[Nuclear DNA in neoplastic pathology of the thyroid gland]. / Il DNA nucleare nella patologia neoplastica della tiroide.

We have analysed the nuclear DNA content in a series of 33 thyroid tumours (18 carcinomas and 15 adenomas) and, for comparison, in 189 nodular strumas and 17 tissue samples of normal thyroids, calculating cell cycle parameters and presence of aneuploid DNA. Cell suspensions were prepared for each of three tissue fragments taken for different areas of the surgical specimens. Cell subpopulations with aneuploid DNA content were present in 39% of carcinomas, 27% of adenomas, 20% of strumas, but were absent in the samples from normal thyroids. The presence of hyperdiploid DNA was much more frequent than that of hypodiploid DNA in all pathologic tissues. The mean proliferation index was 14.1 in the carcinomas, 6.6 in the adenomas, 7.1 in the strumas and 6.1 in normal thyroid tissues. The percent of histograms with a coefficient of variation of the G0-G1 peak greater than 5 was highest in the carcinomas (26%). The significance and implications of the reported data are discussed for the interpretation of thyroid neoplastic pathologies.

[Recent findings on the etiopathogenesis of multinodular goiter. II. Thyroid growth factors]. / Recenti acquisizioni sull'etio-patogenesi dello struma multinodulare. II. I fattori di crescita della tiroide.

In the last ten years several experimental data have suggested that growth factors may play an important role in modulating thyroid growth and function. Epidermal Growth Factor (EGF) is present in large quantities in thyroid tissue, where it appears to stimulate DNA synthesis and inhibit cellular function. Insulin-like Growth Factors (IGFs), Interleukin I (IL-I) and Fibroblast Growth Factor (FGF) show the same stimulation on cell proliferation. On the contrary Transforming Growth Factor beta (TGF-beta) and somatostatin display inhibitory effects on follicular growth. Therefore it is likely that growth factors' altered production and/or their receptors abnormal expression on the thyroid cells surface might be involved in the development of multinodular goiter and some thyroid neoplasms.

Phenotypic analysis of human peripheral blood lymphocytes by automatic sampling flow cytometry after stimulation with mitogens or allogeneic cells.

Human peripheral blood lymphocyte (PBL) phenotypes have been analyzed before and after stimulation with phytohemagglutinin (PHA), concanavalin A (ConA) and pokeweed mitogen (PWM) for 3 days and in mixed lymphocyte culture (MLC) for 7 days. PBL labeled with each of 10 fluorescent monoclonal antibodies were automatically sampled for flow cytometry from 96-well microtiter plates using a microsample delivery system. The reference phenotypic ranges were determined in fresh cells and control cultures. PHA was mostly mitogenic for T PBL bearing the CD3, CD5, CD7, CD8 and CD25 differentiation clusters, and a low density of CD1 and CD4 had a small effect on human natural killer cells (HNK) and also did not stimulate B (CD19) and HLA-DR+ PBL. There was an incomplete phenotypic overlapping between PHA- and ConA-stimulated cultures, ConA being more mitogenic for CD4 and less mitogenic for CD8 PBL. The mitogenic effect of PWM was evident on CD3, CD5, CD7, CD4, CD25 and CD8, but not on HNK, HLA-DR and CD19 B PBL, which presumably had already differentiated into antibody-secreting cells. After MLC stimulation all T, B and HNK PBL subsets tested were increased, but the cells bearing CD1, CD4, CD5, CD7, CD25, HNK, CD19 and HLA-DR had the greatest proliferation with respect to the unmixed control PBL. The present approach to the phenotyping of PBL subsets could offer more complete and accurate data for monitoring and follow-up of patients in transplantation and immunopathology hospital wards.

Heterogeneity of lymphokine-activated killer (LAK) populations at the clonal level: both NK and CD3+, CD4-, CD8- clones efficiently mediate tumor cell killing.

To investigate at the clonal level the phenotypic and functional properties of interleukin 2 (IL-2) activated killer cells (LAK), recombinant IL-2 activated peripheral blood lymphocytes were cultured under limiting conditions. Among 56 clones that lysed P815 in the presence of phytohemagglutinin (PHA) (22% of total proliferating microcultures) 36 clones lysed also the natural killer (NK)-sensitive K562 and the NK-resistant Hu126 glioma cell lines and one clone lysed only the K562 cell line. Several LAK clones were further assayed for both phenotype and functional activity. Of 22 clones, 10 were CD3-, CD4-, CD8-, and expressed the CD16 marker of NK cells; only one clone had the conventional phenotype of cytolytic T cells (CD3+, CD4-, CD8+), while 11 clones were CD3+, CD4-, CD8- and did not express alpha/beta heterodimer of T-cell antigen receptor as identified by WT31 monoclonal antibody. Only one of the latter clones was CD16+. Endogenous production of IL-2 after stimulation with PHA and phorbol myristate acetate was positive in 3/9 CD3- and in 8/8 CD3+, CD4-, CD8- clones. CD3- mediated strong antibody-dependent cellular cytotoxicity, a function exerted also by some CD3+, CD4-, CD8- T-cell clones to a lower extent. CD3+, CD4-, CD8- T-cell clones lysed different major histocompatibility complex unrelated tumor targets; moreover, this lytic activity seems to be CD3 dependent.

