Characteristics of extended-spectrum cephalosporin-resistant Escherichia coli isolated from Swiss and imported poultry meat.

A worrisome phenomenon is the progressive global spread of Enterobacteriaceae in poultry and chicken meat expressing plasmid-mediated enzymes that inactivate ß-lactam antibiotics, suggesting that the food chain might play a role in the epidemiology and the transmission of extended-spectrum cephalosporin-resistant Enterobacteriaceae to humans. The aim of the present study was to further characterize 24 extended-spectrum cephalosporin-resistant Enterobacteriaceae isolated from domestic and imported poultry meat by antibiotic susceptibility testing, identification of the blaESBL/blapAmpC genes, conjugation mating experiments and determination of plasmid incompatibility types, multilocus sequence typing, and analysis of the Escherichia coli phylogenetic groups. On account of their resistance patterns, 21 of the total 24 isolates were classified as multidrug resistant. Eleven isolates carried a blaCMY-2 gene, whereas 13 isolates harbored a blaCTX-M-1 gene. All isolates harbored plasmids that were assigned to 8 of the 18 described plasmid incompatibility groups, the most frequent of which were IncI1, IncFIB, IncB/O, and IncFrepB. The blaESBL/blapAmpC genes were harbored mainly by transferable IncI1 and IncB/O plasmids. Multilocus sequence typing as well as E. coli phylogenetic group typing revealed a high heterogenicity even among different isolates of the same sample.

Detection of adeno- and lentiviral (HIV1) contaminations on laboratory surfaces as a tool for the surveillance of biosafety standards.

AIMS: As a biosafety laboratory, we survey the handling of adenovirus type 5 (Ad5) and HIV1-derived lentivirus in contained-use facilities in Switzerland to identify insufficiencies of the safety precautions taken by the laboratories. METHODS AND RESULTS: In the past 9 years, we took 430 swab samples from various types of surfaces in research laboratories. Samples were examined for Ad5 contaminations by real-time PCR and infectivity assay or for the presence of lentivirus (HIV1) nucleic acids by real-time (RT) PCR. Samples collected from centrifuges did not only contain Ad5 DNA more frequently but also exhibited higher numbers of Ad5 and lentiviral (HIV1) DNA copies than swabs from any other area of sampling. Five of ten samples containing infectious Ad5 particles or lentivirus (HIV1) RNA were found in samples taken from centrifuges. Ad5 contamination rates were higher in the tube holder and lower on the inner wall of the rotor chamber in centrifuges that were fitted with aerosol tight covers compared to centrifuges without covers. CONCLUSIONS: Our results allowed the comparison of hygiene standards of different laboratories and lead to the identification of centrifuges as hotspots for contaminations. SIGNIFICANCE AND IMPACT OF THE STUDY: Based on our results, we propose to use the collected data as a tool for rating future swab results. Furthermore, the amount of Ad5 and HIV1-derived lentivirus DNA could serve as an indicator of the level of good laboratory practice in contained-use laboratories handling these viral vectors.

Contaminations of laboratory surfaces with Staphylococcus aureus are affected by the carrier status of laboratory staff.

AIM: As a biosafety laboratory, we take samples from surfaces in microbiological laboratories to survey the handling of micro-organisms. Whereas contaminations with other micro-organisms were rare, Staphylococcus aureus was found in the working environment of many laboratories. As 20-60% of the healthy population are carriers of S. aureus we wanted to asses the effect of carriers on our sampling results. METHODS AND RESULTS: Nasal swabs of staff members in nonmicrobiological laboratories and offices as well as surface samples from their personal work environment were taken and analysed for S. aureus DNA. In addition S. aureus strains were isolated using S. aureus-specific agar plates and analysed by randomly amplified polymorphic DNA (RAPD)-PCR and multilocus sequence typing (MLST). Our data show that contaminations with S. aureus in nonmicrobiological environments are common with 29% of the surface samples containing S. aureus DNA. In the working environment of carriers, the number of contaminations was significantly increased compared to the environment of noncarriers. CONCLUSION: The carrier status of staff members significantly affects the number of contaminations on laboratory surfaces. Therefore, even in the absence of intentional handling of S. aureus, contaminations can be detected on a substantial amount of surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: Sampling procedures need to be adapted based on these results with respect to the locations where samples are taken and the threshold for significant contaminations. Because of its wide distribution, S. aureus can serve as a marker for hygienic standards in laboratories.

