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Many studies have shown a strong correlation between CIN and HPV infection. Molecular biology has allowed identification of types of HPV which seem to be connected, more frequently than others, to dysplastic lesions. Physical state of HPV-genome seems to play an important role in the development of cervical cancer. In this study the HPV-genome has been searched in tissue specimens obtained from 34 women affected by CIN II and III. All patients underwent laser conization. Immediately before treatment, colposcopically directed biopsies of the cervical lesion and of the areas with no colposcopically apparent disease were taken and on these samples, HPV-DNA has been searched, isolated and analysed for HPV types and physical state. Histologic examination on cones showed 6 cases of CIN II (3 with HPV), 24 cases of CIN III (14 with HPV), 1 microinvasive carcinoma and 3 with no residual lesion. Southern blot analysis detected HPV-DNA in 4 cases of CIN II (16.7%) and in 20 cases of CIN III (70.6%). In 50% of CIN II and 85% of CIN III HPV 16 DNA has been found and in the remaining 50% of CIN II and 15% of CIN III HPV 31 DNA has been detected. All CIN II and 14 cases of CIN III showed episomal HPV-DNA. Integrated HPV-DNA has been found in 3 cases of CIN III and the other 3 cases of CIN III showed both integrated and episomal HPV-genome. Integrated form has been noticed only for HPV 16 type. In no case of colposcopically normal tissue has HPV-DNA been found. These data seem to confirm the strong correlation between HPV 16 type, which often has integrated form, and CIN III strengthening the hypothesis of its potential oncogenic action.

Quantitative analysis of DNA content represents a critical step when only very small amounts of nucleic acids are available. The DNA content of a small RNA-free sample can be measured in a simple and precise way using a two-dimensional approach. DNA samples are spotted on the surface of an agarose gel containing ethidium bromide (EtBr) and the ultraviolet-induced low-light fluorescence emitted by EtBr molecules intercalated into the DNA is evaluated. The high sensitivity and reproducibility of this quantitative method has been obtained using an advanced analysis system capable of distinguishing low-light fluorescent patterns, as in the case of DNA stained with EtBr, from the background. Use of an internal standard is necessary because the intensity of the signal is due to the aperture of camera diaphragm and to gel conditions. Using this two-dimensional analysis system it is possible to obtain rapid and precise quantitation of as little as 2 ng of DNA.

Cytokine-mediated communication between the immune system and the nervous system has been shown in the past few years. The precise cellular sources of these molecules in the brain is still a controversial issue. We have thus immortalized primary cell cultures from mouse embryonic brains to analyze cloned cells involved in cytokine production. The cell clones obtained were identified as microglial cells and shown to produce several monokines. Among these, TNF alpha was detected by molecular analysis and cytotoxicity assays and shown to be expressed by microglial cells, after activation with LPS. Surprisingly, the TNF alpha-mediated cytotoxic activity, which was neutralized by specific antisera, was not detected in the cell supernatants but was mediated through cell-to-cell contact. Using antibodies to TNF alpha in FACS analysis, specific cell membrane staining on live microglial cells was shown. The results suggest that in the brain the form of TNF alpha detectable by standard procedures is the cell bound form and not the most common form, secreted TNF alpha. In addition, the effects of recombinant TNF alpha in vitro and in vivo were evaluated. In vitro, rTNF alpha stimulated beta-endorphin, GH, and PRL release from cultured cells prepared from rat anterior pituitary glands. In vivo, the administration of rTNF alpha to rats was able to modify analgesic responses. The concomitant administration of naloxone, an opiate receptor antagonist, or monoclonal anti-IL-1 antibody decreased the analgesic effects induced by rTNF alpha. This indicates that the analgesic effect might not be mediated directly by rTNF alpha but by other mediators, whose action is under the control of TNF alpha.