Magnetic exchange splitting in Fe above the Curie temperature.

The magnetic exchange splitting of electronic states in a 7 monolayer Fe film on Cu(001) was investigated below and above the Curie temperature T(C), using image-potential surface states as sensor. At T(C), the long-range magnetic order breaks down as reflected by a vanishing spin splitting and vanishing spin polarization. The exchange splitting, in contrast, does not change abruptly at T(C) but persists up to T=1.2T(C). Equally, the spin-integrated linewidth shows no signature of the magnetic phase transition but smoothly decreases with increasing temperature. Our experimental results confirm theoretical expectations that, at T(C), the long-range magnetic order disappears but the local magnetic moments and, in particular, the valence electronic structure are unaffected by the phase transition.

Ultrafast magnon generation in an Fe film on Cu(100).

We report on a combined experimental and theoretical study of the spin-dependent relaxation processes in the electron system of an iron film on Cu(100). Spin-, time-, energy- and angle-resolved two-photon photoemission shows a strong characteristic dependence of the lifetime of photoexcited electrons on their spin and energy. Ab initio calculations as well as a many-body treatment corroborate that the observed properties are determined by relaxation processes involving magnon emission. Thereby we demonstrate that magnon emission by hot electrons occurs on the femtosecond time scale and thus provides a significant source of ultrafast spin-flip processes. Furthermore, engineering of the magnon spectrum paves the way for tuning the dynamic properties of magnetic materials.

Ferromagnetic Fe on Cu(001) throughout the fcc-like phase: arguing from the viewpoint of the electronic structure.

The scientific enthusiasm for ultrathin Fe films on Cu(001) has now lasted for more than 20 years. Is there ferromagnetic iron with a face-centred cubic (fcc) structure? Does ferromagnetism in Fe hinge on the body-centred cubic (bcc) structure? In this contribution, we try to establish that the electron system gives evidence of ferromagnetic behaviour with fcc-like electronic bands. We examine a crystal-induced surface state, which is characteristic of fcc surface order. Furthermore, we compare electronic signatures of fcc and bcc: the d-band exchange splitting, image-potential-state energies and the work function. We conclude that, from the viewpoint of the electronic structure, Fe on Cu(001) is found to be ferromagnetic throughout the fcc-like phase. This result raises a new question: how much deviation from the relaxed fcc order is acceptable without losing the electronic signature of fcc?

Spin-orbit hybridization points in the face-centered-cubic cobalt band structure.

Linear magnetic dichroism is observed in spin-, time-, and energy-resolved two-photon photoemission from valence bands of epitaxial fcc cobalt on Cu(001). With image-potential states as spectator states we identify initial bulk and surface states with minority spin character as the source for dichroic intensities and apparent dichroic lifetimes. Excellent agreement with ab initio fully relativistic calculations of the cobalt fcc band structure allows us to precisely determine spin-orbit hybridization points close to the Fermi level. These spin hot spots enhance spin-flip scattering by several orders of magnitude and are therefore assumed to be crucial in ultrafast demagnetization.

Combination treatment with intralesional cidofovir and viral-DNA vaccination cures large cottontail rabbit papillomavirus-induced papillomas and reduces recurrences.

We used the cottontail rabbit papillomavirus (CRPV) New Zealand White rabbit model to test a combination treatment of large established papillomas with intralesional cidofovir and DNA vaccination to cure sites and reduce recurrences. Intralesional 1% (wt/vol) (0.036 M) cidofovir treatment of rabbit papillomas led to elimination, or "cure," of the papillomas over a 6- to 8-week treatment period (N. D. Christenson, M. D. Pickel, L. R. Budgeon, and J. W. Kreider, Antivir. Res. 48:131-142, 2000). However, recurrences at periods from 1 to 8 weeks after treatment cessation were observed at approximately 50% of cured sites. DNA vaccinations with CRPV E1, E2, E6, and E7 were initiated either after or at the time of intralesional treatments, and the recurrence rates were observed. When DNA vaccinations were started after intralesional cures, recurrence rates were similar to those of vector-vaccinated rabbits. A small proportion of recurrent sites subsequently regressed (4 out of 10, or 40%) in the vaccinated group versus no regression of recurrences in the vector-immunized group (0 out of 19, or 0%), indicating partial effectiveness. In contrast, when DNA vaccinations were conducted during intralesional treatments, a significant reduction of recurrences (from 10 out of 19, or 53%, of sites in vector-immunized rabbits to 3 out of 20, or 15%, of sites in viral-DNA-immunized rabbits) was observed. DNA vaccination without intralesional treatments had a minimal effect on preexisting papillomas. These data indicated that treatment with a combination of antiviral compounds and specific immune stimulation may lead to long-term cures of lesions without the ensuing problem of papilloma recurrence.

