Treatment of complicated skin and skin structure infections in areas with low incidence of antibiotic resistance-a retrospective population based study from Finland and Sweden.

Complicated skin and skin-structure infections (cSSSI) are a common reason for hospitalization and practically all new antimicrobial agents against Gram-positive bacteria are studied in cSSSI. The aim of this population-based observational study was to assess the treatment of patients with cSSSI in areas with a low incidence of antibiotic resistance. The study population consisted of adult residents who were treated because of cSSSI during 2008-2011 from two Nordic cities, Helsinki and Gothenburg. In the final analysis population (460 patients; mean age 60.8 years; 60.9% male) 13.3% of patients had bacteraemia, 15.9% were admitted to an Intensive Care Unit and 51.5% underwent at least one surgical intervention. Treatment failure occurred in 28.2%, initial antibiotic treatment modification to another intravenous drug in 38.5% and streamlining in 5.0% of the cases. Gram-positive bacteria were predominantly isolated, with staphylococci (24.5%) and streptococci (16.0%) being the most common aetiologies. Median overall durations of hospital stay and antimicrobial treatment were 13 and 17 days, respectively, and on average 3.5 (SD 2.1) different antibiotics were used per patient. Oral antimicrobial treatment was continued in 64.3% of patients after discharge. The overall mortality rates in 30 days and in 12 months were 4.1% and 11.8%, respectively, and 16.4% of patients had a recurrence of SSSI within 12 months. In conclusion, in this population-based study antimicrobial treatment modifications were frequent and the treatment time was longer than recommended. However, bacteraemia, clinical failure and recurrences were more common than in previous non-population-based studies.

Emergence of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae during the years 2000 and 2004 in Helsinki, Finland.

The molecular epidemiology of 33 Escherichia coli and 81 Klebsiella pneumoniae extended-spectrum beta-lactamase-producing healthcare-associated and community-acquired isolates collected in the Helsinki district during 2000-2004 was investigated. Clonality studies, antimicrobial susceptibility and genotyping of the isolates were performed. Newly emerging CTX-M-producing E. coli and bla(SHV-12)-producing K. pneumoniae isolates were detected. Clonal clusters of both species persisted throughout the study period.

Effects of noise and filtering on SVD-based morphological parameters of the T wave in the ECG.

Singular value decomposition (SVD) based electrocardiogram (ECG) morphology analysis is a novel method in the assessment of subtle abnormalities in the T wave morphology of 12-lead ECG. As various types of noise contaminate the ECG signal and create a bias for the morphological analyses, this study was designed to estimate the effects of noise on the SVD method in an experimental setup. Ideal signals were generated by filtering real ECG signals several times with the Savitzky-Golay filter. Random and real noise samples were superimposed on the ideal signals. The noisy signals were filtered with a power line interference filter combined with the Savitzky-Golay or the wavelet filter. Results show that noise increased both the dipolar and non-dipolar components significantly unless filtering was applied. R-TWR (relative T wave residuum) and A-TWR (absolute T wave residuum) were four to eight times higher in noisy signals. The experiments with patient data demonstrated that certain types of noise may even lead to erroneous classification of patients. Filtering brings the median values closer to the correct ones and decreases significantly the variance of the values of parameters.

Analysis of tyrosine kinase mRNAs including four FGF receptor mRNAs expressed in MCF-7 breast-cancer cells.

The MCF-7 cell line is a hormone-responsive human breast-cancer cell line, which has been extensively used in studies of estrogen regulation of cell growth. These studies have indicated that the growth stimulation of the MCF-7 cells by estrogens may be effected by an autocrine mechanism involving several growth factors, such as EGF, TGF alpha and IGF-I and their receptors. We have amplified and cloned tyrosine-kinase-related sequences from the MCF-7 cell mRNA using the polymerase chain reaction and characterized the partial cDNAs obtained by nucleic acid sequencing. Nine tyrosine kinase cDNAs and one serine/threonine kinase cDNA were identified among the amplified sequences. Four different tyrosine kinase genes encoding receptors for fibroblast growth factors (FGFs) were found to be expressed by the MCF-7 cells. In addition, differences were observed in the expression of these members of FGF receptor family in different breast-cancer cells. A putative tyrosine-kinase receptor and a novel serine/threonine kinase were preferentially expressed in estrogen-responsive tumor cell lines. However, no estrogen-dependent regulation of any of the novel tyrosine-kinase receptor mRNAs was found in any of the cell lines including the MCF-7 or ZR-75-I cells, where the expression of the neu proto-oncogene mRNA was decreased during estrogen treatment. The expression of several FGF receptors by breast-cancer cells suggests that FGFs may be involved in their growth regulation and tumorigenesis.

A chimeric EGFR/neu receptor in functional analysis of the neu oncoprotein.

