Normal inflammasome activation and low production of IL-23 by monocyte-derived macrophages from subjects with a history of reactive arthritis.

OBJECTIVES: The pathogenesis of reactive arthritis (ReA) is incompletely understood but may involve aberration(s) in the host's innate immune response towards infecting microbes. We therefore studied the production of interleukin (IL)-1ß, a marker of inflammasome activation, and of IL-6, IL-12, IL-23, and tumour necrosis factor (TNF)-α, promoters of T-cell differentiation, by peripheral blood mononuclear cells (PBMNs) and monocyte-derived macrophages from healthy subjects with a history of ReA. METHOD: The study included 10 human leucocyte antigen (HLA)-B27-positive healthy subjects with previous ReA triggered by Yersinia enterocolitica O:3 infection and 20 healthy reference subjects, of whom 10 were HLA-B27 positive. PBMNs and macrophages were cultured for 18 h with bacterial lipopolysaccharide (LPS), muramyl dipeptide (MDP), Yersinia, or their appropriate combinations. PBMNs were also stimulated with monosodium urate (MSU) crystals. Cytokine levels were measured using an enzyme-linked immunosorbent assay (ELISA) and the Luminex system. RESULTS: IL-1ß secretion was similar from cells of the ReA group and from the HLA-B27-positive and -negative reference groups. TNF-α production from macrophages upon co-stimulation of LPS and MDP increased in the order ReA group < HLA-B27-positive reference group < HLA-B27-negative reference group (p for a trend = 0.027). Similarly, Yersinia-induced TNF-α and IL-23 production increased in the same order (p for trend for TNF-α = 0.036; p for trend for IL-23 = 0.026). CONCLUSIONS: PBMNs and macrophages from healthy subjects with previous ReA show normal inflammasome activation and low TNF-α and IL-23 production. This low cytokine production may impair bacterial elimination and thereby contribute to the triggering of ReA.

Potential role of a navigator gene NAV3 in colorectal cancer.

BACKGROUND: The recently described navigator proteins have a multifaceted role in cytoskeletal dynamics. We report here on the relevance of one of them, navigator 3 (NAV3), in colorectal cancer (CRC). METHODS: We analysed changes in chromosome 12 and NAV3 copy number in CRC/adenoma samples of 59 patients and in 6 CRC cell lines, using fluorescence in situ hybridisation, loss of heterozygosity, and array-CGH. NAV3 target genes were identified by siRNA depletion, expression arrays, and immunohistochemistry. RESULTS: NAV3 deletion and chromosome 12 polysomy were detected in 30 and 70% of microsatellite stability (MSS) carcinomas, in 23 and 30% of adenomas and in four of six CRC cell lines. NAV3 amplification was found in 25% of MSS samples. NAV3 alterations correlated with lymph node metastasis. In normal colon cells, NAV3 silencing induced upregulation of interleukin 23 receptor (IL23R) and gonadotropin releasing hormone receptor. In MSS and microsatellite instability tumours, IL23R immunoreactivity correlated with Dukes' staging and lymph node metastases, whereas nuclear beta-catenin correlated with lymph node metastases only. CONCLUSION: NAV3 copy number changes are frequent in CRC and in adenomas, and upregulation of IL23R, following NAV3 silencing, strongly correlates with Dukes' staging and lymph node metastases. This suggests that NAV3 has a role in linking tissue inflammation to cancer development in the colon.

Molecular cytogenetics in the study of cutaneous T-cell lymphomas (CTCL).

The etiology of primary cutaneous T-cell lymphomas (CTCL) has been poorly understood. CTCL patients show a large variety of non-clonal and clonal chromosome aberrations, but no specific aberration has been found until recently. This review describes cytogenetic and molecular cytogenetic findings and their relevance to diagnostics and etiology in two of the most common forms of CTCL, mycosis fungoides (MF) and the leukemic CTCL, Sézary syndrome (SS).

The genetic background of tumour necrosis factor receptor-associated periodic syndrome and other systemic autoinflammatory disorders.

Systemic autoinflammatory disorders are hereditary diseases with symptoms of acute inflammation and a rise in serum acute phase proteins as a consequence, but with no signs of autoimmunity. By the end of the 1990s, four types of hereditary periodic fever had been described in the medical literature: familial Mediterranean fever, hyperimmunoglobulinemia D with periodic fever syndrome (HIDS), tumour necrosis factor receptor-associated periodic fever syndrome (TRAPS) and Muckle-Wells syndrome. Since then, the number of diseases classified as systemic autoinflammatory disorders has increased to eight. In patients of Nordic descent, cases of HIDS and TRAPS have been reported. We provide an overview of the genetic background and main clinical aspects of the different autoinflammatory disorders, with an emphasis on TRAPS.

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.

Allelic variants in genes associated with hereditary periodic fever syndromes as susceptibility factors for reactive systemic AA amyloidosis.

We investigated the hypothesis that low-penetrance mutations in genes (TNFRSF1A, MEFV and NALP3/CIAS1) associated with hereditary periodic fever syndromes (HPFs) might be risk factors for AA amyloidosis among patients with chronic inflammatory disorders, including rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), Crohn's disease, undiagnosed recurrent fevers and HPFs themselves. Four of 67 patients with RA plus amyloidosis had MEFV variants compared with none of 34 RA patients without amyloid (P value=0.03). The E148Q variant of MEFV was present in two of the three patients with TNF receptor-associated periodic syndrome (TRAPS) complicated by amyloid in two separate multiplex TRAPS families containing 5 and 16 affected members respectively, and the single patient with Muckle-Wells syndrome who had amyloidosis was homozygous for this variant. The R92Q variant of TNFRSF1A was present in two of 61 JIA patients with amyloidosis, and none of 31 nonamyloidotic JIA patients. No HPF gene mutations were found in 130 healthy control subjects. Although allelic variants in HPFs genes are not major susceptibility factors for AA amyloidosis in chronic inflammatory disease, low-penetrance variants of MEFV and TNFRSF1A may have clinically significant proinflammatory effects.

