Venezuelan equine encephalitis virus glycoprotein pseudotyping confers neurotropism to lentiviral vectors.

We have produced high-titre HIV-1 green fluorescent protein-expressing lentiviral (LV) vectors pseudotyped with strain 3908 Venezuelan equine encephalitis virus glycoprotein (VEEV-G) and used them to study transduction of: (1) rat embryonic motor neuron (MN) and striatal neuron primary cultures, (2) differentiated MN cell line NSC-34 and (3) adult rat striatum. In primary neuronal cultures, transduction with VEEV-G-pseudotyped LV was more efficient and more neuronal than with vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped LV. In NSC-34 cells clear retrograde transport of VEEV-G vector particles was observed. In the striatum at the injection site, transduction with the VEEV-G vectors driven by cytomegalovirus or phosphoglycerate kinase promoters exhibited a distinct neuronal tropism with no microglial and only a minor astroglial component, superior to that obtained with VSV-G-pseudotyped LV, irrespective of the promoter used. Neuronal transduction efficiency increased over time. Distal to the injection site transduction of mitral cells in the olfactory bulb, thalamic neurons and dopaminergic neurons in the substantia nigra pars compacta was detected. This, together with observations of retrograde axonal trafficking in vitro indicates that these vectors also possess low level of retrograde neuronal transduction capability in vivo. In this study, we demonstrate both strong neurotropism as well as sustainability of expression and minimal host immune response in vivo, making the VEEV-G-pseudotyped LV vectors potentially useful for gene therapy of neurodegenerative diseases.

Enhanced pseudotyping efficiency of HIV-1 lentiviral vectors by a rabies/vesicular stomatitis virus chimeric envelope glycoprotein.

Rabies virus glycoprotein (RVG) can pseudotype lentiviral vectors, although at a lower efficiency to that of vesicular stomatitis virus glycoprotein (VSVG). Transduction with VSVG-pseudotyped vectors of rodent central nervous system (CNS) leads to local neurotropic gene transfer, whereas with RVG-pseudotyped vectors additional disperse transduction of neurons located at distal efferent sites occurs via axonal retrograde transport. Attempts to produce high-titre RVG-pseudotyped lentiviral vectors for preclinical and clinical trials has to date been problematic. We have constructed several chimeric RVG/VSVG glycoproteins and found that a construct bearing the external/transmembrane domain of RVG and the cytoplasmic domain of VSVG shows increased incorporation onto HIV-1 lentiviral particles and has increased infectivity in vitro in 293T cells and in differentiated neuronal cell lines of human, rat and murine origin. Stereotactic application of vector pseudotyped with this RVG/VSVG chimera in the rat striatum resulted in efficient gene transfer at the site of injection showing both neuronal and glial tropism. Distal neuronal transduction in the substantia nigra, thalamus and olfactory bulb via retrograde axonal transport also occurs after intrastriatal administration of chimera-pseudotyped vectors at similar levels to that observed with a RVG-pseudotyped vector. This is the first report of distal transduction in the olfactory bulb. The enhanced pseudotyping with this envelope should enable easier production of higher-titre pseudotyped lentiviral vectors that exhibit efficient local and dispersed neuronal transduction in the CNS.

Inhibition of skin tumorigenesis by rosemary and its constituents carnosol and ursolic acid.

