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Background: Herpes simplex virus infection (HSV) is a common and ubiquitous infection of the skin which causes mucocutaneous lesions called cold sores (herpes labialis) or fever blisters. It is estimated that approximately 80% of the population worldwide are carriers of the Herpes simplex virus, approximately 40% suffer from recurrent recurrent infections. This study evaluates the in vitro skin permeation and penetration of penciclovir and acyclovir from commercialized creams for the treatment of herpes labialis (cold sores), using non viable excised human abdominal skin samples, which were exposed to 5 mg/cm2 of acyclovir 5% cream or penciclovir 1% cream., Methods: After 24 h of cream application, excess cream was washed off and layers of stratum corneum were removed by successive tape stripping. Amounts of active ingredients having penetrated through the skin were measured, as well as the amounts in the washed-off cream, in skin strips and creams remaining in the skin. Molecular modelling was used to evaluate physico-chemical differences between the drugs. Western blot analysis enabled to determine whether the marker of basal cells keratin 5 could be detected in the various tape strips., Results: Application of penciclovir 1% cream yielded higher concentration of drug in the deeper layers of the epidermis as well as a higher drug flux through the skin. Molecular modelling showed two higher hydrophobic moieties for acyclovir. Presence of the basal cell marker keratin 5 was underscored in the deeper tape strips from the skin, giving evidence that both drugs can reach their target cells., Conclusion: Penciclovir 1% cream has the tendency to facilitate the diffusion of the drug through the stratum corneum into the deeper epidermis layers, in which it could reach the target basal cells at effective therapeutical concentration. The small difference in the surface properties between both molecules might also contribute to favour the passage of penciclovir through the epidermis into the deeper basal cells.

In search for new valuable tumor-associated antigens using the AMIDA technique, we identified the KIAA 1273-AAA-TOB3 protein. KIAA 1273 and AAA-TOB3 were considered synonyms for the atad3B gene product. We show that the atad3b gene encodes two distinct proteins, both overexpressed in head and neck carcinomas and required for correct cell division. Both products differ within the N terminus, are generated upon distinct transcription initiation sites, and have been termed AAA-TOB3s and AAA-TOB3l. Both isoforms are early targets of c-myc and are located in mitochondria. A previous report suggested pro-apoptotic properties of the murine homolog of AAA-TOB3l. Here, we did not observe any pro-apoptotic effects in human cell lines, overexpressing h-AAA-TOB3s or h-AAA-TOB3l. By contrast, the specific knock-down of both mRNAs resulted in polynuclear cells and decreased proliferation, along with dysfunctional cell division followed by increased apoptosis. Thus, the present data suggest a role for AAA-TOB3s/l in tumor progression.

We reported the identification of tumour-associated antigens from head and neck carcinomas, including cytokeratin 8 (CK8). These antigens were isolated based on the humoral immune response they elicit in vivo using the antibody-mediated identification of antigens technology. Unlike healthy squamous epithelium, tumour cells displayed CK8 at the plasma membrane. However, the actual presence of CK8 at the plasma membrane is still a matter of debate. Here, we have analyzed the expression of CK8 in detail using confocal laser scanning microscopy and circumstantiated its localization at the plasma membrane of carcinoma cells. Healthy human tissues were devoid of CK8 at the membrane, with the exception of hepatocytes. Moreover, membrane-associated CK8 molecules experienced a re-distribution throughout mitosis, which was associated with phosphorylation at serine 73. Phosphorylated CK8 redistributed into dense speckles and relocated to the plasma membrane upon cytokinesis. Thus, CK8 possesses genuine extracellular epitopes on tumour cells, which may represent valuable targets for therapy.

Recently, we described a new target-identification technology, autoantibody-mediated identification of antigens (AMIDA). AMIDA takes advantage of autologous serum autoantibodies to identify disease-associated antigens. Here, we evaluated the allogenic variant of AMIDA (allo-AMIDA), using permanent cancer cell lines as an antigen-pool rather than primary biopsy samples. Twelve different proteins were retrieved exclusively with antibodies from cancer patients, but not from healthy donors. The expression of three of these antigens, e-FABP, hnRNP H, and Grb2, was evaluated in more detail. All three proteins were strongly overexpressed in primary carcinomas and metastases thereof, as compared to healthy epithelium. Additionally, serum reactivity against e-FABP was detected in 20% of cancer patients but only 2% of healthy volunteers. In summary, we demonstrate that permanent cancer cell lines represent a reliable source for tumour-associated antigens. Moreover, we show that allo-AMIDA is suitable for the identification of tumour-specific antigens overcoming the limitations of autologous screening techniques., (Copyright 2004 Elsevier Inc.)

We describe AMIDA (autoantibody-mediated identification of antigens), a novel target identification technology based on the immunoprecipitation of disease-specific antigens by autologous serum antibodies followed by two-dimensional electrophoretic separation, and their identification via mass spectrometry. Twenty-seven potential carcinoma antigens were identified including proteins of hitherto unknown function. Validation of one of the identified antigens, cytokeratin 8, revealed its de novo expression in hyperplastic tissue, gradual overexpression with increasing malignancy, and ectopic localization on the cell surface. Furthermore, a strong prevalence of CK8-specific antibodies occurred in the serum of cancer patients already at early disease stages. In situ hybridization for one marker of unknown function, KIAA1273/TOB3, demonstrated its strong overexpression in head and neck carcinomas, thus making it a likely tumor antigen candidate. Eventually, AMIDA could foster significant improvements for the diagnosis and therapy of human diseases eliciting a humoral immune response, and allows for the rapid identification of new target molecules.

Paclitaxel concentrations in the brain are very low after intravenous injection. Since paclitaxel is excluded from some tumors by p-glycoprotein (p-gp), the same mechanism may prevent entry into the brain. In vitro, paclitaxel transport was examined in capillaries from rat brains by confocal microscopy using BODIPY Fl-paclitaxel. Western blots and immunostaining demonstrated apical expression of p-gp in isolated endothelial cells, vessels, and tissue. Secretion of BODIPY Fl-paclitaxel into capillary lumens was specific and energy-dependent. Steady state luminal fluorescence significantly exceeded cellular fluorescence and was reduced by NaCN, paclitaxel, and SDZ PSC-833 (valspodar), a p-gp blocker. Leukotriene C(4) (LTC(4)), an Mrp2-substrate, had no effect. Luminal accumulation of NBDL-cyclosporin, a p-gp substrate, was inhibited by paclitaxel. In vivo, paclitaxel levels in the brain, liver, kidney, and plasma of nude mice were determined after intravenous injection. Co-administration of valspodar led to increased paclitaxel levels in brains compared to monotherapy. Therapeutic relevance was proven for nude mice with implanted intracerebral human U-118 MG glioblastoma. Whereas paclitaxel did not affect tumor volume, co-administration of paclitaxel (intravenous) and PSC833 (peroral) reduced tumor volume by 90%. Thus, p-gp is an important obstacle preventing paclitaxel entry into the brain, and inhibition of this transporter allows the drug to reach sensitive tumors within the CNS.