In vivo anti-papillomavirus activity of nucleoside analogues including cidofovir on CRPV-induced rabbit papillomas.

A series of nucleoside analogues were tested for in vivo anti-papillomavirus activity using the cottontail rabbit papillomavirus (CRPV) domestic rabbit model. Compounds were delivered either topically, injected into growing papillomas, or delivered subcutaneously at a site remote from the papillomas. Compounds tested included cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine] (HPMPC); cyclic HPMPC (cHPMPC); cyclopentenylcytosine (CPE-C); lobucavir [1R(1alpha,2beta,3alpha)]-9-[2, 3-bis(hydroxymethyl)cyclobutyl]guanine; 9-((2-phosphonylmethoxy)propyl)adenine (PMPA); adefovir 9-((2-phosphonylmethoxy)ethyl)adenine(PMEA) and cyclopropyl 9-(2-phosphonylmethoxyethyl)-2,6-diaminopurine (cyclopropylPMEDAP). Dose response curves and time-course treatments were included for most compounds tested. Strong anti-viral activity was detected using cidofovir and cHPMPC when delivered either topically or by the intralesional route. Complete cures were obtained using 1% (w/v) topical cidofovir at dosing schedules of twice daily for 8 weeks beginning at 4 weeks after CRPV infection, which represents a time when papillomas were clearly visible. Complete cures of large established papillomas were obtained by intralesional injection of 1% cidofovir three times per week for 8 weeks. Topical treatments with adefovir had strong anti-viral activity, cyclopropyl PMEDAP had moderate anti-viral activity, and CPE-C, PMPA and lobucavir showed no effects. These data indicate that certain nucleoside analogues have strong in vivo anti-papillomavirus activity and that the CRPV/rabbit model is a good model for assessing clinical responses of anti-viral treatments for patients with HPV disease.

DNA vaccination prevents and/or delays carcinoma development of papillomavirus-induced skin papillomas on rabbits.

Malignant progression is a life-threatening consequence of human papillomavirus-associated lesions. In this study, we tested the efficacy of papillomavirus early-gene-based vaccines for prevention of carcinoma development of papillomavirus-induced skin papillomas on rabbits. Rabbit skin papillomas were initiated by infection with cottontail rabbit papillomavirus (CRPV). The papillomas were allowed to grow for 3 months without any treatment intervention. Rabbits were then immunized by gene gun-mediated intracutaneous administration of four DNA plasmids encoding CRPV E1, E2, E6, and E7 genes, respectively. All eight control rabbits receiving vector alone developed invasive carcinoma within 8 to 13 months. In contrast, only two of eight vaccinated rabbits developed carcinoma at 12 and 15 months, respectively. Papilloma growth was suppressed in the majority of vaccinated rabbits but not completely eradicated. These results indicate that gene gun-mediated immunization with papillomavirus early genes may be a promising strategy for prevention of malignant progression of human papillomavirus-associated lesions in humans.

A broad-spectrum microbicide with virucidal activity against sexually transmitted viruses.