Dermal fibroblast-derived growth factors restore the ability of beta(1) integrin-deficient embryonal stem cells to differentiate into keratinocytes.

Embryonal stem (ES) cells that are homozygous null for the beta(1) integrin subunit fail to differentiate into keratinocytes in vitro but do differentiate in teratomas and wild-type/beta(1)-null chimeric mice. The failure of beta(1)-null ES cells to differentiate in culture might be the result of defective extracellular matrix assembly or reduced sensitivity to soluble inducing factors. By culturing embryoid bodies on dead, deepidermized human dermis (DED) we showed that epidermal basement membrane did not induce beta(1)-null ES cells to undergo keratinocyte differentiation and did not stimulate the differentiation of wild-type ES cells. Coculture with epidermal keratinocytes also had no effect. However, when human dermal fibroblasts were incorporated into DED, the number of epidermal cysts formed by wild-type ES cells increased dramatically, and small groups of keratin 14-positive cells differentiated from beta(1)-null ES cells. Fibroblast-conditioned medium stimulated differentiation of K14-positive cells in wild-type and beta(1)-null embryoid bodies. Of a range of growth factors tested, KGF, FGF10, and TGFalpha all stimulated differentiation of keratin 14-positive beta(1)-null cells, and KGF and FGF10 were shown to be produced by the fibroblasts used in coculture experiments. The effects of the growth factors on wild-type ES cells were much less pronounced, suggesting that the concentrations of inducing factors already present in the medium were not limiting for wild-type cells. We conclude that the lack of beta(1) integrins decreases the sensitivity of ES cells to soluble factors that induce keratinocyte differentiation.

Synthesis and receptor binding analysis of thirteen oligomeric alpha-MSH analogs.

Thirteen oligomeric analogs from dimers up to a hexamer of alpha-melanocyte-stimulating hormone (alpha-MSH) were synthesized and tested on melanoma cells for their ability to bind to melanocortin type 1 (MC1) receptors and to stimulate melanin production in the cells. The peptidic oligomers were made by linking several copies of the alpha-MSH fragment analog Nle-Asp-His-[D-Phe]-Arg-Trp-Lys-NH2 to different templates through formation of oxime bonds. They were found to have binding affinities at 37 degrees C up to 8 times higher and melanogenesis-inducing activities up to 4 times higher than those of the native hormone. At 15 degrees C, one dimer showed a binding affinity 20 times higher than that of alpha-MSH. These results are discussed in terms of possible bridging of neighboring receptors which has been suggested to occur in some other systems.

Comparison of integrin, cadherin, and catenin expression in squamous cell carcinomas of the oral cavity.

In addition to their role in maintenance of tissue integrity, cell adhesion molecules regulate the growth and differentiation of stratified squamous epithelia. Reduced expression of E-cadherin and the alpha 2 beta 1, alpha 3 beta 1 and alpha 6 beta 4 integrins is already reported to correlate with poor histological differentiation in oral squamous cell carcinomas. However, it is not clear how closely cadherin and integrin loss are related in any given tumour, nor whether cadherin loss is correlated with changes in expression of the cytoplasmic regulatory proteins known as catenins. Double-label immunofluorescence has been used to stain a panel of 22 oral squamous cell carcinomas with antibodies to ten proteins, including E- and P-cadherin, the major keratinocyte integrin subunits, and alpha-, beta- and gamma-catenin. Overall, E-cadherin expression and integrin expression correlated well with tumour grade, while P-cadherin staining was more variable. All tumours, regardless of differentiation status, showed reduced staining for at least two of the catenins, implying that the adhesive function of E- and P-cadherin could be impaired even when cadherin expression is normal. It is concluded that in all squamous cell carcinomas, regardless of degree of histological differentiation, there is some perturbed expression of cell adhesion molecules and that integrin and E-cadherin loss are closely related.