A methanol extract of the leaves of the plant Rosmarinus officinalis L. (rosemary) was evaluated for its effects on tumor initiation and promotion in mouse skin. Application of rosemary to mouse skin inhibited the covalent binding of benzo(a)pyrene [B(a)P] to epidermal DNA and inhibited tumor initiation by B(a)P and 7,12-dimethylbenz[a]anthracene (DMBA). Topical application of 20 nmol B(a)P to the backs of mice once weekly for 10 weeks, followed 1 week later by promotion with 15 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) twice weekly for 21 weeks, resulted in the formation of 7.1 tumors per mouse. In a parallel group of animals that were treated topically with 1.2 or 3.6 mg of rosemary 5 min prior to each application of B(a)P, the number of tumors per mouse was decreased by 54 or 64%, respectively. Application of rosemary to mouse skin also inhibited TPA-induced ornithine decarboxylase activity, TPA-induced inflammation, arachidonic acid-induced inflammation, TPA-induced hyperplasia, and TPA-induced tumor promotion. Mice initiated with 200 nmol DMBA and promoted with 5 nmol TPA twice weekly for 19 weeks developed an average of 17.2 skin tumors per mouse. Treatment of the DMBA-initiated mice with 0.4, 1.2, or 3.6 mg of rosemary together with 5 nmol TPA twice weekly for 19 weeks inhibited the number of TPA-induced skin tumors per mouse by 40, 68, or 99%, respectively. Topical application of carnosol or ursolic acid isolated from rosemary inhibited TPA-induced ear inflammation, ornithine decarboxylase activity, and tumor promotion. Topical application of 1, 3, or 10 mumol carnosol together with 5 nmol TPA twice weekly for 20 weeks to the backs of mice previously initiated with DMBA inhibited the number of skin tumors per mouse by 38, 63, or 78%, respectively. Topical application of 0.1, 0.3, 1, or 2 mumol ursolic acid together with 5 nmol TPA twice weekly for 20 weeks to DMBA-initiated mice inhibited the number of tumors per mouse by 45-61%.

Differential inhibition of epidermal growth factor binding by photoactivated psoralens and the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate in cells overexpressing protein kinase C.

A rat fibroblast cell line, R6PKC3, that stably overexpresses the beta-1 form of protein kinase C was used to analyze sensitivity to inhibitors of epidermal growth factor (EGF) binding. R6PKC3 cells overexpress protein kinase C activity 53-fold relative to non-overexpressing control R6C1 cells. Inhibition of EGF binding by the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) and photo-activated psoralens was compared in these cells. We found that 125I-EGF bound both of the cell lines and was rapidly internalized in a temperature-dependent process and metabolized. Binding of EGF to the R6 cells overexpressing protein kinase C was markedly less than binding to R6C1 control cells. In both of the cell lines, TPA and photoactivated psoralens inhibited 125I-EGF binding but the response of these cells to these inhibitors was distinct. R6PKC3 cells were markedly more sensitive to TPA and were resistant to recovery from TPA-induced inhibition of 125I-EGF binding when compared to control cells. These differences were not observed in other subclones of cells overexpressing protein kinase C, suggesting that they may be unique to R6PKC3 cells. In contrast, no major differences in sensitivity to photoactivated psoralens were observed in R6C1 and R6PKC3 cells. These data indicate that TPA and photoactivated psoralens inhibit 125I-EGF binding to these cell lines by distinct mechanisms.

Developing a Public Key Infrastructure for a secure regional e-Health environment.

OBJECTIVES: Internet technologies provide an attractive infrastructure for efficient and low cost communications in regional health information networks. The advantages provided by the Internet come however with a significantly greater element of risk to the confidentiality and integrity of information. This is because the Internet has been designed primarily to optimize information sharing and interoperability, not security. The main objective of this paper is to propose the exploitation of public-key cryptography techniques to provide adequate security to enable secure healthcare Internet applications. METHODS: Public-key cryptography techniques can provide the needed security infrastructure in regional health networks. In the regional health-care security framework presented in this paper, we propose the use of state-of-art Public Key Infrastructure (PKI) technology. Such on e-Health PKI consists of regional certification authorities that are implemented within the central hospitals of each region and provide their services to the rest of the healthcare establishments of the same region. RESULTS: Significant experience in this area has been gained from the implementation of the PKI@AUTH project. CONCLUSIONS: The developed PKI infrastructure already successfully provides its security services to the AHEPA university hospital. The same infrastructure is designed to easily support a number of hospitals participating in a regional health information network.

Access control based on attribute certificates for medical intranet applications.

