An improved, sensitive, non-radioactive in situ hybridization method for the detection of cytokine mRNAs.

We established an improved non-radioactive in situ hybridization (ISH) method to detect mRNA of cytokines in cell preparations and tissues. Via this method we could demonstrate various cytokines in stimulated peripheral blood mononuclear cells (PBMC), lymphoid cell lines and human lymphoid tissues. The probes for the in situ hybridization were made by labelling cytokine-specific PCR products with digoxigenin (Dig) in a repeated PCR. This resulted in an intrinsic labelling of the probe with several Dig-UTP molecules. Incorporation of Dig-11-dUTPs was shown on ethidium bromide-stained agarose gels by a higher molecular weight of the PCR products with incorporated Dig-dUTPs when compared to control PCR products without digoxigenin. Cytospin-centrifuged cells of PHA-stimulated PBMC or lymphoid cell lines and frozen sections of various human lymphoid tissues were hybridized with the Dig-labelled cytokine probes and the hybridized probes were detected immuno-histochemically. In this way, we detected and localized cytokine mRNAs (IL-2, IL-4, IL-6, IL-8, IL-10) in PBMC, in the human T-cell line Jurkat, in the follicular lymphoma cell line DoHH2, and in human lymph nodes and tonsils. The in situ hybridization had a high sensitivity as the results correlated closely with the detection of cytokine mRNA by reverse transcriptase-PCR (RT-PCR) data from the same samples. We showed that Jurkat and DoHH2 cells produce several cytokines constitutively and that, after activation with the phorbol ester PMA, expression of several cytokine mRNAS was enhanced.

Encoded latent membrane protein 1 of Epstein-Barr virus on follicular dendritic cells in residual germinal centres in Hodgkin's disease.

AIMS: To determine if there is an association between Epstein-Barr virus (EBV) infection and Hodgkin's disease. METHODS: Fifty cases of Hodgkin's disease and 25 reactive lymph nodes were screened for the presence of EBV-RNA (EBER) using in situ hybridisation, and for the expression of EBV encoded latent membrane protein 1 (LMP-1) by immunohistochemistry. RESULTS: In 42% of the cases of Hodgkin's disease, EBER was detected in the nuclei of the malignant cells, and in LMP-1 expression was found 36%. Both EBER and LMP-1 positivity were seen in 34% of the cases. An additional finding was the presence of LMP-1 on follicular dendritic cells in residual germinal centres in two cases of Hodgkin's disease. EBER was not detected in these germinal centres. In reactive lymph nodes only occasional EBER positive, small, lymphoid cells were found, without LMP-1 expression. CONCLUSIONS: These results show a strong correlation between the presence of EBER and the LMP-1 expression in the Reed-Sternberg cells. They corroborate a role for EBV in at least some cases of Hodgkin's disease. LMP-1 is probably presented as an immune complex in the germinal centres, as part of an immune response against EBV.

In situ hybridization: a valuable tool in diagnostic pathology.

In situ hybridization or hybridohistochemistry has evolved in recent years in a new histologic modality. In situ hybridization (ISH) can be used for the detection of DNA (DISH) or RNA (RISH). The potential diagnostic value within a pathologic setting are well recognized. In this review paper, we summarize the use of DISH in a pathologic setting for the detection of chromosomal aberrations and localization of DNA-viruses like cytomegalovirus and Epstein Barr virus. RISH which is still in a more experimental stage can be applied for the localization of RNA-virus, like human immunodeficiency virus. However, the most important application of RISH will be the detection of gene-expression at the level of mRNA. Potentially this has many applications especially in early diagnostics of neoplastic tissues. Finally, we have summarized some pitfalls which may hamper the introduction of in situ hybridization for diagnostic purposes and some future developments in ISH.