Inability to culture the dominant T-cell clone from the skin of primary cutaneous T-cell lymphoma as proven by TCR gamma-chain gene sequencing.

Molecular analysis of T-cell receptor (TCR) chain rearrangement has recently become an attractive tool for demonstrating the clonal origin of cutaneous T-cell lymphoma (CTCL) and for identifying the malignant clone at the molecular level. Over the past decade a number of attempts have been made to culture malignant CTCL cells using standard procedures and these attempts have resulted in several cell lines from the peripheral blood of Sézary syndrome, mycosis fungoides and CD30+ lymphoma patients. However, so far it has not been proven by sequence analysis that the cultured T cells truly represent the malignant cells. Aiming to functionally analyze the malignant T cells at a clonal level, we generated a total of 150 T-cell clones (TCC) from lesional skin and peripheral blood of three patients with mycosis fungoides and one patient with a CD30+ lymphoma. Cells were grown either in the presence of autologous irradiated peripheral blood feeder cells using various conditions for T-cell stimulation by direct outgrowth or from skin specimens with various cytokine combinations. In order to identify the malignant TCC we used N-region-specific PCR and compared TCR gamma-chain sequences from clones of lesional skin with in vitro-generated TCC. With the methods employed, none of the 150 established cell lines was found to be identical to the malignant TCC which was readily detected in lesional skin. Our results indicate that standard cell culture methods are not suitable for growing low-grade CTCL cells from the skin but give rise only to benign infiltrating T cells.

T-cell clones from early-stage cutaneous T-cell lymphoma show no polarized Th-1 or Th-2 cytokine profile.

Recent studies of the cytokine pattern in skin lesions of patients with cutaneous T-cell lymphoma (CTCL) have shown that interleukin-4 (IL-4) and Il-10, both cytokines produced by T-helper type 2 cells, dominate in these lesions. Also, in single studies, interferon-gamma (IFN-gamma), a major cytokine of Th-1-cells, has been found to be absent. Consequently, it has been hypothesized that immune-suppressive Th-2 cytokines may promote local growth of the malignant lymphocyte clone. However, there is so far no evidence for T-cells as the source of the Th-2 cytokines in CTCL skin lesions nor have these cytokines been investigated at a clonal T-cell level. We established a total of 120 T-cell clones (TCCs) from lesional skin and 54 TCCs from the blood of four patients with mycosis fungoides. Epidermal TCCs (mostly CD8-positive) and dermal TCCs (mostly CD4-positive) were stimulated by the mitogen concanavalin A and, seeking a polarized cytokine pattern, the supernatants were assessed by ELISA. We showed that the vast majority of TCCs were able to secrete IFN-gamma and IL-4. IFN-gamma-deficient TCCs occurred only in the epidermis. Some (18) TCCs were found to be either negative for IL-10 production or to produce low levels only. No significant differences were observed between blood- and skin-derived TCCs. Thus a polarized Th-2 cytokine pattern was not detectable among cultured skin-infiltrating nonmalignant T-cells (TILs) isolated from early mycosis fungoides. It therefore appears unlikely that Th-2-mediated immune suppression is a major mechanism operating in early CTCL. However, this does not exclude its role in late-stage disease.

IFN-gamma is only partially restored by co-stimulation with IL-12, IL-2, IL-15, IL-18 or engagement of CD28.

