Combined chemotherapy of murine mammary tumors by local activation of the prodrugs ifosfamide and 5-fluorocytosine.

The success of chemotherapeutic intervention is limited because the necessary high local drug doses cannot be achieved without systemic toxicity. Application of suicide genes (SGs) and direct conversion of prodrugs (PDs) to toxic metabolites in situ by SGs may enhance the efficacy of chemotherapy. To evaluate this strategy in two murine breast cancer models, TS/A and GR, we injected cellulose sulfate capsules harboring cat kidney cells expressing the SGs cytosine deaminase and cytochrome P450 2B1 (CYP2B1) intratumorally. The PDs 5-fluorocytosine and ifosfamide were administered in 3-day intervals. The effect of in situ chemotherapy with each PD alone and the combination was analyzed over a period of 100 days. The results reveal that for TS/A tumors, the antitumoral effect mediated by CYP2B1 is more efficient than that of cytosine deaminase, whereas for GR tumors, both systems worked equally well. Furthermore, we find additive toxicity using both SG/PD systems for both TS/A and GR tumors.

Rapid and sensitive detection of enhanced green fluorescent protein expression in paraffin sections by confocal laser scanning microscopy.

Various forms of green fluorescent protein (GFP) have become important reporters of gene transfer and expression after transfection or infection of cells in cell culture. Frequently, molecular biological assays (Northern blots, PCR) are applied to detect reporter gene expression in target organs. However, these methods are not suitable for evaluation of tissue- or cell-specific expression which would be of great interest especially in case of using tissue-specific promoters. Therefore, organs of transgenic mice with the enhanced green fluorescent protein (EGFP) gene under control of the cytomegalovirus (CMV) promoter were processed for histology by formaldehyde fixation and embedding in paraffin. Sections were deparaffinized, mounted and evaluated for fluorescence in a confocal laser scanning microscope. This method combines the advantages of direct exploitation of tissue sections without further staining procedures with evaluable tissue-, cell-, and even subcellular-specific distribution patterns of EGFP expression in tissues. Results obtained by direct evaluation of EGFP fluorescence in paraffin sections were confirmed by immunohistochemical staining with anti-EGFP. In the present report, we demonstrate that application of confocal microscopy on routinely processed histological preparations is very suitable for determining gene transfer efficiency and promotor activities.

Diagnosis of feline herpesvirus infection by immunohistochemistry, polymerase chain reaction, and in situ hybridization.

An adult domestic shorthair cat had severe chemosis due to purulent and necrotizing blepharitis and conjunctivitis. Purulent rhinitis, necrotizing glossitis, and dermatitis were also diagnosed. The cat was positive for feline immunodeficiency virus and feline leukemia virus. Histologically, intranuclear Cowdry type A inclusions were found within numerous epithelial cells adjacent to the lesions in skin, conjunctiva, and tongue. Electron microscopic examination revealed herpesviral particles within the lesions. Paraffin-embedded skin and tongue tissues were processed in a polymerase chain reaction, using primers to amplify a 306-bp region of the thymidine kinase gene of feline herpesvirus type 1, resulting in a distinct amplification product of the predicted size. The distribution of feline herpesvirus was demonstrated by immunohistochemistry and nonradioactive in situ hybridization. Positive immunostaining was found in nuclei and cytoplasm of numerous epithelial cells within and next to the lesions, whereas in situ hybridization, performed with a digoxigenin-labeled double-stranded DNA probe, revealed hybridization signal only in nuclei of intact epithelial cells. Neither immunohistochemistry nor in situ hybridization showed feline herpesvirus type 1 in tissues of lungs, liver, spleen, intestine, or brain.

Puumala virus and two genetic variants of Tula virus are present in Austrian rodents.

Puumala and Tula viruses are hantaviruses found in Europe and are associated with the rodents Clethrionomys glareolus and Microtus arvalis, respectively. Puumala virus is associated with the human disease nephropathia epidemica. In Austria, ten clinically diagnosed cases of nephropathia epidemica, presumably caused by Puumala virus infection, have been reported but not virologically confirmed [Leschinskaya et al., 1991; Aberle et al., 1996]. To identify the hantaviruses that are present in Austria, five species of rodents were trapped and screened for virus antibodies, antigen, and RNA. Hantaviruses were detected in two species, Cl. glareolus and M. arvalis, by reverse transcription-polymerase chain reaction (RT-PCR). RT-PCR products from Cl. glareolus tissues yielded a unique Puumala virus sequence distinct from Puumala virus sequences reported from other parts of Europe. RT-PCR products from M. arvalis tissues yielded two genetically distinct Tula virus sequences, one similar to sequences reported from Slovakia and the Czech Republic and another that appears to be a novel genetic variant of Tula virus. This is the first confirmed report of hantaviruses in Austria.