The value of immunohistochemistry and in situ hybridization in detecting cytomegalovirus in bone marrow transplant recipients.

Autopsy tissues of 19 patients with complications after bone marrow transplantation (BMT) were analysed for the presence of cytomegalovirus (CMV) using histochemical methods. CMV antigens were detected by antibodies to CMV Immediate Early Antigen (IEA) or CMV Late Antigen (LA). CMV-DNA was detected by DNA in situ hybridization (DISH). IEA was detected in one or more tissues in 79% of 14 patients from whom frozen tissue was available. CMV-DNA was detected on paraffin sections in 84% of all 19 patients. CMV components were present in all organs studied; the highest incidence was found in lung, gastrointestinal tract and kidney. In histology, only 37% of patients showed signs of CMV infection by the presence of cytomegalic cells with nuclear inclusions (or so called "owl eye cells"). In tissue culture, only 33% of 15 patients were CMV positive. Serologically, 68% of all patients had active CMV infection, as indicated by a rise in antibody titres. We conclude that the quick detection of CMV IEA and CMV-DNA has a high sensitivity and predictive value, which is comparable to or exceeds the serological detection of CMV.

Detection of calcitonin-encoding mRNA by radioactive and non-radioactive in situ hybridization: improved colorimetric detection and cellular localization of mRNA in thyroid sections.

The localization of mRNA encoding calcitonin was studied by in situ hybridization using 35S-labeled RNA probes and biotin-labeled DNA probes. Radiolabeled probes were detected by autoradiography and biotin-labeled probes by streptavidin-biotin-peroxidase. To intensify the colorimetric signal, the indirect avidin-biotin complex (ABC) method was performed. However, the results were often variable. To improve the sensitivity, the peroxidase reaction signal was enhanced with a gold-silver deposit intensification reaction. To shorten the incubation times and to enhance the colorimetric reaction, several reaction steps were performed in a microwave oven. The localization of calcitonin mRNA in thyroid tissue, as detected with in situ hybridization, was confirmed by immunohistochemical localization of the calcitonin polypeptide. The results of in situ hybridization using biotinylated probes were compared to in situ hybridization using radioactive probes. Our data show that the results of in situ hybridization applied on frozen and paraffin-embedded sections using biotinylated DNA probes, detected with an indirect streptavidin-biotin-peroxidase reaction and intensified by silver-gold enhancement, were comparable to those obtained with radioactive probes. The localization of calcitonin encoding mRNA was in agreement with the localization of the calcitonin polypeptide.

Ultrastructural cytochemistry of non-specific esterase in murine peritoneal macrophages.

Non-specific esterase (NSE) activity was demonstrated in glutaraldehyde-fixed monolayers of murine peritoneal macrophages. Using 2-naphthylthiol acetate (NTA) as substrate and Fast Blue BB as coupling agent a strong osmiophilic reaction product was obtained. The reaction product was observed as electron-dense dots covering cisterns of the rough endoplasmic reticulum, Golgi saccules and vesicles, or as large aggregates in lysosomes. Using alpha-naphthyl butyrate (ANB) as substrate and hexazotized pararosaniline as coupling agent the osmiophilic reaction product was observed extracellularly on the plasma membrane as an electron-dense continuous layer, whereas intracytoplasmic staining of lysosomes was rare. Substitution of the coupling agents in the respective media resulted in a slight reaction with the ANB medium whereas with the NTA medium reaction product was observed only in lysosomal structures. The substrate specificity of the different types of esterases was confirmed after isoelectric focusing on thin-layer polyacrylamide gels. The results indicate that in murine peritoneal macrophages different types on NSE are detected with NTA and ANB, having distinct ultrastructural localizations.

Tumor growth stimulatory macrophages induced by a subclinical bacterial infection in vivo.

Nonelicited peritoneal macrophages obtained from normal mice from our animal house unexpectedly expressed a strong tumor growth stimulatory effect in vitro. Macrophages expressing this stimulatory effect had an aberrant morphology compared to the morphology of normal macrophages as observed by electron microscopy. The results of immunization of these affected mice with tumour cells led to the usual lymphocyte sensitization. No external symptoms were observed, and the mice looked healthy. Treatment of the affected macrophage donors with antibiotics resulted in the abolishment of the tumor growth stimulatory effect by the macrophages. Thus, this tumor cell growth stimulation by macrophages was probably due to a subclinical infection of the mice.