MSH receptors and the response of human A375 melanoma cells to interleukin-1 beta.

alpha-Melanocyte-stimulating hormone (alpha-MSH, alpha-melanotropin) has been shown to be an inhibitory factor in many immunologic and inflammatory processes involving the cytokine interleukin-1 (IL-1). As the mechanism of the interaction between IL-1 and alpha-MSH at the receptor level is unknown, we have studied the role of MC1 melanocortin receptors in two variants of the human melanoma cell line A375 differing in their sensitivity to the cytostatic effects of IL-1 beta. Both IL-1 sensitive (A375r-) and resistant cells (A375r+) carry specific high affinity receptors for IL-1, albeit their concentration is 10-fold higher in A375r+ cells. In A375r- cells, MC1 receptors are absent or below the level for reliable detection in the binding assay. Conversion of A375r- to A375r+ cells by prolonged culture in medium not depleted of endotoxin led to the appearance of MC1 receptors (KD 0.4 +/- 0.123 nmol/l; 608 +/- 134 receptors/cell). Stable transfection of A375r- cells with the human MC1 receptor did not, however, render them resistant to the cytostatic effect of IL-1 beta on concomitant treatment with alpha-MSH or result in the production of IL-6 on treatment with IL-1 beta. Therefore, the presence of MC1 receptors on the surface of A375 cells or their binding to alpha-MSH does not seem to be a factor in cytokine resistance or IL-6 secretion. No interaction between IL-1 beta and alpha-MSH could be demonstrated at the cellular level in this melanoma cell line.

Differentiation of embryonal stem cells into keratinocytes: comparison of wild-type and beta 1 integrin-deficient cells.

beta 1 Integrins are known to regulate terminal differentiation and morphogenesis in the adult epidermis. We have investigated their role in the embryonic development of keratinocytes by comparing the differentiation of wild-type and beta 1-null mouse embryonal stem (ES) cells. By 12-15 days in culture, differentiation of embryonic or simple epithelial cells occurred in both ES cell populations, as detected by expression of keratins 8, 18, and 19. From 21 days, expression of keratins 10 and 14 and of the cornified envelope precursor involucrin indicated that some of the wild-type cells had differentiated into keratinocytes. In contrast, keratinocyte markers were not expressed in beta 1-null cultures. The beta 1-null cells failed to express the alpha 2 and alpha 3 integrin subunits on the cell surface, consistent with the association of these a subunits with beta 1. Furthermore, alpha 6 and beta 4 expression was reduced in the beta 1-null cultures. Although beta 1-null ES cells failed to undergo differentiation into keratinocytes in vitro, they did form keratinocyte cysts expressing alpha 6 beta 4, keratins 1 and 14, and involucrin when allowed to form teratomas by subcutaneous injection in mice; furthermore, beta 1-null keratinocytes were found in the epidermis of a wild-type/beta 1-null chimeric mouse. As judged by immunofluorescence microscopy, extracellular matrix assembly was severely impaired in beta 1-null ES cell cultures, but not in the teratomas or chimeric mouse skin. We therefore speculate that the failure of beta 1-null cells to differentiate into keratinocytes in vitro may reflect an inability to assemble a basement membrane.

Induction of constitutive melanogenesis in amelanotic mouse melanoma cells by transfection of the human melanocortin-1 receptor gene.

The human melanocortin-1 (MC1) receptor was stably expressed in the amelanotic mouse melanoma cell clone B16-G4F which does not express its own (mouse) MC1 receptor and hence is unresponsive to alpha melanocyte stimulating hormone (alpha MSH). From several stable transfectant cell lines expressing the human MC1 receptor in relatively high numbers, three melanin producing clones (G4F-12, 14, and 15) and one amelanotic clone (G4F-7) were further analyzed in competition binding experiments and in cAMP and melanin assays. The dissociation constants (KD) for [Nle4, D-Phe7]-alpha MSH in all four clones ranged from 0.187 to 0.705 nmol/l, thus corresponding to the KD observed with the different human melanoma cell lines so far studied. Intracellular cAMP content was 3- to 5-fold higher than that of control cells, and alpha MSH induced an additional 1.5- to 1.7-fold increase. G4F-15 cells secreted melanin into the medium whereas the other clones did not secrete melanin. The extent of melanin secretion was similar to that of fully alpha MSH-stimulated B16-F1 mouse melanoma cells but the onset of secretion was delayed. alpha MSH induced an additional dose-related increase (up to 1.3-fold) in melanin production which could be suppressed by the addition of specific alpha MSH antibodies without altering the constitutive part of melanogenesis. Human and mouse agouti proteins, which inhibit basal and alpha MSH-induced melanogenesis in B16-F1 cells, both reduced alpha MSH-induced melanin production in G4F-15 cells but did not affect the constitutive melanogenesis. These results indicate that human MC1 receptor expressed in mouse B16-G4F cells induces constitutive activation of the signalling pathway controlling melanogenesis, most likely by tightly coupling to Gs alpha, in a similar manner to that reported for constitutively active receptor mutants in other systems.

alpha-MSH receptor autoradiography on mouse and human melanoma tissue sections and biopsies.

