Characterization of a monoclonal antibody, HTA28, recognizing a histone H3 phosphorylation site as a useful marker of M-phase cells.

Mitosis is a valuable indicator of active tissue proliferation but, other than morphological characteristics, there have hitherto been no markers available to detect only M-phase cells. However, a newly established monoclonal antibody (MAb), HTA28, recognizing histone H3 (H3) harboring phosphoserine 28, allows visualization with mitotic chromosomal condensation. In this study we investigated the use of HTA28 for immunohistochemical (IHC) detection of M-phase cells in the regenerating rat liver after partial hepatectomy (PH). Groups of three to five rats were sacrificed at intervals up to 72 hr after PH and proliferation was then assessed by IHC staining using HTA28 and other markers. The temporal pattern of the HTA28 staining index (SI) was very similar to that for the mitotic index (MI), also showing similarities to the bromodeoxyuridine (BrdU) labeling index (LI) with a time lag. The HTA28 SI proved to be higher than MI at every time point in line with HTA28 immunoreactivity maintained for all stages of M-phase. The spatial distribution of HTA28-positive cells corresponded with those of other proliferative cell markers. These therefore provide strong evidence for the applicability of HTA28 as an M-phase marker. We also showed that antigenicity for HTA28 is lost if tissue is not immediately fixed after sampling.     

Abnormal increase in multinuclear macrophages in primary cultures of BUF/Mna rat thymomas.

The in vitro characteristics of spontaneous thymomas from BUF/Mna rats, a strain with a high incidence of these tumors, were studied. In primary cultures, the adherent cells consisted of mononuclear macrophages, mono- and multi-nuclear epithelial cells and some fibroblastic cells on day 3. The macrophages rapidly increased in number with the formation of large multinuclear cells by day 9. A modest increase in the number and nuclearity of macrophages was also noted in adherent cultures of normal thymuses from 5-week-old BUF/Mna rats. On the other hand, in cultures of thymic cells from 1-year-old or 5-week-old ACI/NMs rats, a normal control rat strain, macrophages did not increase in number and only rarely formed multinuclear cells in adherent cell cultures. These results suggest that abnormal proliferation signal(s) to thymic macrophages and/or their progenitor cells accompanies and may be involved in the development of thymomas in BUF/Mna rats.