Proteins and immunohistochemical markers of breast diseases

In this work, we have compared malignant and non-malignant diseases of the mammary gland using 8 proteins: HRG, MUC1, PAI-1, HSP90αA1, CDH1, ERα, PGR and IL-12. Their concentrations in the supernatants of blood cells and breast biopsies were compared in terms of spontaneous production, induced by a polyclonal activator and after exposure to biopsy samples of the HLDF differentiation factor, as well as the indices of the effect of the polyclonal activator and HLDF on the protein production. In addition, the correlation relationships of the above indicators with the expression of markers of the epithelial-mesenchymal transition: collagen type II (CII), β-1 integrin (CD29) and cadherin-E (CDH1) were studied. The study revealed statistically significant differences in the concentration of HRG in the supernatant of blood cells, IL-12 during spontaneous production by biopsy specimens, PGR production of biopsy specimens induced by the polyclonal activator, CDH1 and IL-12 production biopsy specimens exposed to HLDF. According to the influence index of the polyclonal activator and HLDF, statistically significant differences were found for CDH1production. Comparison of non-specific invasive carcinoma biopsy specimens and non-malignant breast diseases by means of the markers of the epithelial-mesenchymal transition revealed statistically significant differences in CD29 expression and the lack of differences in the expression of CDH1 and CII. This indicates the presence of cell atypia in samples of non-malignant breast diseases; it is confirmed by the recognized correlation between the production of certain proteins and the expression of the epithelial-mesenchymal transition markers.

CYTOKINE-PRODUCING RESOURCE OF IMMUNOCOMPETENT BLOOD CELLS IN BREAST TUMORS AND PRECANCEROUS CHANGES OF MAMMARY GLAND

At present, only ductal carcinoma in situ is included into the group of precancerous lesions of mammary ducts, according to International Agency for the Study of Cancer. However, based on recent publications, in addition to ductal carcinoma in situ, sclerosing adenosis, intraductal proliferative lesions and radial scar may be also attributed to precancerous changes. A variety of both benign and malignant events in mammary gland, the features of neoplastic growth and age of the patients require new approaches to study of carcinogenic events in mammary gland. As based on the known role of cytokines in genesis of malignancies, the aim of the study was to evaluate the cytokine-producing resource of immunocompetent blood cells in malignant, benign and precancerous mammary disorders. To assess the cytokine-producing resource of immunocompetent blood cells in the patients, we studied quantitative effects of polyclonal activators upon production of cytokines by immunocompetent blood cells of patients with invasive ductal cancer representing a histological type of adenocarcinoma (group I), and patients with non-malignant breast neoplasias (group II). At subsequent step, the patients with non-malignant neoplasms of the breast were divided into a subgroup of patients with only fibroadenoma and mastopathy (group III), and a group which included patients with precancerous diseases, i.e., sclerosing adenosis and interductal proliferates (group IV). Concentrations of IL-2, IL-6, IL-8, IL-10, IL-17, IL-18, IL-1β, IL-1ra, TNFα, IFNγ, G-CSF, GM-CSF, VEGF, and MCP-1 were determined by solid-phase enzyme immunoassay. When comparing groups I and II, we revealed higher influence of polyclonal activators upon production of G-CSF and GM-CSF in patients with invasive ductal cancer. When comparing the influence of polyclonal activation for cytokine production in patients of I and III groups, higher values were registered in patients with invasive ductal cancer (production of IL-2, G-CSF, and GM-CSF), and in patients with fibroadenoma and mastopathy (IL-18, and TNFαproduction). When comparing patients of groups I and IV, higher indexes of the polyclonal activator effects were found only for IL-1ra, G-CSF, and VEGF production in invasive ductal cancer. When comparing the indexes of polyclonal activator influence upon cytokine production of groups III and IV, higher values were obtained in patients with benign changes for the following cytokines: IL-8, IL-18, IL-1β, IL-1ra and TNFα, in contrast to patients with sclerosing adenosis and proliferates. The lower indexes of polyclonal activating effects upon the production of a number of cytokines in patients with precancerous changes, as compared to patients with malignant and benign breast tumors, do not indicate a decreased functional activity of immunocompetent blood cells. However, those may be due to high level of spontaneous cytokine production in sclerosing adenosis and interductal proliferates.

