Tissue localization of Toll-like receptors in biopsy specimens of liver from children infected with hepatitis C virus.

Toll-like receptors (TLR) are important tools of innate immunity, localized mainly on cells of the immune system, but also have been shown on cells of other origin. In the current study, they have been searched in biopsy specimens of liver from children bearing chronic viral hepatitis of C type (HCV). TLR2, TLR3 and TLR4 were traced by means of polyclonal antibodies and avidin-biotin complex (ABC) immunohistochemistry. Besides, mRNA for TLR was looked for using specific primers and polymerase chain reaction. Several controls, including neutralization of primary antibody with respective blocking peptide, confirmed the specificity of the immunohistochemical reaction. All TLR tested could be visualized in a focal distribution in single hepatocytes and some cells of inflammatory infiltrates. There was no reaction whatsoever in liver samples not infected with hepatotropic virus. In molecular studies, mRNA for TLR2 and TLR4 was detected in both noninfected and hepatitis B virus-infected established cell lines of human hepatoma as well as in HCV(+) biopsy samples. These data indicate that TLR can be traced in liver cells, both at the protein and at the mRNA level. Their irregular and focal distribution in HCV(+), but not in HCV(-), liver suggests some role of TLR in the pathogenesis of chronic viral hepatitis, at least in children.

Protein and mRNA expression of CD56/N-CAM on follicular epithelial cells of the human thyroid.

In order to confirm CD56/N-CAM antigen prevalence in the human thyroid and to compare its expression on thyrocytes and NK cells, an expression of CD56/N-CAM antigen was searched for on isolated thyroid follicular cells and NK cells by flow cytometry. In addition, mRNA for CD56 was searched for in RNA isolated from human thyroid samples and few other organs using dot blot hybridization assay to prove the existence of mechanisms for active synthesis of the protein in question. The isolated cells from the follicular epithelium of 22 various pathological thyroid tissue specimens were examined for the expression of CD56/N-CAM in terms of the percentage of positive cells and the mean fluorescence intensity (MFI). Blood lymphocytes were tested in parallel. The total RNA isolated from thyroid and control tissue specimens was subjected to dot-blot hybridization assay using CD56/N-CAM cDNA probe. All thyroid specimens expressed CD56/N-CAM, but the obtained values differed depending on the tissue examined and the CD56 antibody used. There were no significant differences between the non-malignant thyroid cells of various histology, while cells in the carcinoma group had a much lower MFI, especially the median value. CD56 expression on NK cells from the donors' blood had a homogeneous distribution but the mean and median values of FI were almost three times lower than those on the thyroid cells. Dot blot hybridization came out positive with the RNA isolated from the thyroid specimens and also from the RNA isolated from the tonsil and lymph nodes, but came out negative with the RNA isolated from human and rat kidneys. These results strongly suggest that thyroid follicular epithelial cells express both protein and mRNA of CD56/N-CAM, thus being able to synthesis the relevant antigen. The protein expression seems to be affected by the malignant transformation of the thyroid cells. NK cells have apparently lower CD56/NCAM expression than thyroid cells.

Expression of CD56/N-CAM antigen and some other adhesion molecules in various human endocrine glands.

CD56/N-CAM antigen, 140 kDa isoform of neural cell adhesion molecule (N-CAM) has been previously traced by some of us in follicular epithelium of human thyroid by immunohistochemistry. The reaction product was cell membrane bound, being stronger in hyperactive thyroid as compared to colloid goiter. In the current study, CD56 was searched in other endocrine glands and their tumors including parathyroids, adrenal cortex and parafollicular C cells of the thyroid (TT cell line). The antigen was also examined in the tissue extracts of endocrine and nonendocrine organs by dot blot immunoassay and anti CD56 monoclonal antibody. Besides, some other cell adhesion molecules (CAMs) were looked for in the tissues and cells tested. It has been found that CD56 is expressed in all zones of adrenal cortex, albeit in various intensity. The reaction was cell membrane bound in cortical hyperplasia and adenoma but cytoplasmic in the carcinoma of adrenal cortex. Other endocrine tissues and cells tested were devoid of CD56. Presence of CD56 antigen could be confirmed by dot blot assay with 3M KCl and NP40 extracts of both, thyroid and adrenal glands. Apart from CD56 some other CAMs could be traced in thyroid cell membranes including CD44, VLA-3 integrin and E-cadherin, what was not the case in the adrenal cortex. In parathyroids and parathyroid adenoma, diffuse immunostaining of E-cadherin and irregular, focal expression of CD44 was observed. These results show, apart from CD56, abundance of other CAMs in the thyroid gland and their relative scarcity in other endocrine tissues tested.