BACKGROUND: Clinical information systems frequently use intranet and Internet technologies. However these technologies have emphasized sharing and not security, despite the sensitive and private nature of much health information. Digital certificates (electronic documents which recognize an entity or its attributes) can be used to control access in clinical intranet applications. OBJECTIVES: To outline the need for access control in distributed clinical database systems, to describe the use of digital certificates and security policies, and to propose the architecture for a system using digital certificates, cryptography and security policy to control access to clinical intranet applications. METHODS: We have previously developed a security policy, DIMEDAC (Distributed Medical Database Access Control), which is compatible with emerging public key and privilege management infrastructure. In our implementation approach we propose the use of digital certificates, to be used in conjunction with DIMEDAC. RESULTS: Our proposed access control system consists of two phases: the ways users gain their security credentials; and how these credentials are used to access medical data. Three types of digital certificates are used: identity certificates for authentication; attribute certificates for authorization; and access-rule certificates for propagation of access control policy. Once a user is identified and authenticated, subsequent access decisions are based on a combination of identity and attribute certificates, with access-rule certificates providing the policy framework. CONCLUSIONS: Access control in clinical intranet applications can be successfully and securely managed through the use of digital certificates and the DIMEDAC security policy.

Integrating the lightweight authentication protocol (LAP) with access control mechanisms in wireless health care information systems.

Health information networks are expected to support information exchange that is authentic, accurate, private and available when, where and to whom is needed. With the increase of the shared medical information and resources in healthcare wireless information systems, unauthorized access to the information by illegal users also increases. The security of the transmitted information is a vital issue. In this paper, we report on the development of the Lightweight Authentication Protocol (LAP), which makes a mobile and distributed system more secure and flexible and we implement it in a Health Care Environment where the clinicians use mobile and wireless devices like PDAs. We also provide an indicative example of integrating the LAP with access control mechanisms. Context-based Team Access Control (C-TMAC) model is used in this example, since it provides great flexibility on user-permissions management in collaborative healthcare environments. LAP is indeed capable to support efficiently the advanced authorization procedures of such demanding active security models.

Programming secure mobile agents in healthcare environments using role-based permissions.

The healthcare environment consists of vast amounts of dynamic and unstructured information, distributed over a large number of information systems. Mobile agent technology is having an ever-growing impact on the delivery of medical information. It supports acquiring and manipulating information distributed in a large number of information systems. Moreover is suitable for the computer untrained medical stuff. But the introduction of mobile agents generates advanced threads to the sensitive healthcare information, unless the proper countermeasures are taken. By applying the role-based approach to the authorization problem, we ease the sharing of information between hospital information systems and we reduce the administering part. The different initiative of the agent's migration method, results in different methods of assigning roles to the agent.

Inhibitory effects of curcumin on tumor initiation by benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene.

The effects of topical administration of curcumin on the formation of benzo[a]pyrene (B[a]P)-DNA adducts and the tumorigenic activities of B[a]P and 7,12-dimethylbenz[a]anthracene (DMBA) in epidermis were evaluated in female CD-1 mice. Topical application of 3 or 10 mumol curcumin 5 min prior to the application of 20 nmol [3H]B[a]P inhibited the formation of [3H]B[a]P-DNA adducts in epidermis by 39 or 61% respectively. In a two-stage skin tumorigenesis model, topical application of 20 nmol B[a]P to the backs of mice once weekly for 10 weeks followed a week later by promotion with 15 nmol 12-O-tetradecanoylphorbol-13-acetate (TPA) twice weekly for 21 weeks resulted in the formation of 7.1 skin tumors per mouse, and 100% of the mice had tumors. In a parallel group of mice, in which the animals were treated with 3 or 10 mumol curcumin 5 min prior to each application of B[a]P, the number of tumors per mouse was decreased by 58 or 62% respectively. The percentage of tumor-bearing mice was decreased by 18-25%. In an additional study, topical application of 3 or 10 mumol curcumin 5 min prior to each application of 2 nmol DMBA once weekly for 10 weeks followed a week later by promotion with 15 nmol TPA twice weekly for 15 weeks decreased the number of tumors per mouse by 37 or 41% respectively.