Establishment of a reliable in-vivo model of implant-associated infection to investigate innovative treatment options.

The increasing number of implant-associated infections and of multiresistant pathogens is a major problem in the daily routine. In the field of osteomyelitis, it is difficult to manage a valid clinical study because of multiple influencing factors. Therefore, models of osteomyelitis with a simulation of the pathophysiology to evaluate treatment options for implant-associated infections are necessary. The aim of this study is to develop a standardized and reproducible osteomyelitis model in-vivo to improve treatment options. This study analyses the influence of a post-infectious implant exchange one week after infection and the infection progress afterward in combination with a systemic versus a local antibiotic treatment in-vivo. Therefore, the implant exchange, the exchange to a local drug-delivery system with gentamicin, and the implant removal are examined. Furthermore, the influence of an additional systemic antibiotic therapy is evaluated. An in-vivo model concerning the implant exchange is established that analyzes clinic, radiologic, microbiologic, histologic, and immunohistochemical diagnostics to obtain detailed evaluation and clinical reproducibility. Our study shows a clear advantage of the combined local and systemic antibiotic treatment in contrast to the implant removal and to a non-combined antibiotic therapy. Group genta/syst. showed the lowest infection rate with a percentage of 62.5% concerning microbiologic analysis, which is in accordance with the immunohistochemical, cytochemical, histologic, and radiologic analysis. Our in-vivo rat model has shown valid and reproducible results, which will lead to further investigations regarding treatment options and influencing factors concerning the therapy of osteomyelitis and implant-associated infections.

Neurotensin receptor type 1: Escherichia coli expression, purification, characterization and biophysical study.

NT (neurotensin) is an endogenous tridecapeptide neurotransmitter found in the central nervous system and gastrointestinal tract. One receptor for NT, NTS1, belongs to the GPCR (G-protein-coupled receptor) superfamily, has seven putative transmembrane domains, and is being studied by a range of single-molecule, functional and structural approaches. To enable biophysical characterization, sufficient quantities of the receptor need to be expressed and purified in an active form. To this end, rat NTS1 has been expressed in Escherichia coli in an active ligand-binding form at the cell membrane and purified in sufficient amounts for structural biology studies either with or without fluorescent protein [YFP (yellow fluorescent protein) and CFP (cyan fluorescent protein)] fusions. Ligand binding has been demonstrated in a novel SPR (surface plasmon resonance) approach, as well as by conventional radioligand binding measurements. These improvements in production of NTS1 now open up the possibility of direct structural studies, such as solid-state NMR to interrogate the NT-binding site, EM (electron microscopy), and X-ray crystallography and NMR.

Correlation of tumor markers p53, bcl-2, CD34, CD44H, CD44v6, and Ki-67 with survival and metastasis in laryngeal squamous cell carcinoma.

BACKGROUND: Recent basic discoveries about the biological significance of nuclear and cell-surface marker proteins have opened new areas of research into head and neck cancer. However, the clinical significance of these markers is not yet understood. OBJECTIVE: To perform a historical prospective study of 70 patients with squamous cell carcinoma of the larynx who were treated at our institution between 1979 and 1989 to correlate tumor marker expression with survival and metastasis. DESIGN: Archival tissue was immunohistochemically stained for the p53 tumor suppressor gene product, the inhibitor of apoptosis (bcl-2), the stem cell marker CD34, the cell adhesion molecules CD44H and CD44v6, and a marker of cellular proliferation (Ki-67). The slides were examined using a light microscope and scored according to intensity and percentage of cells labeled. The patients were stratified by tumor stage, and survival and metastatic data were correlated with staining scores. RESULTS: For the stage IV group, increased expression of p53 and decreased expression of CD44H and CD44v6 correlated with a decreased survival (P = .03, P = .03, and P = .02, respectively), and decreased expression of CD44H correlated with an increase in metastasis (P = .01). For all stages, excluding metastatic cases, increased p53 expression was consistent with a shorter survival (P < .03), while increased CD44v6 expression was consistent with a longer survival (P < .02). CONCLUSIONS: The present study suggests that a loss of cell proliferation control implied by overexpression of p53 and loss of cell adhesion implied by decreased expression of CD44 may be determinants of survival in patients with carcinoma of the larynx. The tumor markers bcl-2 and Ki-67 were not prognostic discriminators in this limited series. This study also indicates that the stem cell marker CD34 is rarely expressed by laryngeal carcinoma cells.

