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The handling of infectious viral vectors such as adenovirus type 5 (Ad5), lentivirus (HIV1), and vaccinia virus (VV) requires biosafety level 2 (BSL-2) containment. This also applies to modified vaccinia Ankara (MVA) and adeno-associated virus type 2 (AAV2) provided that the insert involves additional hazard. To transfer infected cells to the lower BSL-1 containment, viral particles have to be demonstrably cleared from the supernatant as specified by biosafety guidelines. With the objective to provide data about the washout kinetics using common culture techniques, the authors infected different adherent mammalian cells with the aforementioned viral vectors. The supernatant of the cell culture was subsequently monitored for the presence of viral nucleic acids during various steps of washing and cell passaging. Complete clearance could be demonstrated for Ad5 when infecting Hela cells and for lentivirus (HIV1) in HEK293T cells. No or an undefined clearance was detected for AAV2, VV, and MVA in Vero-B4 cells and Ad5 after infection of HEK293 cells. Results demonstrated that virus persistence in the supernatant was greatly influenced by the washing procedure, the number of passages, and the vector titer, as well as the type of host cell line. The authors therefore conclude that procedures for clearance cannot be predefined for given virus-host systems. An analysis of the supernatant should be performed for each individual experimental setup prior to downgrading the risk class and subsequently the containment level used.

The handling of infectious viral vectors such as adenovirus type 5 (Ad5), lentivirus (HIV1), and vaccinia virus (VV) requires biosafety level 2 (BSL-2) containment. This also applies to modified vaccinia Ankara (MVA) and adeno-associated virus type 2 (AAV2) provided that the insert involves additional hazard. To transfer infected cells to the lower BSL-1 containment, viral particles have to be demonstrably cleared from the supernatant as specified by biosafety guidelines. With the objective to provide data about the washout kinetics using common culture techniques, the authors infected different adherent mammalian cells with the aforementioned viral vectors. The supernatant of the cell culture was subsequently monitored for the presence of viral nucleic acids during various steps of washing and cell passaging. Complete clearance could be demonstrated for Ad5 when infecting Hela cells and for lentivirus (HIV1) in HEK293T cells. No or an undefined clearance was detected for AAV2, VV, and MVA in Vero-B4 cells and Ad5 after infection of HEK293 cells. Results demonstrated that virus persistence in the supernatant was greatly influenced by the washing procedure, the number of passages, and the vector titer, as well as the type of host cell line. The authors therefore conclude that procedures for clearance cannot be predefined for given virus-host systems. An analysis of the supernatant should be performed for each individual experimental setup prior to downgrading the risk class and subsequently the containment level used.

Treatment of dead bacteria with propidium monoazide (PMA) prior to DNA extraction has been described to inhibit subsequent amplification of DNA by polymerase chain reaction (PCR) methods. As an enforcement authority involved in the surveillance of compliance with biosafety regulations, the State Laboratory of Basel City swabs surfaces to check for contaminations with microorganisms. To evaluate the PMA-PCR method for its use in distinguishing DNA from living and dead bacteria in swab samples, we used Staphylococcus aureus as a model organism. In this study PMA treatment was able to reduce amplification of DNA from dead bacteria by a factor of 1,000. However, PMA also entered living S. aureus to some extent, resulting in a reduction of DNA amplification by a factor of 10. When cells were spotted on a dry surface and collected again by wiping, cells were still viable on agar plates, but a distinction between DNA from living or dead bacteria was no longer possible using the PMA-PCR method. The process of drying and wiping seemed to interfere with the membrane of S. aureus resulting in diffusion of PMA into still viable cells. The authors conclude that this method offers a limited capacity to distinguish between living and dead S. aureus in liquid culture but does not allow determining the amount of living S. aureuscells in wipe-samples.

Treatment of dead bacteria with propidium monoazide (PMA) prior to DNA extraction has been described to inhibit subsequent amplification of DNA by polymerase chain reaction (PCR) methods. As an enforcement authority involved in the surveillance of compliance with biosafety regulations, the State Laboratory of Basel City swabs surfaces to check for contaminations with microorganisms. To evaluate the PMA-PCR method for its use in distinguishing DNA from living and dead bacteria in swab samples, we used Staphylococcus aureus as a model organism. In this study PMA treatment was able to reduce amplification of DNA from dead bacteria by a factor of 1,000. However, PMA also entered living S. aureus to some extent, resulting in a reduction of DNA amplification by a factor of 10. When cells were spotted on a dry surface and collected again by wiping, cells were still viable on agar plates, but a distinction between DNA from living or dead bacteria was no longer possible using the PMA-PCR method. The process of drying and wiping seemed to interfere with the membrane of S. aureus resulting in diffusion of PMA into still viable cells. The authors conclude that this method offers a limited capacity to distinguish between living and dead S. aureus in liquid culture but does not allow determining the amount of living S. aureuscells in wipe-samples.