Sodium dodecyl sulfate (SDS), an alkyl sulfate surfactant derived from an organic alcohol, possesses surfactant properties but also denatures and unfolds both monomeric and subunit proteins. In preliminary experiments, we demonstrated that SDS is a potent inactivator of herpes simplex virus type 2 and human immunodeficiency virus type 1 at concentrations comparable to those used for the surfactant nonoxynol-9. We hypothesized that SDS might be capable of denaturing the capsid proteins of nonenveloped viruses. In this report, we demonstrate inactivation of rabbit, bovine, and human papillomaviruses after brief treatment with dilute solutions of SDS. Effective concentrations were nontoxic to rabbit skin and to split-thickness grafts of human foreskin epithelium. This is the first report of a microbicidal surfactant that will inactivate papillomaviruses. We propose that SDS is now a candidate microbicide for formulation and testing with humans.

Different utilization of neutral lipids by Malassezia furfur and Malassezia sympodialis.

In recent years, the genus Malassezia has been expanded based on molecular data; in addition to M. furfur and M. pachydermatis, five new species (M. sympodialis, M. globosa, M. obtusa, M. restricta, M. slooffiae) have been described. Apart from their lipid dependence, little is known about the metabolism and nutritional requirements of these new species. Defined inocula of Malassezia reference strains were cultured on selective agar for pathogenic fungi which was overlaid with olive oil. Samples of the olive oil overlay were taken at regular intervals and the lipid fractions were analysed by high performance thin layer chromatography. Depending on the time of incubation and the number of cells, M. sympodialis and the other recently described species produced a significant increase in free fatty acids. In addition, a band of an apolar substance was identified as a mixture of fatty acid ethyl esters. While showing growth, strains of M. furfur produced only small amounts of ethyl esters and free fatty acids. The growth kinetics of M. furfur and M. sympodialis were also different: for M. sympodialis, a clear lag phase was observed, possibly indicating the necessity of extracellular hydrolysis of the triglycerides. The significance of the synthesis of ethyl esters could not be clarified. For routine differentiation, this metabolic difference is only of limited usefulness because slight contamination of M. furfur strains with other lipophilic Malassezia species may lead to misinterpretation due to the high metabolic activity. These metabolic differences might be important in the pathogenesis of Malassezia infections.

Differentiation of Malassezia species: selectivity of cremophor EL, castor oil and ricinoleic acid for M. furfur.

In recent years, the genus Malassezia has been reclassified based on molecular data. In addition to M. furfur, M. pachydermatis and M. sympodialis, four new species, M. globosa, M. obtusa, M. restricta and M. slooffiiae, have been described. However, apart from their lipid dependence, little is known about the metabolism and nutritional requirements of all the seven species. Further to recent studies, 10 hydrophilic emulsifiers (HLB > 10) were examined in an agar diffusion test to determine their growth-promoting effect on reference strains of the different Malassezia species. Polyethylene glycol (PEG) 7 glyceryl monoalcanoate (Cetiol HE). PEG-glyceryl stearate (Tagat S2) and macrogol-50 stearate (Myrj 53) were metabolized by all strains, while PEG-35 castor oil (Cremophor EL) was metabolized only by M. furfur. The latter observation is due to a different metabolism of castor oil and its main component, ricinoleic acid (12-hydroxy oleic acid), which may also give an insight into the pathogenesis of diseases that are associated with Malassezia spp. As hydroxy fatty acids are important in maintaining the epidermal structure and function, their metabolism specifically by M. furfur might clarify some clinical aspects of pityriasis versicolor. Apart from this speculation, use of Cremophor EL, with splitting of esculin as an additional key character, improves the distinction of the species M. furfur, M. slooffiae and M. sympodialis.

Polidocanol sensitivity--a possible tool in the differentiation of Malassezia spp.

In recent years, the genus Malassezia was reclassified based on molecular data; in addition to M. furfur, M. pachydermatis and M. sympodialis, four new species (M. globosa, M. obtusa, M. restricta, M. slooffiae) were described. Primary keys for routine identification have recently been presented. Polidocanol was shown to have specific inhibitory effects against Malassezia spp. In an agar diffusion test, type strains of all Malassezia species were incubated with polidocanol concentrations between 0.01% and 10%. M. furfur strains were most resistant, with minimal inhibitory concentrations (MIC) ranging from 7.5% to 10%. Inhibitory concentrations of the other strains were lower by at least one factor of ten. Most sensitive were strains of M. pachydermatis (0.05%). In a further test, polidocanol-containing olive oil was used to determine the sensitivity of Malassezia furfur and M. sympodialis. Again, the inhibitory concentrations for strains of M. sympodialis were one tenth of those found for M. furfur. In addition to its antifungal effects, polidocanol might therefore be a useful tool in differentiating Malassezia species.

