Mariusz Stasiolek

Glucocorticoid effects on endothelial barrier function in the murine brain endothelial cell line cEND incubated with sera from patients with multiple sclerosis

Compromised blood—brain barrier integrity is a major hallmark of active multiple sclerosis (MS). Alterations in brain endothelial tight junction protein and gene expression occur early during neuroinflammation but there is little known about the underlying mechanisms. In this study, we analysed barrier compromising effects of sera from MS patients and barrier restoring effects of glucocorticoids on blood—brain barrier integrity in vitro. cEND murine brain microvascular endothelial cell monolayers were incubated with sera from patients in active phase of disease or in relapse. Data were compared with effects of the glucocorticoid dexamethasone alone or in combination with MS sera on barrier integrity. Tight junction protein levels and gene expression were evaluated concomitant with barrier integrity. We reveal downregulation of claudin-5 and occludin protein and mRNA and an accompanying upregulation in expression of matrix metalloproteinase MMP-9 after incubation with serum from active disease and remission and also a minor reconstitution of barrier functions related to dexamethasone treatment. Moreover, we for the first time describe downregulation of claudin-5 and occludin protein after incubation of cEND cells with sera from patients in remission phase of MS. Our findings reveal direct and differential effects of MS sera on blood-brain barrier integrity.

Thyroid Hormones Influence Human Dendritic Cells' Phenotype, Function, and Subsets Distribution

BACKGROUND Dendritic cells (DCs) are the most effective antigen-presenting cells and key regulators of immune response. The immunoregulatory properties of DCs strongly depend on the microenvironment in which DCs have been matured and activated. Thyroid hormones are an important part of this environment and regulate many vital processes including growth and cellular metabolism. The aim of the study was an analysis of the influence of thyroid hormones on blood DC subtypes ex vivo, including the surface expression of molecules involved in antigen presentation, costimulation, and maturation, as well as on functional properties of DCs in vitro. METHODS Blood samples for the quantitative and phenotypic analysis of peripheral blood plasmacytoid and myeloid DC subtypes were collected from thyroidectomized patients at two time points: (i) at the time of the so-called stimulation with endogenous thyrotropin-a group of hypothyroid patients after l-thyroxine (L-T(4)) withdrawal (pretreatment group)-and (ii) after 2 months of L-T(4) administration for thyrotropin suppression-a posttreatment group. The phenotype of DCs including HLA-DR, costimulatory molecules (CD40, CD80, and CD86), and maturation marker CD83 was assessed by flow cytometry. The influence of isolated peripheral blood DCs on autologous peripheral blood mononuclear cell proliferation and cytokine secretion (interferon alpha, interleukin-12) under triiodothyronine (T(3)) deficiency or T(3) excess was investigated in culture experiments. RESULTS The percentage of peripheral blood plasmacytoid and myeloid DCs was higher after L-T(4) administration when compared with the pretreatment group. Moreover, the expression of CD86 on both DC subtypes was higher in the L-T(4) treated than in the hypothyroid patients. In the in vitro experiments, T(3) stimulation increased CD86 expression on cultured DCs. The phenotypic difference was paralleled by enhanced ability of T(3)-stimulated DCs to activate interleukin-12 secretion and proliferation of autologous peripheral blood mononuclear cells (PBMLs) in coculture experiments. CONCLUSIONS In the present study, we provide for the first time an evidence that the thyrometabolic status has an influence on the phenotype and function of human peripheral blood DCs. This observation may be of potential importance for the understanding of the pathogenesis of immune and endocrine disorders.