As the factor binding to the neu protein has been unknown, it has not been possible to confirm experimentally the proposed growth-factor receptor like functions of the neu protein. To approach this problem we constructed a recombinant receptor which enabled ligand regulation of the neu tyrosine kinase. The hybrid receptor consisted of the extracellular ligand binding, transmembrane and protein kinase C-substrate domains joined to the intracellular tyrosine kinase and carboxyl-terminal domains of the neu protein. Several properties of NIH3T3 cells carrying this construct were tested. We obtained the first experimental evidence that the neu proto-oncogene has mitogenic and transforming activities only in the presence of a ligand stimulating its tyrosine kinase activity. Various cellular and molecular biological parameters indicated that the chimeric receptor behaved very similarly to the EGFR. Also, this chimeric receptor has allowed us to compare the constitutive oncogenic and the ligand-activated non-oncogenic activities of the neu tyrosine kinase. In the future we plan to focus on characterization of possible differences between EGFR and neu signalling in more differentiated cellular backgrounds.

Down-regulation of cellular platelet-derived growth factor receptors induced by an activated neu receptor tyrosine kinase.

The functional integration of growth factor signaling occurs at several levels in target cells. One of the most proximal mechanisms is receptor transmodulation, by which one activated receptor can regulate the expression of other receptors in the same cells. Well-established transregulatory loops involve platelet-derived growth factor (PDGF) down-regulation of epidermal growth factor (EGF) receptors and beta-type transforming growth factors modulation of PDGF receptors. We have studied the relationship between neu tyrosine kinase activation and the expression of the PDGF receptors in transfected NIH/3T3 cells. Expression of the neu oncogene, but not of the neu proto-oncogene, was associated with a decrease of PDGF alpha- and beta-receptors on the cell surface, as measured by [125-I]PDGF-AA and -BB binding. These results were corroborated by metabolic labeling and immunoprecipitation of the PDGF beta-receptors. PDGF alpha- and beta-receptor mRNAs were strongly decreased in the neu oncogene-transformed cells in comparison with control cells expressing the neu proto-oncogene. Down-regulation of the PDGF receptors and their mRNAs was also observed after EGF treatment of cells expressing a chimeric EGF receptor/neu receptor, where the neu tyrosine kinase is activated by EGF binding. These results show that the neu tyrosine kinase can down-modulate PDGF receptor expression, and the effect is mediated via decreased PDGF receptor mRNA levels.

Constitutively activated neu oncoprotein tyrosine kinase interferes with growth factor-induced signals for gene activation.

The neu receptor oncoprotein tyrosine kinase, capable of transforming cultured fibroblasts and causing mammary carcinomas in transgenic mice, carries a point mutation in its transmembrane domain and shows a constitutive tyrosine kinase activity. We analyzed the neu tyrosine kinase and its substrates in transfected NIH 3T3 fibroblasts by phosphotyrosine immunoblotting. Tyrosine phosphorylated proteins were similar but not identical in epidermal growth factor (EGF)-stimulated cells expressing the human EGF receptor (EGFR) or a chimeric EGFR/neu receptor but differed from phosphotyrosyl proteins constitutively expressed in neu oncogene-transformed cells. The neu oncoprotein in the latter cells was phosphorylated in tyrosine in a ligand-independent manner and had a shortened half-life in comparison with the normal neu protein. Tumor promoter pretreatment inhibited ligand-induced receptor tyrosine phosphorylation and decreased tyrosine phosphorylated neu oncoprotein. Prolonged pretreatment with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) also prevented the induction of immediate early growth factor-regulated genes in response to neu activation. Expression of the neu oncogene but not the protooncogene in NIH 3T3 cells was associated with enhanced levels of the jun and fos oncoproteins and loss of serum growth factor induction of immediate early mRNA responses. The constitutively activated neu oncoprotein tyrosine kinase thus deregulates cellular genomic responses to growth factors.

Downregulation of the early genomic growth factor response in neu oncogene-transformed cells.

Peptide growth factor-induced signal transduction leads to a long-term adjustment of the genetic programs of responding cells. A point mutation in the transmembrane domain of the neu receptor has been found to activate its tyrosine kinase and oncogenic potential. Our previous studies show that ligand stimulation of a chimeric epidermal growth factor receptor-neu proto-oncogene (EGF-R/neu) induces the neu tyrosine kinase and leads to the programmed activation of cell growth-regulated genes. We have now studied the effect of the neu oncoprotein on the genomic growth factor response in cells expressing the EGF-regulated neu tyrosine kinase. Expression of the neu oncogene in these cells inhibited 75-90% of the EGF-stimulated mRNA induction of the immediate early serum response genes, such as junB encoding a transcription factor, N10 encoding a putative nuclear hormone binding receptor for an as yet undefined ligand, and B10, the protein product of which is still unknown. The relative lack of mRNA induction was not due to a loss of the chimeric EGF-R/neu receptors from the cell surface. Also, the neu oncogene decreased serum- and tumor promoter induction of these genes. Our results suggest that the neu oncogene is capable of deregulating mRNA responses to extracellular signalling, similar to the effects of the c-Ha-ras oncogene. Knowledge of the mechanisms responsible for these changes in gene regulation will help to define oncogenic transformation of cells in molecular terms.