Diffusion ability of endotoxin through dentinal tubules

The aim of this study was to evaluate the ability of endotoxin to diffuse through dentinal tubules towards the cement and to observe the period of time needed for it to reach the external root surface. Thirty single-rooted human teeth had their crowns and apices removed in order to standardize the root length to 15 mm. Teeth were instrumented until #30 K-file and made externally impermeable with epoxy adhesive, leaving 10 mm of the exposed root (middle third). The specimens were placed in plastic vials and irradiated (60Co gamma-rays). Then, they were divided into 2 groups (n = 15): G1) Escherichia coli endotoxin was inoculated into the root canal of the specimens and 1 ml of pyrogen-free water was put in the tubes; G2) (control): pyrogen-free water was inoculated into the root canals and 1 ml of pyrogen-free water was put in each tube. After 30 min, 2 h, 6 h, 12 h, 24 h, 48 h, 72 h and 7 days, the water of the tubes was removed and replaced. The removed aliquot was tested for the presence of endotoxin. Considering that the endotoxin is a B-lymphocyte polyclonal activator, at each experimental period, B-lymphocyte culture was stimulated with a sample of water removed from each tube and antibody (IgM) production was detected by ELISA technique. The results of IgM production were higher in groups of 24 h, 48 h, 72 h and 7 days in relation to the other studied groups, with statistically significant differences (ANOVA and Tukey's test p < 0.05). Endotoxin was able to diffuse through the dentinal tubules towards the cement, reaching the external root surface after the period of 24 h.

The autoantigen-binding B cell repertoires of normal and of chronically graft-versus-host-diseased mice.

A quantitative analysis of the frequencies of autoantibody-producing B cells in GVHD and in normal mice has been undertaken by generating collections of hybridomas of activated B cells. These hybridomas secreted sufficient quantities of Ig to allow binding analyses on a panel of autoantigens. B cells have been activated in a variety of ways. In vivo they were activated by injection of alloreactive T cells of one parent, leading to GVHD by a foreign antigen, sheep erythrocytes, in a secondary response, or by the polyclonal activator LPS. B cells from an experimentally unstimulated animal were used for an analysis of the normal background. In vitro B cells were activated by alloreactive T cells or by LPS. The frequencies of hybridomas and, therefore, of activated B cells producing autoantibodies to DNA or to kidney were not significantly different in mice activated by a graft-vs.-host T cell response as compared with B cell populations activated by any of the other procedures. They were found to compose 7.1-17.1% of the total repertoire of activated B cells. Moreover, the frequencies of autoantibody-producing activated B cells does not change with time after induction of the graft-vs.-host reaction. The pattern and frequencies of autoantigen-binding specificities to cytoskeleton, smooth muscle, nuclei, mitochondria, and DNA were not found to be different in any of the groups of hybridomas. The single notable exception, found in GVHD mice, were hybridomas producing autoantibodies to kidney proximal tubular brush border. These results allow the conclusion that autoantigen-binding B cells exist in an activated state in GVHD mice, as well as in mice activated by a foreign antigen or by a polyclonal activator, in B cell populations activated in vitro either by alloreactive T cells or by a polyclonal activator, and even in the background of experimentally unstimulated animals. T cell-mediated graft-vs.-host activation, in large part, does not lead to a selective expansion of autoantigen-binding B cells. The main difference between the graft-vs.-host-activated B cell repertoire and all others is that approximately 90% of teh autoantibodies were of the IgG class, whereas al autoantibodies found in the other groups were IgM.

Synthetic Fc peptide-mediated regulation of the immune response. I. Characterization of the immunomodulating properties of a synthetic 23-amino acid peptide derived from the sequence of the CH3 domain of human IgG1.

A synthetic 23-amino acid peptide derived from the CH3 domain of human IgG1 was found to be a potent adjuvant as well as a polyclonal activator. The Fc peptide was found to enhance human and murine humoral, and T cell-mediated immune responses. Moreover, in vivo administration of Fc peptide enhanced murine natural killer cell activity. The synthetic Fc peptide was found to be more potent, on a molar basis, than native Fc fragments in inducing polyclonal antibody production and potentiating immune responses.