Flow cytometry in the differential diagnostics of Hashimoto’s thyroiditis and MALT lymphoma of the thyroid

INTRODUCTION A combination of traditional cytology methods with fluorescence activated cell sorting (FACS) analysis of fine-needle aspiration biopsy (FNAB) material is considered a powerful diagnostic tool in the differential diagnosis of thyroid lesions suspected of mucosa-associated lymphoid tissue lymphoma (MALT-L). The aim of this study was to demonstrate the FACS-based diagnostic process of thyroid lesions in a clinical situation where ultrasound and cytological examinations did not allow differentiation between Hashimotos thyroiditis (HT) and MALT-L. MATERIAL AND METHODS The patients analysed in this study presented significantly different clinical courses of thyroid disease: quickly enlarging painless tumour of the thyroid right lobe in the first case, and chronic HT with palpable tumour in the thyroid isthmus in the second patient. Due to the suspicion of MALT-L resulting from indeterminate ultrasound and FNAB-cytology results, FNAB material was obtained from all the previously examined thyroid lesions and directly subjected to FACS assessment, encompassing κ/λ light chain restriction analysis, as well as measurements of B and T cell surface antigens. RESULTS The FACS analysis of FNAB material obtained from our patients did not show any definite signs of light chain restriction. Although one of the samples showed a borderline value of κ/λ ratio (κ/λ = 0.31), further immunophenotyping confirmed clonal expansion in none of the examined thyroid regions. Histopathological findings documented the diagnosis of HT in both clinical cases. CONCLUSION We believe that FACS represents a useful and reliable complementary diagnostic measure in FNAB-based differential diagnosis of lymphoproliferative thyroid disorders.

The Effect of Diagnostic Absorbed Doses from 131I on Human Thyrocytes in Vitro

Background: Administration of diagnostic activities of 131I, performed in order to detect thyroid remnants after surgery and/or thyroid cancer recurrence/metastases, may lead to reduction of iodine uptake. This phenomenon is called “thyroid stunning”. We estimated radiation absorbed dose-dependent changes in genetic material, in particular in sodium iodide symporter (NIS) gene promoter, and NIS protein level in human thyrocytes (HT). Materials and Methods: We used unmodified HT isolated from patients subjected to thyroidectomy exposed to 131I in culture. The different 131I activities applied were calculated to result in absorbed doses of 5, 10, and 20 Gy. Results: According to flow cytometry analysis and comet assay, 131I did not influence the HT viability in culture. Temporary increase of 8-oxo-dG concentration in HT directly after 24 h (p < 0.05) and increase in the number of AP-sites 72 h after termination of exposition to 20 Gy dose (p < 0.0001) were observed. The signs of dose-dependent DNA damage were not associated with essential changes in the NIS expression on mRNA and protein levels. Conclusions: Our observation constitutes a first attempt to evaluate the effect of the absorbed dose of 131I on HT. The results have not confirmed the theory that the “thyroid stunning” reduces the NIS protein synthesis.

The Molecular Effect of Diagnostic Absorbed Doses from 131I on Papillary Thyroid Cancer Cells In Vitro

Diagnostic whole-body scan is a standard procedure in patients with thyroid cancer prior to the application of a therapeutic dose of 131I. Unfortunately, administration of the radioisotope in a diagnostic dose may decrease further radioiodine uptake—the phenomenon called “thyroid stunning”. We estimated radiation absorbed dose-dependent changes in genetic material, in particular in the sodium iodide symporter (NIS) gene promoter, and the NIS protein level in a K1 cell line derived from the metastasis of a human papillary thyroid carcinoma exposed to 131I in culture. The different activities applied were calculated to result in absorbed doses of 5, 10 and 20 Gy. Radioiodine did not affect the expression of the NIS gene at the mRNA level, however, we observed significant changes in the NIS protein level in K1 cells. The decrease of the NIS protein level observed in the cells subjected to the lowest absorbed dose was paralleled by a significant increase in 8-oxo-dG concentrations (p < 0.01) and followed by late activation of the DNA repair pathways. Our findings suggest that the impact of 131I radiation on thyroid cells, in the range compared to doses absorbed during diagnostic procedures, is not linear and depends on various factors including the cellular components of thyroid pathology.