Photoaffinity labeling of the hemolymph juvenile hormone binding protein of Manduca sexta.

A synthetic analogue of the insect juvenile hormone (JH) III, 10,11-epoxy[10-3H]farnesyl diazoacetate [( 3H]-EFDA), binds to several proteins in a partially purified preparation of hemolymph protein from fourth instar larvae of Manduca sexta when irradiated with UV light. Approximately 80% of this binding could be inhibited by the addition of excess unlabeled JH I. To compare the relative affinity of EFDA for the juvenile hormone binding protein (JHBP) with that of the various JH homologues, the ability of unlabeled EFDA and JH homologues to displace [3H]JH I from binding sites was measured. The relative affinities were EFDA greater than JH I greater than JH II greater than JH III. When Scatchard analysis of the binding of [3H]EFDA or [3H]JH I to the larval JHBP was performed, an estimated apparent KD of 4.5 X 10(-8)M was found for EFDA, whereas for JH I a slightly higher KD of 8.8 X 10(-8) M was calculated. To determine if [3H]EFDA bound at the JH I binding site, displacement of [3H]JH I from the JHBP complex with unlabeled JH I, JH II, and JH III was compared to the displacement of [3H]EFDA with the same homologues. The results demonstrated that the photoaffinity label bound covalently at the JH I binding site on the hemolymph binding protein of Manduca sexta. Fluorescence autoradiography of [3H]EFDA photoaffinity labeled proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that [3H]EFDA bound covalently to two major proteins in the absence of JH I.(ABSTRACT TRUNCATED AT 250 WORDS)

A specific photoaffinity label for hemolymph and ovarian juvenile hormone-binding proteins in Leucophaea maderae.

A tritium-labeled diazocarbonyl juvenile hormone (JH) analog, (10-[10,11-3H]epoxyfarnesyl diazoacetate, [3H]EFDA), covalently bound to proteins in both hemolymph and ovarian extracts when reaction mixtures were irradiated with UV light. The addition of various concentrations of unlabeled JH III selectively inhibited [3H]EFDA photoattachment to proteins. Using the Scatchard method of analysis, [3H]EFDA bound specifically and with relatively high affinity (KD = 1.5 X 10(-6) M) to a macromolecule in each extract, although nonspecific binding to other molecules was also present (20-50%). To determine if [3H]EFDA bound at the JH III-binding site on the binding proteins, radioactive [3H]JH III or [3H]EFDA was complexed with proteins in the presence of various concentrations of either unlabeled JH III or JH I under equilibrium conditions. The results demonstrated that the natural hormone, JH III, displaced both bound labeled ligands 4.1 +/- 0.5 times better than the homolog JH I. Thus, the photoaffinity label [3H]EFDA bound at the same site on the protein as [3H] JH III. Fluorescent autoradiography of [3H]EFDA-labeled proteins separated by sodium dodecyl sulfate electrophoresis revealed that several proteins in both hemolymph and ovarian extracts bound [3H]EFDA. To determine the specificity of binding, extracts were irradiated with UV light in the presence of unlabeled JH III and [3H]EFDA. The results demonstrated that JH III prevented photoattachment of [3H]EFDA to a major protein in each extract. The molecular weight of these proteins was estimated at approximately 200,000 for both the hemolymph protein and the ovarian protein.

Assay and identification of juvenile hormone binding proteins in Leucophaea maderae.

We modified a binding assay using polyethylene glycol (PEG) to precipitate bound hormone. Optimum precipitation occurred when reaction mixtures were incubated with 10-40% PEG and 1.25-2.5 mg/ml gamma-globulins for 2-90 min at 4 or 23 degrees C. Results from this assay and from the dextran-coated charcoal assay were similar. Addition of phenylmethylsulfonyl fluoride eliminated nonspecific esterase activity in extracts. JH III-binding macromolecules were identified in hemolymph and ovaries of Leucophaea maderae. These molecules were pronase- and heat-sensitive and saturable. Using Scatchard analysis an average KD of 2.04 (+/- 0.32) X 10(-8) M and 1.91 (+/- 0.80) X 10(-8) M was calculated for hemolymph and ovarian binding proteins. JH III had the highest affinity for binding sites, followed by JH I and JH 0. Various extraction procedures caused changes in JH affinity for both binding proteins. At high concentrations the (+) isomer and mixed isomer preparations of methoprene and hydroprene competed for binding sites. Binding proteins had no affinity for the (-) isomer or for the JH III acid.