BACKGROUND: Although it is well established that T cells derived from patients with atopic diseases produce low levels of interferon-gamma (IFN-gamma), the mechanisms responsible for this phenomenon are poorly understood. OBJECTIVES: To elucidate whether IFN-gamma production may be restored by co-stimulatory molecules known to increase IFN-gamma production in vitro. Further, to investigate whether deficient IFN-gamma production is associated with disease activity. METHODS: Purified peripheral T cells obtained from patients with severe atopic dermatitis (AD), individuals with a history but no symptoms of AD and healthy control subjects were activated with anti-CD3 MoAbs in the presence or absence of anti-CD28 MoAbs, interleukin (IL-) 12, IL-2, IL-15 or IL-18. IFN-gamma production was determined at the single cell level by flow cytometry, as well as by ELISA. RESULTS: Activated T cells from patients with severe AD produced less IFN-gamma than T cells from healthy control individuals. IL-12 or engagement of CD28 enhanced IFN-gamma production in both healthy and atopic T cells. However, absolute values of IFN-gamma were still different. IL-2, IL-15 and IL-18 did not restore IFN-gamma production. T cells from individuals with a history of AD produced more IFN-gamma than those from subjects with severe AD, but less than T cells from healthy individuals. Atopic T cells expressed regular levels of CD3, CD28 and Stat4, the main signal transducer and activator of transcription for IL-12. IL-4, IL-10 and TGF-beta production by T cells were not different between healthy and atopic individuals. CONCLUSION: IFN-gamma deficiency in atopic T cells is not due to a lack of responsiveness to CD28, IL-12, IL-2, IL-15 or IL-18. T cell-derived cytokines able to antagonize IFN-gamma do not contribute to decreased IFN-gamma production. The extent of IFN-gamma deficiency seems to be dependent on disease activity.

A microsomal ATP-binding protein involved in efficient protein transport into the mammalian endoplasmic reticulum.

Protein transport into the mammalian endoplasmic reticulum depends on nucleoside triphosphates. Photoaffinity labelling of microsomes with azido-ATP prevents protein transport at the level of association of precursor proteins with the components of the transport machinery, Sec61alpha and TRAM proteins. The same phenotype of inactivation was observed after depleting a microsomal detergent extract of ATP-binding proteins by passage through ATP-agarose and subsequent reconstitution of the pass-through into proteoliposomes. Transport was restored by co-reconstitution of the ATP eluate. This eluate showed eight distinct bands in SDS gels. We identified five lumenal proteins (Grp170, Grp94, BiP/Grp78, calreticulin and protein disulfide isomerase), one membrane protein (ribophorin I) and two ribosomal proteins (L4 and L5). In addition to BiP (Grp78), Grp170 was most efficiently retained on ATP-agarose. Purified BiP did not stimulate transport activity. Sequence analysis revealed a striking similarity of Grp170 and the yeast microsomal protein Lhs1p which was recently shown to be involved in protein transport into yeast microsomes. We suggest that Grp170 mediates efficient insertion of polypeptides into the microsomal membrane at the expense of nucleoside triphosphates.

Basic FGF-like immunoreactivity in the developing and adult rat brainstem.

Although a variety of in vitro and in vivo actions of basic fibroblast growth factor (bFGF) on neuronal cells have been documented, the physiological role of this protein in the nervous system is still contested. Since the distribution of a molecule in the nervous system may provide cues for an understanding of its possible roles, we have begun to study its cellular localization in the central and peripheral nervous system using immunocytochemistry with an anti-bFGF-specific antibody. Here we provide an account on the distribution of bFGF-like immunoreactivity (bFGF-IR) in the brainstem of the developing and adult rat. Basic FGF-IR was found to be widely distributed in motor and sensory nuclei. In all nuclei examined, only subpopulations of neurons were stained. Different staining patterns were found. For example, in the red nucleus weakly or unstained perikarya were surrounded by numerous immunoreactive fibers, often in close contact with the neuronal surface. In the reticular formation and facial nerve, many neuronal cell bodies showed a strong IR that extended into the processes. Glial cells were consistently unstained. During early postnatal development changes of the distribution of bFGF IR were found. From this wide distribution pattern of bFGF-IR, we conclude that bFGF may have more general and, possibly, diverse functions rather than a restricted role for a particular subset of neurons. Variations in the staining pattern of nerve cell bodies in a single nucleus may suggest a function related to neuronal activity.

High molecular weight forms of basic fibroblast growth factor recognized by a new anti-bFGF antibody.

An antibody against basic fibroblasts growth factor (bFGF) was raised using purified bovine pituitary bFGF. Western blot analysis revealed immunoreactive bands at 18, 24, 30-33 and 46 kDa in immunoaffinity purified extracts of pituitary and adrenal gland using this antibody. A similar staining pattern was obtained with ovary extracts with the exception of the missing 18 kDa band. A second anti-bFGF antibody raised against a synthetic peptide comprising the 24 N-terminal amino acids of bFGF reacted with the 18 kDa and the 46 kDa band of immunoaffinity purified ovary and adrenal gland extracts.