MSH receptors and their binding characteristics of [125I]-labelled derivatives of alpha-MSH have been studied extensively on various mouse and human melanoma cell lines in culture. The aim of this study was to determine the binding characteristics of alpha-MSH radioligands to MSH receptors occurring in experimental mouse and human melanoma tumours as well as in human melanoma biopsies. For this reason, solid tumours were grown on experimental animals by inoculation of murine B16-F1 and human D10 and HBL melanoma cells. After excision and cryosectioning of the tumours, frozen tissue sections were incubated with [(125I)Tyr2]-alpha-MSH or [(125I)Tyr2,Nle4,D-Phe7]-alpha-MSH and specific alpha-MSH binding sites were visualized by subsequent autoradiography. The presence of increasing concentrations of unlabelled alpha-MSH during incubation with tracer led to a dose-dependent displacement of the radioligand. Quantitative analysis of the autoradiograms produced dissociation constants which were comparable with those obtained with cell binding assays: KD = 1.87 and 1.31 nmol/l for B16 tumours and cells, respectively; 0.32 and 0.33 nmol/l for D10, and 2.24 and 1.36 nmol/l for HBL tumours and cells, respectively. This indicates similar binding properties of alpha-MSH radioligands to both cultured melanoma cells and tissue sections of melanoma tumours from experimental animals. Similar binding characteristics were also observed with human melanoma tissue sections originating from biopsies of melanoma patients.

Stable expression of the human MSH receptor in a mouse melanoma cell line.

Stable expression of the MSH receptor in a homologous system is important for the study of the function and mechanism of signalling of this receptor. This is the first report on the stable expression of the human alpha-MSH receptor in the mouse melanoma G4F clone which lacks an endogenous MSH receptor. Several stable transfectant cell lines were obtained all of which express the human MSH receptor in high numbers. Human MSH receptor mRNA expression was detected by Northern blot analysis. Competition binding experiments showed that the MSH receptors expressed in these cells have the same affinity for [Nle4,D-Phe7]-alpha-MSH as the MSH receptors of the human HBL melanoma cell line. Several of the transfectant cell lines produced melanin constitutively, some of them secreting melanin into the medium whereas other clones did not secrete melanin. MSH and cholera toxin did not or only marginally increase melanogenesis in these clones, and forskolin had an opposite effect. These results suggest that the human MSH receptor may be constitutively active in these transfected mouse melanoma cells.

[111In]-DTPA-labeled analogues of alpha-melanocyte-stimulating hormone for melanoma targeting: receptor binding in vitro and in vivo.

Six alpha-MSH(4-10) [Nle-Asp-His-D-Phe-Arg-Trp-Lys-amide] derivatives carrying 2 or 1 or no 2,3-dihydroxy-(2S)-propyl (DHP) groups on the Lys10 amino side chain were coupled to diethylene-triaminopentaacetic acid (DTPA, a chelator for 111In) in monomeric and dimeric forms and tested for their binding activity and bioactivity in vitro with mouse and human melanoma cell lines and by receptor autoradiography to tumor sections, as well as in vivo with normal and melanoma-bearing mice: DTPA-[Nle4,Asp5,D-Phe7,Lys(bis-DHP)10]-alpha-MSH(4-10),DTPA-[Nle4, Asp5, D-Phe7,Lys(mono-DHP)10]-alpha-MSH(4-10), DTPA[Nle4,Asp5,D-Phe7,Lys10]-alpha-MSH(4-10), DTPA-bis-([Nle4,Asp5,D-Phe7,Lys(bis-DHP)10]-alpha-MSH(4-10)), DTPA-bis[([Nle4,Asp5,D-Phe7,Lys(mono-DHP)10]-alpha-MSH(4-10)) and DTPA-bis-([Nle4,Asp5,D-Phe7,Lys10]-alpha-MSH(4-10)). In the receptor-binding assays with B16-F1 mouse and D10 human melanoma cells, the KD values ranged between 0.76 and 31.17 nM and in the melanin bioassay the results were similar (EC50 values between 0.15 and 4.40 nM). The tissue distribution of the 111In-labeled compounds in C57Bl/6J mice showed that the dimeric [111In]-DTPA-bis([Nle4,Asp5,D-Phe7,Lys10]-alpha-MSH(4-10)) and the monomeric [111In]-DTPA-[Nle4,Asp5,D-Phe7,Lys(bis-DHP)10]-alpha-MSH(4-10) exhibited the lowest non-specific binding. In mice carrying B16-F1 melanoma tumors, the monomeric compound displayed 2-fold higher 111In uptake by the tumor and a much lower non-specific uptake by the liver (12-fold) and the kidneys (2.5-fold) than the dimeric derivative. This demonstrates that modification of the Lys10 side chain by DHP is a promising lead for new MSH radiopharmaceuticals for melanoma targeting.

