The application of in-situ hybridisation and immuno-cytochemistry to problem resolution in drug development.

Localization of specific mRNA and protein molecules within cells and tissues can provide important information on the effects of a drug within a biological test system and help elucidate mechanisms of drug-induced toxicity and organ dysfunction. The most widely used techniques for measuring the cellular and tissue distribution of mRNA and protein are in-situ hybridization (ISH) and immunocytochemistry (ICC), respectively. These can be applied alongside quantitative measurements to provide an integrated picture of gene expression. In some cases, for example when the gene expression of interest is confined to a small subset of cells within a tissue, histochemical techniques may be the preferred method of analysis.

Pancreatic and nephrotoxicity of immunomodulator compounds.

Increasing clinical experience of FK 506 in transplantation therapy has revealed a number of potentially restrictive adverse effects associated with its use. The mechanisms of action underlying 2 prominent toxic effects of FK 506, namely diabetogenesis and renal dysfunction, were investigated. A simple model system based on the effect of FK 506 on isolated rat pancreatic islets was utilised to study the relationship between inhibition of insulin biosynthesis, inhibition of interleukin 2 (IL-2) activation and FK binding protein (FKBP-12) binding of FK 506 and a number of FK 506 analogues. Results indicate that the action of these compounds on inhibition of insulin biosynthesis (and by implication, diabetogenesis) may be related to their immunosuppressive potential. Observations on the FK 506-induced release of endothelin-1 from isolated rat kidney mesangial cells suggest that this cell may be an important target associated with the nephrotoxic potential of the drug, and that this action may be mediated via the FKBP.

FK506-induced endothelin release by cultured rat mesangial cells.

Renal damage is a major side effect of the macrolide immunosuppressant FK 506. Although little is known about the underlying mechanism of FK 506 nephrotoxicity, available data suggest that it may be associated with a disturbance in renal hemodynamics, possibly brought about by alterations in the production of vasoactive substances such as endothelin-1 (ET-1). We have investigated the release of ET-1 from primary renal mesangial cells in culture. Mesangial cells derived from rat renal cortex were exposed to a range of FK 506 concentrations (10(-10) to 10(-6) M) or vehicle for up to 6 h. FK 506 caused a significant dose-related increase in ET release. This effect was dependent on cell density and was blocked by co-incubation with FPL 65620, an FK 506 analogue that binds to the FK binding protein (FKBP) and inhibits the immunosuppressive activity of FK 506. These data suggest that mesangial cell ET may play a role in FK 506 nephrotoxicity and that the effect of FK 506 on ET release may be mediated through the FKBP.

Detection and cellular localization of stress-induced 72 kD heat shock protein in cultured Swiss 3T3 mouse fibroblasts.

Cellular expression of the 72 kD heat shock protein (72 kD hsp) is increased following exposure to a wide range of physical and chemical stimuli and may be useful as a marker of cell toxicity. The primary objective of this work was to develop a cytochemical method suitable for the detection and cellular localization of stress-inducible 72 kD hsp mRNA and protein in cell cultures. Swiss 3T3 mouse fibroblasts, grown on multi-chamber glass slides, were transiently exposed to an elevated incubation temperature or various chemical agents and cellular 72 kD hsp expression visualized using immunocytochemical and in situ hybridization detection methods. Expression of 72 kD hsp was found to be dependent on the applied stimulus and recovery time. The combined culture system and cytochemical assay for 72 kD hsp provides a convenient method for studying cellular responses to a variety of toxins.

Effect of intravenous iron-dextran (Imferon) infusion on antigen induced monarticular arthritis in rabbits.

The effect of intravenously infused iron-dextran (Imferon) on the progression of antigen induced monarticular arthritis in rabbits was studied. A rapid deposition of iron and apoferritin in the synovia of arthritis joints occurred after infusion of iron-dextran during either the acute or chronic phases of the disease. This coincided with the appearance of catalytic (bleomycin reactive) iron in the synovial fluid. There was no evidence, however, for an exacerbation of the antigen induced arthritis as a result of the iron-dextran, and synovial and bleomycin reactive iron concentrations decreased with time after administration, indicating a redistribution of the synovial iron load. Thus although intravenously infused iron-dextran appears to 'prime' the rabbit arthritic joint transiently with the potential for iron stimulated oxygen free radical damage, other factors may determine its occurrence.

Localization of immunoglobulin light chain mRNA expression in Hodgkin's disease by in situ hybridization.

In situ hybridization techniques were used to detect immunoglobulin light chain messenger RNA (mRNA) in 28 formalin-fixed, paraffin-embedded samples of Hodgkin's disease. Cocktails of biotinylated oligonucleotide probes specific for the constant regions of kappa and lambda light chain mRNA were used. None of the Reed-Sternberg cells or their variants in any of the cases studied showed positive staining with either probe, in contrast to normal plasma cells which showed strong staining in the same sections. It was concluded, therefore, that the cytoplasmic immunoglobulin frequently detected within these cells by immunocytochemistry is present not as a result of synthesis, but as a result of some other mechanism.

In situ hybridization demonstration of poly-adenylated RNA sequences in formalin-fixed paraffin sections using a biotinylated oligonucleotide poly d(T) probe.

An in situ hybridization technique has been developed for assessing poly(A)+ RNA preservation in routine pathology specimens. The method detects poly-adenylated RNA sequences in tissue sections using a biotinylated polydeoxythymidine (poly d(T)) probe. The probe was prepared from single-stranded 25-30 base oligo d(T) and was biotinylated using the enzyme terminal deoxynucleotide transferase with biotin-11-dUTP and dTTP in the ratio 1:4. The hybridization protocol uses varying concentrations of proteinase K to unmask mRNA sequences and the biotin-labelled hybrids are demonstrated after hybridization under standard conditions by the application of streptavidin and biotinylated alkaline phosphatase. Alkaline phosphatase was visualized using a Fast Red naphthol-capture method and the sections were counterstained with haematoxylin. The results have confirmed that the method is specific for poly(A)+ RNA and shows that poly(A)+ RNA can be demonstrated in routine formalin-fixed sections using non-radioactive techniques with retention of morphology. It also provides a means of optimizing the hybridization conditions for specific mRNA probes and produces a staining pattern demonstrating the relative level of poly(A)+ RNA per cell which may reveal new information about cell activity and tissue function.

The measurement of serum unsaturated iron-binding capacity in the presence of iron-dextran.

The standard colorimetric methods most often used for the measurement of serum unsaturated iron binding capacity (UIBC) are subject to gross interference by iron dextran. This paper describes a brief evaluation of an alternative radiometric assay for serum UIBC, based on a commercially available kit method, but incorporating a modification to the manufacturer's protocol. The effects of iron dextran on the assay were determined.