Embryonal stem (ES) cells that are homozygous null for the β1integrin subunit fail to differentiate into keratinocytes in vitrobut do differentiate in teratomas and wild-type/β1-null chimeric mice. The failure of β1-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 β1-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 β1-null ES cells. Fibroblast-conditioned medium stimulated differentiation of K14-positive cells in wild-type and β1-null embryoid bodies. Of a range of growth factors tested, KGF, FGF10, and TGFα all stimulated differentiation of keratin 14-positive β1-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 β1integrins decreases the sensitivity of ES cells to soluble factors that induce keratinocyte differentiation.

Three different monolodinated radioligands of α-MSH (α-melanocyte-stimulating hormone) were compared in a binding assay with human D10 melanoma cells: [Tyr(125l)2]-α-MSH, [Tyr(125l)2, Nle4]-α-MSH, and [Tyr(125l)2, Nle4, D-Phe7]-α-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(125l)2-α-MSH led to a high degree of non-specific binding to the cells which could not be displaced by excess α-MSH and only partially by [Nle4]α-MSH. The [Tyr(125l)2, Nle4, D-Phe7]-α-MSH tracer gave similar results but with a much lower proportion of non-specific binding. On the other hand, [Tyr(1251)2]-a-MSH proved to be an excellent radioligand whose non-specific binding to the D10 cells was not higher than 20% of the total binding.

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 α-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]-α-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.

A biotinylated derivative of [β-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-β-Ala-OH was then coupled to the terminal amino group and the resulting [Nα-(biotinyl-β-alanyl)-β-Ala1, Lys17]-ACTH1-17-NH-(CH2)4-NH2 (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]-α-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 α-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

Abstractα-Melanocyte-stimulating hormone (α-MSH, α-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 α-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-1β. 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 (KD0.4 ± 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-1β on concomitant treatment with α-MSH or result in the production of IL-6 on treatment with IL-1β Therefore, the presence of MC1 receptors on the surface of A375 cells or their binding to α-MSH does not seem to be a factor in cytokine resistance or IL-6 secretion. No interaction between IL-1β and α-MSH could be demonstrated at the cellular level in this melanoma cell line.

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 α2β1, α3β1and α6β4integrins 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 α‐, β‐ and γ‐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. © 1998 John Wiley & Sons, Ltd.

MSH receptors and their binding characteristics of [125I]-labelled derivatives of α-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 α-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]-α-MSH or [(125I)Tyr2,Nle4,D-Phe7]-α-MSH and specific α-MSH binding sites were visualized by subsequent autoradiography. The presence of increasing concentrations of unlabelled α-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 α-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.

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 α2β1, α3β1 and α6β4 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 α-, β- and γ-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. © 1998 John Wiley & Sons, Ltd.

β1Integrins 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 β1-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 β1-null cultures. The β1-null cells failed to express the α2and α3integrin subunits on the cell surface, consistent with the association of these α subunits with β1. Furthermore, α6and β4expression was reduced in the β1-null cultures. Although β1-null ES cells failed to undergo differentiation into keratinocytesin vitro,they did form keratinocyte cysts expressing α6β4, keratins 1 and 14, and involucrin when allowed to form teratomas by subcutaneous injection in mice; furthermore, β1-null keratinocytes were found in the epidermis of a wild-type/β1-null chimeric mouse. As judged by immunofluorescence microscopy, extracellular matrix assembly was severely impaired in β1-null ES cell cultures, but not in the teratomas or chimeric mouse skin. We therefore speculate that the failure of β1-null cells to differentiate into keratinocytesin vitromay reflect an inability to assemble a basement membrane.

AbstractThirteen oligomeric analogs from dimers up to a hexamer of α-melanocyte-stimulating hormone (α-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 α-MSH fragment analog Nle-Asp-His-[D-Phe]-Arg-Trp-Lys-NH2to different templates through formation of oxime bonds. They were found to have binding affinities at 37°C up to 8 times higher and melanogenesis-inducing activities up to 4 times higher than those of the native hormone. At 15°C, one dimer showed a binding affinity 20 times higher than that of α-MSH. These results are discussed in terms of possible bridging of neighboring receptors which has been suggested to occur in some other systems.