Extracellular matrix proteins expression and incidence of tumor infiltrating cells in laryngeal carcinoma.

Cryosections from surgical tissue blocks of primary laryngeal carcinoma were examined by immunohistochemistry for the distribution of several extracellular matrix (ECM) proteins and of tumor infiltrating cells (TIC). For the assessment of ECM proteins 8 monoclonal antibodies (Moabs) versus fibronectin, its neoplastic isoforms, tenascin, type IV collagen, laminin were used. TIC were evaluated by means of 9 Moabs versus various lymphocyte subsets, NK cells and macrophages. Marked differences were found in overall normal ECM proteins expression between tissues free from neoplastic invasion, those of closest tumor vicinity and of tumor mass. ECM proteins were found to be the most abundant in remote, apparently normal tissue compartments. They were less expressed in direct tumor proximity and almost absent from tumor mass. On the contrary, fibronectin isoforms were absent in tissue areas free from tumor but became demonstrable in tumor mass and its close vicinity. Tumor infiltrating cells were quite abundant in direct tumor vicinity. While comparing an intensity of expression of ECM proteins and accumulation of TIC in laryngeal carcinoma, reverse correlations were noticed. Strong expression of normal ECM proteins was accompanied by relatively scarce lymphocytic infiltrates in areas distant from primary tumor, while in the tissues abundant in TIC accumulation, such as in direct tumor vicinity, only weak ECM protein expression was seen. This was not the case when fibronectin neoplastic isoforms were studied. The latter could be shown in proximity or within tumor mass exclusively.

Alloantibodies, autoantibodies, and immune complexes in patients with lung cancer.

The sera of patients with lung cancer, nonmalignant lung disease, and blood donors were subjected to various immunologic assays. Nine assays, based on immunoradiometric (IRMA) and immunoenzymatic (ELISA) principles, included 3 types of fetal cell antibodies, 2 established lung cancer cell antibodies, anti-DNA, anti-IgG autoantibodies, and immune complex assays based on C1q binding and anti-C3 activity. Antitumor cell antibody level was significantly lower in patients with lung cancer compared to blood donors. In the remaining 7 assays, the lung cancer patients tended towards higher median values compared to both control patients and blood donors, but without statistical significance, with the exception of anti-DNA antibodies. Statistical analysis of all 9 assays taken together has shown significant differences between the 3 groups. When only 5 assays were used to assess 3 types of fetal cell antibodies, anti-DNA antibodies, and immune complexes by means of ELISA anti-C3, the margins between groups increased. A range of values for the selected assays was established that may discriminate 70% of tested individuals of the 3 groups. These results suggest the existence of a characteristic profile of deranged humoral immunity in lung cancer patients.

The influence of high-dose prednisone medication on autoantibody specific activity and on circulating immune complex level in cerebrospinal fluid of multiple sclerosis patients.

In agreement with the close correlation between intrathecal IgG production and anti-MBP (myelin basic protein) and anti-MAG (myelin-associated glycoprotein) antibody activity in the CSF of active MS cases, and parallel to the reduction of intrathecal IgG synthesis resulting from corticosteroids medication, we have found a significant reduction of anti-MBP and anti-MAG antibody activity expressed per 0.5 micrograms of CSF IgG in the same group of 40 MS patients subjected to high-dose prednisone therapy. Every patient received 3980 mg of prednisone over 54 days. In native CSF of 30% (21/70) of active MS cases, circulating immune complexes (CIC) were detected by C1q binding solid-phase RIA. There was no correlation between CIC level in the CSF or MS patients and 1. IgG index which was used as an indicator of intrathecal IgG synthesis, or 2. CSF anti-MBP specific antibody activity, or 3. CSF anti-MAG specific antibody activity. High-dose prednisone therapy resulted in a highly significant reduction of the CSF CIC level. CIC were also found in the CSF of patients affected with various chronic diseases of the CNS.