Ultrastructure of pre- and postcolostral enterocytes of the newborn calf.

Ten calves were used to elucidate the ultrastructure of enterocytes before and 24 h after colostral intake. Tissue samples were obtained from duodenum, jejunum (5 locations) and ileum. Protein A-gold technique was applied to immunoelectron-microscopically demonstrate colostral IgA. The prominent feature of the precolostral enterocytes are intracytoplasmic vacuoles. The frequency of vacuoles increases from cranial jejunum to ileum and from the villi bases to the tips. The appearance of absorptive vacuoles after colostral administration correlates with the incidence of precolostral empty vacuoles. Bovine IgA was detected in absorptive vacuoles and within the intestinal lumen of postcolostral calves. In addition to a diffuse IgA labelling of most vacuoles, a few corresponding enterocytic vacuoles labelled inhomogenously or negatively. This study demonstrates morphologically that the main site of colostral absorption is the middle-to-caudal region of the small intestine. Immunoelectron microscopy of IgA labelling provides indications of a selective IgA absorption in addition to pinocytosis.

Association of tumor necrosis factor-alpha gene expression and apoptotic cell death with regression of Shope papillomas.

The objective of this study was to test the hypothesis that spontaneous regression of Shope papillomas involves tumor necrosis factor-alpha and apoptotic cell death of the papilloma cells. In situ hybridization using RNA probes of rabbit tumor necrosis factor-alpha revealed tumor necrosis factor-alpha mRNA in most of the numerous mononuclear cells infiltrating the upper dermis of regressing papillomas and at the dermoepidermal junction. Such cells in progressing papillomas were much fewer in number and were located in the deeper dermis. In situ terminal deoxynucleotidyl transferase assay demonstrated DNA strand breaks in many scattered epidermal keratinocytes of regressing papillomas but in only a few thin layers just beneath the horny layer in progressing papillomas. Electron microscopy demonstrated that regressing papillomas contained many apoptotic bodies and keratinocytes showing apoptotic changes such as chromatin condensation, degradation of condensed nuclei, surface protuberances, and a filamentous degeneration, as well as infiltrating lymphocytes and macrophages. We propose that tumor necrosis factor-alpha produced by infiltrating mononuclear cells probably plays a role in the papilloma regression.

An immunoelectron microscopic investigation of colostral IgG absorption across the intestine of newborn calves.

The protein A-gold was used to examine the transport of colostral IgG from the lumen of the gut to the circulation in four newborn calves and one 24-hour-old calf. The absorptive enterocytes of the duodenum, jejunum and ileum were investigated five to 60 minutes after administering colostrum, and 24 hours after birth. In the newborn calves, an intracellular micropinocytotic transport of IgG molecules was dominant throughout the entire small intestine. The amount transported increased from the duodenum to the ileum. In addition, evidence of a selective, receptor-mediated transport of IgG during the first few hours of life was provided by the presence of bovine clathrin at the microvillous membrane of the duodenal and jejunal enterocytes, indicating the existence of specialised vesicles for transport, the so-called 'coated' vesicles. No sign of paracellular transport was detected. Intestinal closure was interpreted as a multifactorial event comprising the replacement of the fetal intestinal epithelial cells by more mature populations, the initial cessation of transport at the basal and lateral cell membrane of the absorptive enterocytes, and an increase in intracellular proteolytic activity by lysosomes.

Podofilox-induced regression of Shope papillomas may be independent of host immunity.