Synthesis and biological properties of a biotinylated derivative of ACTH1-17 for MSH receptor studies.

A biotinylated derivative of [beta-Ala1,Lys17]-ACTH1-17-NH-(CH2)4-NH2 (ACTH1-17) was synthesized and biologically characterized. The heptadecapeptide with free N-terminus and blocked side-chains was prepared by the solid-phase method using TentaGel resin and a 4-aminobutylamide linker. Biotinyl-beta-Ala-OH was then coupled to the terminal amino group and the resulting [N alpha-(biotinyl-beta-alanyl)-beta-Ala1,Lys17]-ACTH1-17-NH-(CH2)4-N H2 (Bio-ACTH1-17) cleaved from the resin, purified and analyzed. Competition binding assays with mouse B16-F1 and human D10 and HBL melanoma cells using [125I]-alpha-MSH as radioligand gave dissociation constants for Bio-ACTH1-17 of 1.67 +/- 0.07 nM (B16-F1), 0.02 +/- 0.005 nM (D10) and 0.21 +/- 0.02 nM (HBL). The EC50 for Bio-ACTH1-17 in the B16 melanin assay was 4.15 +/- 1.0 nM. Analysis of the binding characteristics of [125I]-Bio-ACTH1-17 demonstrated that in human melanoma cells this radioligand was displaced by ACTH1-17 as well as alpha-MSH whereas in B16-F1 cells the tracer was only displaced from the binding site by ACTH1-17. Studies of Bio-ACTH1-17 with streptavidin showed that the peptide is to a large extent trapped specifically through reaction with biotin. These results demonstrate that (1) the biological characteristics of Bio-ACTH1-17 are almost identical to those of ACTH1-17, (2) Bio-ACTH1-17 is bound by avidin, and (3) Bio-ACTH1-17 may become a useful tool for MSH receptor targeting.

Biologically active monoiodinated alpha-MSH derivatives for receptor binding studies using human melanoma cells.

Three different monoiodinated radioligands of alpha-MSH (alpha-melanocyte-stimulating hormone) were compared in a binding assay with human D10 melanoma cells: [Tyr(125I)2]-alpha-MSH, [Tyr(125I)2,NIe4]-alpha-MSH, and [Tyr(125I)2,NIe4,D-Phe7]-alpha-MSH. They were prepared either by the classical chloramine T method or by the Enzymobead method. A simple and rapid purification scheme was developed consisting of a primary separation on reversed-phase C18 silica cartridges immediately after the iodination, followed by HPLC purification before each binding experiment. Biological testing of the three radioligands showed that they all retained high melanotropic activity in the B16 melanin assay and the Anolis melanophore assay. However, in human D10 melanoma cells, [Tyr(125I)2,NIe4]-alpha-MSH led to a high degree of non-specific binding to the cells which could not be displaced by excess alpha-MSH and only partially by [NIe4]-alpha-MSH. The [Tyr(125I)2,NIe4,D-Phe7]-alpha-MSH tracer gave similar results but with a much lower proportion of non-specific binding. On the other hand, [Tyr(125I)2]-alpha-MSH proved to be an excellent radioligand whose non-specific binding to the D10 cells was not higher than 20% of the total binding.