Paraffin sections from animal or human tissues fixed in different fixatives were submitted to immunostaining with the mouse monoclonal antibody 19A2, developed by Ogata et al. (1987a) against cyclin/PCNA. Detection of the bound antibody was performed by the indirect method with biotinylated sheep antibody and streptavidin‐biotin‐peroxidase complexes. No, or faint, nuclear staining was seen in material fixed in ethanol, Bouin, Bouin‐Hollande, Carnoy or formaldehyde, whereas readily detectable immunocytochemical reaction was constantly observed over nuclei of methanol‐fixed tissues. Hydrolysis with 2 N HCl prior to immunocytochemistry (as currently performed to render incorporated BrdU accessible to antibodies) somewhat improved the results with Bouin or Carnoy and markedly augmented the intensity of the peroxidase reactions in formaldehyde and in methanol‐fixed tissues. The distribution of the positive nuclei in the two latter cases coincided with the proliferative compartment. On the other hand, double labelling with [3H]‐thymidine and with the cyclin/PCNA antibody revealed that in methanol‐fixed tissues the cyclin/PCNA labelling index did not differ by more than 6% from the [3H]‐thymidine index. Besides the two labels overlapped in a proportion of labelled cells that was in reasonable agreement with expectation considering cells flow in and out of S phase since the time of [3H]‐thymidine injection. This indicates that both labels recognize the same cells in this material. In contrast, in formaldehyde‐fixed tissues, the cyclin/PCNA labelling index markedly exceeded the [3H]‐thymidine labelling index. From this it is concluded that cyclin/PCNA immunostaining can be used: In formaldehyde‐fixed tissues (including existing material stored as paraffin blocks): for defining and mapping the proliferative (or germinative) compartment. In methanol‐fixed tissues as a substitute to the [3H]‐thymidine autoradiographic labelling index. From this, a method is proposed (derived from classical ‘double‐labelling’technique) for measuring S phase duration in tissues fixed at a known interval time after a single labelling with [3H]‐thymidine (or BrdU) and submitted to cyclin/PCNA immunocytochemical detection and to autoradiography (or to BrdU immunostaining).
Background: Previous studies have described apoptosis in the stratum granulosum and in the stratum corneum, but not in the germinative compartment in normal skin. In psoriasis, an increased epidermal apoptosis has been observed in the differentiated compartment, suggesting that apoptosis has a key role in the pathogenesis of psoriasis, as a counteracting factor to the overproduction of cells. Little is known on apoptosis in the germinative compartment. Methods: Apoptosis was studied on biopsies of normal skin, established lesions of psoriasis and PUVA-treated psoriasis using the transferase-mediated uridine nick end labelling method, which detects fragmented DNA, and electron microscopy. Counting of apoptotic cells was restricted to the germinative compartment as defined by Mib1 staining to evaluate the impact of cell loss on cell production and tissue architecture. Results: The apoptotic index was 0.12% in normal epidermis, 0.035% in established psoriasis and 0.31% in regressive psoriasis. Conclusion: These results have three implications: (1) they show the physiological presence of apoptosis in the germinative compartment in normal epidermis; (2) they suggest that induction of apoptosis is involved in the regression of psoriatic hyperplasia after PUVA therapy; (3) the decrease of physiological apoptosis in the psoriatic lesion suggests that this phenomenon could play a role in the induction of psoriatic hyperplasia.
Cell population kinetics was studied by bromodeoxyuridine (BrdU) histochemical and 3H thymidine radioautographic labelling in dog thyroids. In-vivo labelling with BrdU and in-vitro labelling of incubated slices with 3H thymidine gave similar results. This validates the use of in-vitro labelling of slices for the study of cell kinetics in the thyroid. In-vitro labelling of human thyroid slices demonstrated a labelling index of 13.4 x 10(-5) for follicular cells; assuming an S phase of 10 h, this corresponds to a turnover time of the order of 8.5 years for the follicular cells. Stromal cells appear to turn over faster. These results show for the first time that human thyroid cells divide about five times during adulthood and therefore that the steady state level of thyroid cell mass results from a balance between cell division and cell loss. A shorter turnover time was found as expected in the thyroid of an adolescent and in follicular colloid nodules.
SUMMARY The duration of the phase of DNA synthesis (S-phase) and turnover times were measured by autoradiography in the vaginal and endo-uterine epithelium of spayed mice at different times after a single oestradiol injection. With three doses of oestradiol (0·3, 1·2 and 10·0 μg), changes of turnover time followed similar patterns in uterine epithelium, but maximal activation and subsequent levelling off of proliferation rates were reached sooner with higher doses. In the vagina, no significant dose-dependence was apparent with the same doses of oestradiol; maximal activation of cell proliferation was observed in every case 12 h after hormone injection. Transient shortening of S-phase was observed in both tissues under oestrogen stimulation. The morphological changes in uterine epithelium appeared to be independent of mitotic stimulation. For vaginal epithelium, the results may be interpreted to indicate that oestrogen-induced keratinization starts by a differentiating action on pre-existing G1 cells.
Monoclonal antibodies specific to human T lymphocyte receptors are currently being used to define the biochemical structure of these proteins as well as of functionally distinct cell subsets. Since one of the antibodies (OKT3) recognizing the T3 (CD3) receptor mimics vital physiological processes involved in the activation of the immune system and has been successfully used as a therapeutical agent, we investigated one of the mechanisms underlying this antibody-receptor interaction. Our results show that after binding of OKT3, the complex (OKT3-T3) disappears rapidly from the cell surface. Using electron microscopy, we found that this down-regulation is due to the internalization of the complex. Parallel experiments performed on the T11 (CD2) and T4 (CD4)/AIDS retrovirus receptor indicate that the same mechanism applies for the down-regulation of those molecules. These data suggest that the T3, T11 and T4 receptors have a behavior comparable to other well characterized, hormonal and viral receptors; they provide information on the metabolization pathway of surface receptors and on the possible intracellular penetration of ligands like the HTLV-III/LAV agent in human T lymphocytes.
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