Activation of the neu tyrosine kinase induces the fos/jun transcription factor complex, the glucose transporter and ornithine decarboxylase.

We have studied the ability of the neu tyrosine kinase to induce a signal for the activation of cell growth-regulated genes. Serum-starved NIH 3T3 cells expressing an epidermal growth factor receptor (EGF-R)/neu construct encoding a hybrid receptor protein were stimulated with EGF and the activation of the neu tyrosine kinase and stimulation of growth factor inducible genes were followed at the mRNA, protein, and activity levels, and compared to the corresponding responses in the neu proto-oncogene and oncogene expressing cells. Induction of the expression of jun mRNAs was an immediate early effect of EGF stimulation, followed by a marked increase in the biosynthesis of the fos/jun transcription factor complex and an increased transcription factor activity as measured by a recombinant transcription unit using chloramphenicol acetyltransferase assays. In distinction, elevated AP-1/PEA-1 activity in the absence of a significant increase in jun and fos expression was characteristic of the neu oncogene-expressing cells. The glucose transporter mRNA increased at 2 h of EGF stimulation and was associated with enhanced glucose transport of the EGF-treated cells. An increase of ornithine decarboxylase (ODC) mRNA and activity followed these changes. In contrast, serum-starved, EGF-treated neu proto-oncogene- and oncogene-expressing cells showed constitutively low and high glucose transporter and ODC activities, respectively. These findings demonstrate that the chimeric EGF-R/neu receptor is capable of activating the expression of both immediate early genes and biochemical activities associated with cell growth stimulation.

A chimeric EGF-R-neu proto-oncogene allows EGF to regulate neu tyrosine kinase and cell transformation.

The neu oncogene, characterized by Weinberg and colleagues, is a transforming gene found in ethylnitrosourea-induced rat neuro/glioblastomas; its human proto-oncogene homologue has been termed erbB2 or HER2 because of its close homology with the epidermal growth factor receptor (EGF-R) gene (c-erbB1). Expression of the rat neu oncogene is sufficient for transformation of mouse NIH 3T3 fibroblasts in culture and for the development of mammary carcinomas in transgenic mice, but the neu proto-oncogene has not been associated with cell transformation. We constructed a vector for expression of a chimeric cDNA and hybrid protein consisting of the EGF-R extracellular, transmembrane and protein kinase C-substrate domains linked to the intracellular tyrosine kinase and carboxyl terminal domain of the rat neu cDNA. Upon transfection with the construct, NIH 3T3 cells gave rise to EGF-R antigen-positive cell clones with varying amounts of specific EGF binding. Immunofluorescence and immunoprecipitation using neu- and EGF-receptor specific antibodies demonstrated a correctly oriented and positioned chimeric EGF-R-neu protein of the expected apparent mol. wt on the surface of these cells. EGF or TGF alpha induced tyrosine phosphorylation of the chimeric receptor protein, stimulated DNA synthesis of EGF-R-neu expressing cells and led to a transformed cell morphology and growth in soft agar. In contrast, the neu proto-oncogene did not show kinase activity or transforming properties when expressed at similar levels in NIH 3T3 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Receptor downregulation and DNA synthesis are modulated by EGF and TPA in cells expressing an EGFR/neu chimera.

EGF was used to stimulate a chimeric receptor consisting of the epidermal growth factor receptor (EGFR) extracellular, transmembrane, and protein kinase C-substrate domains linked to the intracellular tyrosine kinase and carboxyl terminal domains of the rat neu protein in NIH/3T3 cells. EGF-induced rapid and delayed morphological changes consisted of membrane ruffling, increased pinocytosis, extension of lamellar actin-containing footpads at the cell periphery and partial reorganization of the actin stress fibers in the cells. EGF bound to the cells was rapidly internalized in a complex with the EGFR/neu protein, as shown by loss of EGF binding and EGFR antigens from the cell surface. The movement of the EGFR/neu protein was followed with indirect immunofluorescence into a vesicular intracellular compartment using antibodies against both EGFR and neu protein domains. Metabolic labeling and pulse-chase experiments indicated that the receptor was degraded soon after its internalization. EGF treatment also induced the junB transcription factor mRNA and a dose-dependent stimulation of DNA synthesis in cultures expressing the chimeric receptor. The tumor promoter TPA led to a transient loss of cell surface receptors and prevented EGF stimulation of DNA synthesis but did not completely abolish junB mRNA induction or increase degradation of the chimeric receptor. These results show that the chimeric EGFR/neu receptor undergoes typical downregulation upon ligand binding and TPA pretreatment and is capable of transducing an EGF-induced mitogenic signal.