DNA analysis of stimulated lymphocytes by automatic sampling for flow cytometry.

A microsample delivery system (MSDS) was tested for automatic flow cytometry (FCM) analysis of DNA synthesis in stimulated human peripheral blood lymphocytes (PBL) cultivated in wells of microtiter plates. After incubation, either for 1-3 days with phytohemagglutinin, concanavalin A, and pokeweed mitogen, or for 7 days with allogenic PBL, the cells, while in the wells, were washed in hypotonic Tris buffer and stained with ethidium bromide-RNAse solution. The results obtained from quintuplicate replicated wells, each of the five containing the same control or stimulated cultures, were reproducible in terms of the number of nuclei counted in each histogram of control, mitogen-stimulated PBL, and mixed lymphocyte cultures (MLC). Using a computer program that superimposes histograms and calculates their differences on the scale of fluorescence intensity, it was possible to quantify the intensity of the response to the mitogenic stimuli. This approach to the study of lymphocyte proliferation offers not only a simpler and faster analysis of DNA synthesis than the method of 3H-thymidine incorporation, but it also allows for the analysis of other FCM parameters, such as forward and 90 degrees light scatter and double fluorescence labelling of PBL nuclei.

[Immunological aspects of dental transplants]. / Aspetti immunobiologici dei trapianti dentari.

The recent literature on dental transplants in man and experimental models is reviewed. The histopathological and immunological aspects of the methods and results are critically analysed in function of the clinical applicability of this alternative to prosthesis. The different effects of mature tooth and germ transplants are considered in the light of experiments on various mammals, together with the contribution made by the various dental tissues to the taking of the transplant, and its acceptance or rejection by the host. Finally, questions associated with the establishment of a teeth bank and its potential creation of a sufficient supply of organs for transplantation are discussed.

Carcinoembryonic antigen and cancer: a comparative study.

The sera of 212 patients with malignant and non-malignant diseases have been radioimmunoassayed for the presence of the carcinoembryonic antigen (CEA) using 3 different kits produced of Hoffman La Roche, Switzerland (RCK), BY Sorin-IRE, Italy and Belgium (SCK), and by the Istituto Sieroterapico Milanese, Italy (ICK). In the presence of endodermically-derived system carcinomas, the RCK gave more positive results (72.6%) than did the SCK (63.1%) or ICK (56.2%). With regard to other carcinomas, ICK (50.0%) and SCK (47.1%) gave better results than did RCK (30.6%). The results are discussed in terms of clinical usefulness of the CEA assay and as regards reproducibility, procedural advantages, and economical cost of each kit. It is concluded that the CEA assay cannot be used for the diagnosis of gastrointestinal cancers, although it is useful as a measure of "cancerosity" for prognostic purposes. In this sense the double antibody method employed by SCK and ICK is clinically more advantageous than is the perchloric acid extraction-zirconyl phosphate gel precipitation method of RCK.

Cyclicity and memory in the humoral immune response of the marine toad Bufo marinus.

Bufo marinus toads immunized with a single dose of 100 mug of polymerized flagellin (POL) produce a longlasting cyclic humoral response at 22 degrees C in which the serum antibody titers rise and fall within periods of 2-3 weeks. This seems to be regulated by the catabolism of immunoglobulins, since passive 125I-labeled IgM in the serum has a half-life of 17 days, corresponding to the cyclicity of the titers of active antibodies. In toads injected with between 10 ng and 10 mug antigen the serum antibody titer peaks between 4 and 7 weeks, depending on the antigen dose, and declines by the 9th week. At this time a second stimulation with an equal dose of POL induces a secondary response during which higher titers of antibodies rise faster than in the primary response. A dose of 10 mug POL stimulates an optimal primary response and a second equal dose, given when the serum antibodies are disappearing, induces a secondary response enhanced in time but not in antibody titers.

A primary immune response of Bufo marinus spleen cells in vitro.

Single-cell suspensions of Bufo marinus spleen have been induced to produce a primary immune response to a soluble purified protein. Using polymerized flagellin from Salmonella adelaide as antigen and culture conditions commonly available in most laboratories but new for amphibian cells, it has been found that in vitro at 37 degree C, toad spleen cells produce an antibody-forming cell response optimal at day 6. The response depends on the number of cultured cells and dose of antigen, and parallels that obtained in vivo. The optimal immune response is preceded at day 4 by the peak uptake of tritiated thymidine. The antibody-forming cell response is suppressed in the presence of allogeneic serum.