Evaluation of the selected barrier properties of retinal pigment epithelial cell line ARPE-19 for an in-vitro blood-brain barrier model.

In-vitro models that maintain complex transport mechanisms and structural properties associated with the blood-brain barrier in vivo would be useful in drug permeability and neurotoxicological studies. To evaluate the suitability of a human retinal pigment epithelial cell line for a blood-brain barrier model, we have compared the barrier properties of the human retinal pigment epithelial cell line ARPE-19, the human colonic adenocarcinoma cell line Caco-2, and primary porcine microvessel endothelial cells. The tight junction proteins occludin and ZO-1 were stained immunocytochemically. The paracellular ionic permeability was evaluated by measuring the trans-epithelial or trans-endothelial electric resistance. To evaluate the active transport mechanisms, the existence and the activity of the efflux transporters, P-glycoprotein and multidrug resistance-associated proteins, were studied. All the cell types in this study stained positively for occludin and ZO-1. However, the trans-endothelial electric resistance of ARPE-19 cells was low compared with that of primary porcine microvessel endothelial cell and Caco-2 cells. In addition, both the P-glycoprotein expression and its activity in ARPE-19 cells were low. In conclusion, the barrier properties of the human ARPE-19 cell line were not satisfactory for a blood-brain barrier model. For future studies, it is important to develop a human brain endothelial cell line with expression of the complex in-vivo properties of the blood-brain barrier.

A novel tumour necrosis factor receptor mutation in a Finnish family with periodic fever syndrome.

OBJECTIVE: To report a novel mutation of the TNF receptor type 1 gene (TNFRSF1A) in a Finnish patient and her mother, both suffering from periodic fever. METHODS: Soluble TNFRSF1A in serum was measured by enzyme-linked immunoabsorbancy, and induced TNFRSF1A shedding from monocyte cell surfaces was determined using fluorescence-activated cell sorter. Mutation detection was performed using PCR amplification and sequencing of the ten exons of TNFRSF1A. RESULTS: Low levels of soluble TNFRSF1A were detected in both patients between attacks. Sequencing revealed a missense mutation in exon 3 in the second extracellular domain of TNFRSF1A, resulting in a substitution of cysteine with arginine at residue 73 (C73R), confirming the diagnosis of TNF receptor-associated periodic syndrome (TRAPS). We were unable to demonstrate a distinct TNFRSF1A shedding defect. CONCLUSION: In patients of Nordic descent, affected by dominantly inherited recurrent fever, TRAPS is a diagnosis worthy of attention. All TNFRSF1A mutations hitherto described in the Nordic countries have been different.

Chromosomally clonal T cells in the skin, blood, or lymph nodes of two Sezary syndrome patients express CD45RA, CD45RO, CDw150, and interleukin-4, but no interleukin-2 or interferon-gamma.

Cutaneous T cell lymphomas are considered to represent a clonal malignancy of mature T lymphocytes of the T helper memory subtype. A method enabling the direct identification of clonal malignant cells in tissue and, at the same time, identification of the surface molecules they express has not been available, however. We have developed an application of the FICTION technique (simultaneous fluorescence immunophenotyping and interphase cytogenetics) to be used on fresh blood, skin, and lymph node samples. A prerequisite for this method is the characterization of a moleculocytogenetic clone in order to select the proper probes. With this method, we demonstrate that the true malignant cells express CD3, CD4, and CD45RO in the blood, skin, and lymph nodes of two Sezary syndrome patients. The majority of these cells express also CD45RA (albeit of varying intensity) and CDw150. The cytokine expression pattern of the clonal cells in skin and lymph nodes was interleukin-2 and interferon-gamma negative and interleukin-4 positive. Interleukin-10 expression varied. The malignant cells did not express granzyme B, thus indicating that they do not have cytotoxic properties. Clonal cells with the same constant phenotype could be found even in lymph nodes with not yet morphologically identifiable malignant cells. This is the first report of the FICTION method applied directly on skin tissue. With this method we demonstrated that the chromosomally clonal cells in these two cases of Sezary syndrome could be intermediate forms between naïve CD45RA+ and CD45RO+ Th2 cells.

Proapoptotic and antiapoptotic markers in cutaneous T-cell lymphoma skin infiltrates and lymphomatoid papulosis.

BACKGROUND: In cutaneous T-cell lymphoma (CTCL) lesions, both reactive T cells and malignant T cells intermingle. The disease progression is mostly slow. Recent evidence suggests that even if clinical remission is reached, malignant cells persist and a relapse follows sooner or later. To wha extent tumour cell apoptosis occurs in the skin lesions either due to the reactive T cells or t therapeutic efforts is not known. OBJECTIVES: To determine the extent of tumour cell apoptosis and the expression of proapoptotic an antiapoptotic markers in serial skin lesion samples from patients with CTCL, and to compare th findings with those in patients with lymphomatoid papulosis (LyP). METHODS: Thirty-four skin samples were obtained from 12 patients with CTCL at the time o diagnosis and at a mean of 1.6, 3 and 6 years later. The patients received psoralen plus ultraviolet (PUVA), electron beam or cytostatic treatments. In addition, fresh post-treatment samples fro three patients with CTCL undergoing PUVA therapy were obtained. For comparison, skin biopsies o five patients with LyP were studied. Immunohistochemical demonstration of the expression of th following markers was performed on formalin-fixed skin sections: Fas (CD95), Fas ligand (FasL) bcl-2, granzyme B, the tumour-suppressor protein PTEN and the effector caspase, caspase-3. Th malignant cells were identified morphologically, and apoptotic cells were identified with th terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling method on parallel sections. RESULTS: In untreated CTCL lesions, apoptotic lymphocytes were extremely rare, and no increase in the number of apoptotic cells was observed after any of the treatments used. In LyP, apoptotic cell were more frequent, comprising on average 5% of the infiltrate. The apoptosis-associated marker Fas, FasL, caspase-3 and granzyme B were expressed by morphologically neoplastic cells in CTCL and by large atypical cells in LyP, with no significant differences. However, only a few reactive cell in CTCL infiltrates expressed granzyme B while about 10% of the corresponding cells were positive in LyP. The expression of antiapoptotic bcl-2 was more frequent in CTCL than in LyP, while PTE expression was high in both instances. The number of bcl-2 + cells tended to decrease after therapy When comparing the findings between the first and the last samples, a decrease in the number of bcl-2+ cells and an increase in Fas+ cells was associated with disease progression, despite therapy, while the opposite was true for remissions. CONCLUSIONS: Apoptosis was found to be a rare event in CTCL lesions irrespective of precedin therapy During patient follow-up, no significant differences in the expression of apoptotic marker was observed while in most cases a lower level of antiapoptotic bcl-2 expression was observed after all types of therapies and in association with disease progression when compared with high expression in the untreated lesions. The absence of apoptosis and high expression of bcl-2 together with a low expression of apoptosis-inducing granzyme B in the reactive lymphocytes in CTC could explain the chronic nature of the disease and the poor response to therapy, while th more frequent occurrence of granzyme B and apoptosis together with a lower level of expressio of bcl-2 by the large atypical cells in LyP could contribute to the favourable outcome of the latter.