Detection of nucleic acids in cells and tissues by in situ polymerase chain reaction.

The solution-based polymerase chain reaction (PCR) method for amplification of defined gene sequences has proved a valuable tool not only for basic researchers but also for clinical scientists. Using even a minute amount of DNA or RNA and choosing a thermostable enzyme from a large variety of sources, one can enlarge the amount of the gene of interest, which can be analyzed and sequenced. Therefore, genes, or segments of gene sequences present only in a small sample of cells or small fraction of mixed cellular populations can be examined. One of the major drawbacks of the solution-based PCR technique is that the procedure does not allow for the association of amplified signals of a specific gene segment with the histological cell type(s) (1-2). For example, it would be advantageous to determine what types of cells in the peripheral blood circulation or in pathological specimens carry HIV-1 gene sequences, a vector used for gene therapy, an aberrant gene in a leukemia patient, or to determine the percentage of leukemia cells present following antitumor therapy.

Localization of human herpesvirus type 8 (HHV-8) in the Kaposi's sarcoma tissues and the semen specimens of HIV-1 infected and uninfected individuals by utilizing in situ polymerase chain reaction.

Kaposi's sarcoma (KS) is a form of skin cancer, most commonly found in individuals suffering from acquired immunodeficiency syndrome, or AIDS. However, before the worldwide infection of human immunodeficiency virus (HIV), the rare occurrence of KS was confined to two distinct groups of individuals. In the Western world, the classical form of KS was often found in older men (60-70 years of age) from the Mediterranean area. Another form called endemic KS, was found in Equatorial Africa. Currently, the most common cases of KS are found in individuals suffering from AIDS. This is called AIDS-associated KS. Between 30 and 40% of male, homosexual AIDS patients suffer from AIDS-associated KS. KS is also occasionally diagnosed in transplant patients receiving immunosuppressive drugs (to keep their body from rejecting the foreign organ). As opposed to cases of classic and endemic KS, the KS in AIDS patients progresses very quickly, often with a fatal outcome. Human herpesvirus type 8 (HHV-8) has been implicated as the cause of Kaposi's sarcoma (KS), but the exact connection of the virus to the neoplasm is not known. The virus has been detected within the sarcoma skin lesions, but has additionally been seen in peripheral blood cells, semen samples, prostate tissue, and other types of soft tissue tumors. In this study, we evaluated HHV-8 within the skin lesion of KS as well as in semen specimens obtained from HIV-1 infected and uninfected specimens from HIV-1-seronegative individuals. Twenty-eight tissue samples representing AIDS-associated, endemic KS, and six non-KS patients were collected for observation from different centers throughout the world. The tissues were examined utilizing in situ polymerase chain reaction (ISPCR) and hybridization to identify and localize the herpesvirus within the KS lesions. With the use of the sensitive ISPCR technique, HHV-8 DNA was detected in the spindle cells within the nodular skin lesions, as well as in the microvascular endothelial cells which line small vessels within the lesions in all forms of KS. In addition, we analysed semen specimens from HIV-1 infected and uninfected men, our analyses revealed that HHV-8 was present in the significant proportions of the HIV-1-infected-individuals' sperm, as well as in the mononuclear cells of the semen specimens. HHV-8 DNA was demonstrated, by ISPCR, in KS lesions as well as in seminal mononuclear cells and sperm of significantly high proportion of HIV-1-infected men. What role the presence of HHV-8 in the sperm cells plays in the sexual transmission of this herpesvirus will require further study. However, the reports which demonstrate that KS lesions can develop in infants of only a few weeks of age, increases the possibility that this agent may be vertically transmitted. It can be suggested that HHV-8 is relatively ubiquitous and its frequency increases with the increasing immunosuppression.

Adenovirus vector-mediated gene transfer into human epileptogenic brain slices: prospects for gene therapy in epilepsy.

As a first step in the development of a gene therapy approach to epilepsy, we evaluated the ability of adenovirus vectors to direct the transfer into and expression of a marker gene in human brain slices obtained from patients undergoing surgery for medically intractable epilepsy. Following injection of adenovirus vectors containing the Escherichia coli lacZ gene into hippocampal and cortical brain slices, lacZ mRNA, beta-galactosidase protein, and enzymatic activity were detected, confirming successful gene transfer, transcription, and translation into a functional protein. Transfected cells were predominantly glial, with some neurons expressing beta-galactosidase as well. These results support the potential of adenovirus vectors to transfer genetic information into human epileptogenic brain, resulting in expression of the gene into a functional protein. These findings also have implications for the development of gene therapy approaches to certain seizure disorders. A number of potential therapeutic approaches are discussed, including the elevation of inhibitory neurotransmitter or neuropeptide levels, expression or modulation of postsynaptic receptors, and manipulation of signal transduction systems.

Prospects for gene therapy in Parkinson's disease.

Numerous advances in in vivo and ex vivo gene-therapy approaches to Parkinson's disease offer promise for direct clinical trials in patients in the next several years. These systems are predicated on introducing gene that encode enzymes responsible for dopamine biosynthesis or neurotrophic factors that may delay nigrostriatal degeneration or facilitate regeneration. We review the current status of experimental approaches to gene therapy for Parkinson's disease. Comparative advantages and disadvantages of each system are enumerated, and preclinical trials of some of the systems are evaluated. Although the specific in vivo or ex vivo methods used for gene transfer into the brain are likely to be supplanted by newer technology over the next decade, the principles and approaches developed in current studies likely will remain the same.