Chromosomal abnormalities in relation to clinical disease in patients with cutaneous T-cell lymphoma: a 5-year follow-up study.

BACKGROUND: Patients with cutaneous T-cell lymphoma (CTCL) show chromosomal aberrations in skin and blood lymphocytes. OBJECTIVES: To evaluate the significance of peripheral blood clonal or non-clonal chromosomal abnormalities in comparison with the clinical course of cutaneous T-cell lymphoma patients. PATIENTS/METHODS: Five patients with large-plaque parapsoriasis (LPP) or with follicular mucinosis, eight with mycosis fungoides and two with Sézary syndrome were followed for an average of 54 months. G-banding and enzyme-detected in situ hybridization (EDISH) were used to identify aberrations in chromosomes 1, 6, 8, 9, 11, 13/21, 15 or 17, that had previously showed frequent aberrations. RESULTS: The aberration rates of all chromosomes studied differed between patients with active disease and healthy or photochemotherapy-treated controls by EDISH or G-banding (P < 0.01 to P < 0.05). Patients in complete remission differed from healthy controls for aberrations of chromosomes 1, 6 and 11, and from patients with active, progressing disease for chromosomes 1, 6, 8, 11 and 17 (P < 0.01 to P < 0.05, EDISH or G-banding). All 11 samples representing active, progressing disease showed elevated levels of chromosome 8 aberrations in EDISH. The change in chromosomal aberration rate and clinical condition between two consecutive samples agreed for chromosomes 1, 8, 9 and 15 (G-banding) and for chromosome 17 (G-banding and EDISH; kappa > 0.5-0.6). Six of seven patients (five CTCL, one LPP patient) with clonal chromosomal aberrations by G-banding showed continuously active disease and four of them, but none of the other patients, died within 30 months of the detection of the clone. CONCLUSIONS: The rate of chromosomal aberrations associates with the activity of CTCL, and has prognostic significance. Aberrations of chromosomes 1, 6 and 11, although increasing with activity of the disease, seem to be a hallmark of existing disease, detectable even in remission. Aberrations of chromosomes 8 and 17 especially associate with active or progressive disease.

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.

Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define a family of dominantly inherited autoinflammatory syndromes.

Autosomal dominant periodic fever syndromes are characterized by unexplained episodes of fever and severe localized inflammation. In seven affected families, we found six different missense mutations of the 55 kDa tumor necrosis factor receptor (TNFR1), five of which disrupt conserved extracellular disulfide bonds. Soluble plasma TNFR1 levels in patients were approximately half normal. Leukocytes bearing a C52F mutation showed increased membrane TNFR1 and reduced receptor cleavage following stimulation. We propose that the autoinflammatory phenotype results from impaired downregulation of membrane TNFR1 and diminished shedding of potentially antagonistic soluble receptor. TNFR1-associated periodic syndromes (TRAPS) establish an important class of mutations in TNF receptors. Detailed analysis of one such mutation suggests impaired cytokine receptor clearance as a novel mechanism of disease.

Chromosomal abnormalities in cutaneous T-cell lymphoma and in its premalignant conditions as detected by G-banding and interphase cytogenetic methods.

The etiology of cutaneous T-cell lymphomas (CTCL) is unknown. We studied the pattern of chromosomal abnormalities with G-banding and interphase in situ hybridization methods in blood mononuclear cells in 17 patients representing the different phases of CTCL or the premalignant condition, parapsoriasis en plaque, and in 10 control persons. We used biotinylated centromere-specific probes with fluorescent detection (FISH) for chromosomes 1, 11, 8, and 17 and similar, enzymatically detectable, digoxigenin-labeled probes for chromosomes 1, 6, 12, 17, and 18. In G-banding, all patients showed numerical and structural chromosome aberrations. Numerical aberrations of chromosomes 6, 13, 15, and 17, marker chromosomes, and structural aberrations of chromosomes 3, 9, and 13 were increased in mycosis fungoides (MF) compared with healthy controls. In four of five patients the detection of a chromosomal clone preceded relapse or progression of the disease. In FISH of interphase cells, the cells abnormal for chromosomes 8 or 11, and for all four chromosomes collectively, were increased in MF and in Sezary Syndrome (SS). FISH and G-banding methods agreed statistically significantly for the detection of monosomy. Also, digoxigenin-labeled probes hybridized to interphases or mitoses detected aberrations corresponding to those observed with G-banding. Thus, chromosomally abnormal cells can be found in the peripheral blood of both parapsoriasis en plaque and CTCL patients. They can be detected with interphase cytogenetical methods, which obviates the need for dividing cells, often difficult to accomplish in CTCL.

Autosomal dominant 'Mediterranean fever' in a Finnish family.

A 23-year-old Finnish man was examined because of an 8-year history of recurrent bouts of fever and abdominal pain. His father had been repeatedly investigated because of similar episodes since he was 24 years old, and one of the father's sisters was reported to have had recurrent periods of fever. The clinical features closely resembled those of familial Mediterranean fever (FMF), a syndrome rarely described in families of European descent. Unlike typical FMF, which is inherited as an autosomal recessive trait, the mode of inheritance of the syndrome in our family may be regarded as dominant. During a recent attack, serum concentrations of interleukin-1-beta, interleukin-6 and acute phase reactants, including serum amyloid A protein, were high. No signs of amyloidosis were detected in our patients.