by dipeptidase activities of plasma into leukotriene E4. The transition state inhibitor of gamma-glutamyl transpeptidase L-serine-borate decreased the metabolism of leukotriene C4 in plasma. Dilution of plasma demonstrated that the dipeptidase was more active compared to the gamma-glutamyl transpeptidase. The metabolizing activities of plasma were functionally characterized by fractionating the plasma proteins.

Functional characteristics of leukotriene C4- and D4-metabolizing enzymes (gamma-glutamyl transpeptidase, dipeptidase) within human plasma.

Several properties of the leukotriene C4- and leukotriene D4-metabolizing enzymes within human plasma were studied after fractionation of the plasma proteins using ammonium sulfate precipitation. Leukotriene D4-metabolizing enzymes were widely distributed among the fractions obtained. They showed different pH optima (pH 6.5, pH 7.0 and pH greater than or equal to 8.5) and revealed a different degree of thermal stability. The results indicate the presence of more than one enzyme in plasma which interacts with leukotriene D4. EDTA and L-cysteine inhibited the metabolism of leukotriene D4. Two leukotriene C4-metabolizing activities (gamma-glutamyl transpeptidases) differing in their molecular weights were detected after gel filtration. Their molecular weights were estimated to be Mr greater than or equal to 150 000 and Mr between 55 000 and 100 000.

Structural and membrane modifying properties of suzukacillin, a peptide antibiotic related to alamethicin. Part A. Sequence and conformation.

The primary structure and conformation of the polypeptide antibiotic suzukacillin A are investigated. Suzukacillin A is isolated from the Trichoderma viride strain 1037 and exhibits membrane modifying and lysing properties similar to those of alamethicin. A combined gas chromatographic mass spectrometric analysis of the trifluoroacetylated peptide methyl esters of partial hydrolysates revealed a tentative sequence of 23 residues including 10 2-methylalanines and one phenylalaninol, which shows many fragments known from alamethicin: Ac-Aib-Pro-Val-Aib-Val-Ala-Aib-Ala-Aib-Aib-Gln-Aib-Leu-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu(Pheol)-Gln-OH. All chiral amino acids and phenylalainol have L-configuration. Ultraviolet and infrared spectroscopy, circular dichroism in various solvents and in particular 13C nuclear magnetic resonance have been used for a comparative study of suzukacillin with alamethicin. Suzukacillin has a partially alpha-helical structure and the helix content increases largely from polar to lipophilic solvents. Suzukacillin aggregates more strongly than alamethicin in aqueous medis due to a longer alpha-helical part and higher number of aliphatic residues. A part of the alpha-helix is exceptionally stabilized due to 2-methylalanine residues shielding the peptide bonds from interactions with polar solvents. In lipophilic solvents and lecithin vesicles particularly large temperature induced reductions of the high alpha-helix content are found for alamethicin. Suzukacillin shows similar temperature coefficients in lipophilic media, however, in contrast to alamethicin a more linear change in intensity of the Cotton effects is observed.

Structural and membrane modifying porperties of suzukacillin, a peptide antibiotic related to alamethicin. Part B. Pore formation in black lipid films.

Suzukacillin, a polypeptide consisting of presumably 23 amino acids and 1 phenylalaninol, is produced by a Trichoderma viride strain No. 1037 and it can be isolated from the culture medium. It shows membrane-modifying properties similar to those of alamethicin. Discrete condustance fluctuations indicate the formation of oligomer pores of varying diameter. On the basis of voltage jump relaxation experiments evidence is given that the dimer is the nucleation state from which pore formation starts and the oligomer disappears. According to the voltage-current characteristics, voltage-dependent and voltage-independent conductances are observed. A slow process is involved, which can be interpreted as a change in the equilibrium distribution between different conformations of the suzukacillin monomer at the membrane interphase. This change results from its interaction with the lipid matrix. Differences in experimental observations between suzukacillin and alamethicin are attributed to the relatively larger alpha-helix and higher number of aliphatic side chains of the suzukacillin monomer and to a more intense interaction with the lipid membrane. This leads to a higher probability of forming dimers from monomers and to the occurrence of "inactivation".

ICAM-1 expression and low-molecular-weight G-protein activation of human bronchial epithelial cells (A549) infected with RSV.

The airway epithelial cell of the lower respiratory tract is the primary host target cell for respiratory syncytial virus (RSV) infection. To estimate whether infected epithelial cells contribute to the inflammatory host response observed during the acute infection phase we analyzed the cell surface expression of intercellular adhesion molecule-1 (ICAM-1, CD54) on human bronchial epithelial cells (A549) following RSV infection and cytokine priming. The epithelial cells constitutively expressed ICAM-1. The ICAM-1 surface expression was significantly up-regulated up to 72 h post RSV infection. In addition, cytokine priming with tumor necrosis factor alpha (TNF-alpha), interferon-gammma (IFN-gamma), or interleukin-1 alpha/beta (IL-1alpha/beta) induced an enhanced ICAM-1 expression on noninfected as well as on RSV-infected epithelial cells. As early as 24 h post RSV infection and cytokine priming, respectively, the maximum of ICAM-1-expressing cells was observed. In contrast, the maximal ICAM-1 up-regulation per cell was measured 24 h later, e.g., after 48 h of culture. Cytokine priming with IL-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor (G-CSF) did not lead to a significant increase of ICAM-1 expression either on RSV-infected or on noninfected A549 cells up to 72 h of culture time. Performed signal transduction experiments, e.g. [alpha-32P]GTP-binding blot analysis, revealed that low-molecular-weight GTP-binding proteins possess an enhanced GTP-binding capacity in RSV-infected A549 cells.

Metabolism of leukotriene B4 via reduction into dihydro-leukotriene B4 in human monocytes, alveolar macrophages, and U-937 cells.

The biological effects of leukotriene B4 (LTB4) within the microenvironment are controlled by rapid inactivation. In this regard human granulocytes convert LTB4 into omega-oxidated products (20-OH-LTB4 and 20-COOH-LTB4); moreover, we recently described the formation of unpolar metabolites of LTB4 in human tonsillar and lung macrophages. By means of high performance liquid chromatography (HPLC) we identified the main metabolite of LTB4 as dihydro-LTB4 (5,12-dihydroxyeicosatrienoic acid). Studies on a lymphocyte (74-78%), monocyte (19-22%), and basophil (less than 4%) containing cell fraction isolated from peripheral blood as well as peripheral monocytes purified by elutriation centrifugation revealed evidence that these cells metabolize LTB4 to a very low degree if incubated immediately after isolation. However, after culture for 24-72 h these cells showed a strongly increased capacity to metabolize LTB4. The pattern of metabolites in this cell fraction was identical to bronchoalveolar macrophages (purity greater than 95%). Similarly, the LTB4-reductase was expressed in differentiated human monocytic U-937 cells almost 5-7 h after the addition of dimethylsulfoxide (1.3%) or phorbol-myristate-acetate (16 nM). The expression of this pathway was blocked in the presence of cycloheximide (10 micrograms/ml) whereas actinomycin (3.8 micrograms/ml) had no effects. Dihydro-LTB4 was further metabolized by granulocytes probably via omega-oxidation; therefore, several metabolites could be detected by radioactive high performance liquid chromatography (HPLC) after incubation of bronchoalveolar cells consisting of macrophages and granulocytes with 3H-LTB4. Our data provide evidence for a unique role of macrophages to control the level of LTB4 by generation as well as metabolism into dihydro-LTB4.