We tested the hypothesis that infiltrating leukocytes might contribute to papilloma destruction following podofilox treatment. New Zealand White (NZW) rabbits were inoculated with cottontail rabbit papillomavirus (CRPV) onto abraded areas of the dorsal skin. At 21 d after viral inoculation, 5.0% podofilox solution was applied to some papillomas, whereas others were used as controls. Three rabbits were sacrificed at each of three different periods after treatment initiation (1, 4, and 7 d). Four monoclonal antibodies (MoAbs), RG-16 (for B cells), L11/135 (specific for T cells), 2C4 (specific for class II antigen), and Ki67 (specific for proliferating cells), were used in an immunohistochemical study. All positive cells and total cells in the field were counted with an ocular grid. After 1 d of treatment, proliferation of papilloma cells was strongly suppressed in treated papillomas, but leukocytic infiltration was not altered. At 4 d and 7 d of treatment, there were substantial increases (about two to three times) in the numbers of B and T cells and class II-expressing leukocytes. The upper layers of the papillomas were highly necrotic and cell proliferation was absent in all layers. These data support the view that podofilox has a direct toxic effect on papilloma tissue. Leukocyte infiltration is not strongly associated with papilloma tissue and may not contribute to papilloma destruction.

Influence of schedule and mode of administration on effectiveness of podofilox treatment of papillomas.

We investigated the timing of podofilox delivery to see how it correlated with papilloma size. We looked at times ranging from once per week (morning and afternoon) to five times per week (morning and afternoon). We also looked at delivery systems that might enhance the effectiveness of the drug by increasing penetration of the overlying cutaneous horn. These included soaking prior to drug administration, the use of a grooved needle subsequent to drug administration, and the various possible combinations of these techniques. We found that the timing of treatments had relatively little effect on the size of the papillomas. For example, at the end of ten weeks, the geometric mean diameter (GMD) (mm) of the papillomas treated five times per week and induced by a 10(-1) dilution of the virus (group B) was 18. Likewise the GMD (mm) of papillomas treated once per week was 18 (group D). On the other hand, we found that soaking plus the use of a grooved needle plus podofilox resulted in the curing of all lesions induced by a 10(-2) virus dilution and of most induced by the 10(-1) dilution, whereas soak plus podofilox or podofilox alone were not as effective in curing the lesions. Podofilox plus needle approached the soak plus podofilox plus needle in effectiveness. Treatment schedule was not a critical determinant of podofilox effectiveness. Therapeutic benefits were enhanced by hydration of the overlying cutaneous horn and penetration with a grooved needle.

Preclinical system for evaluating topical podofilox treatment of papillomas: dose-response and duration of growth prior to treatment.

The objective of the present study was to assess the utility of Shope rabbit papillomas as an animal model system for studying topical podofilox treatment and to evaluate dose-response relations and influence of duration of papilloma growth prior to treatment. New Zealand White rabbits received inoculations of cottontail rabbit papillomavirus (CRPV) virions of two dilutions at four sites total on the dorsum. Two papillomas on the left side were treated with podofilox (Oclassen Pharmaceuticals, Inc., San Rafael, CA). The drug was given topically twice each day, 5 d per week, for 21 d. We evaluated the effects of drug dose and the duration of papilloma growth prior to treatment. Results indicated that treatment beginning on day 28 with both 0.5 and 2.5% (w/v) podofilox inhibited papilloma growth, but 5.0% was more effective. In a separate experiment, papillomas were treated at 7, 21, or 60 d after virus inoculation. At 7 d, the untreated lesions were latent (not visible). At 21 d after infection, they were about 2.5 mm in diameter. At 60 d, papillomas were about 25 mm. Treatment with 5.0% podofilox beginning on any of those days strongly inhibited papilloma growth. Neither Southern blots nor PCR detected CRPV DNA in cured sites of previous virus infection. Antibody production to CRPV virion was not affected by drug treatment. 5.0% podofilox irritated normal skin adjacent to papillomas as evidenced by inflammation, induration, and superficial erosion. However, healing was satisfactory and no scarring resulted. We concluded that the Shope papilloma was a good model system for studying podofilox treatment because the lesions responded to drug across a broad range of drug concentrations and papilloma sizes.

Caring for families: what we see is what they get.

Working with families of preterm infants challenges the ability of professionals to address several issues and to provide continuity of care. A clinical example is used to illustrate why important data fail to be assessed and communicated and how this compromises care.