Prospective study of high-dose chemotherapy and autologous peripheral stem cell transplantation in adult patients with advanced desmoplastic small round-cell tumour.

A total of 10 desmoplastic small round-cell tumour patients were treated by high-dose chemotherapy with stem cell support. After high-dose chemotherapy, no complete response conversion was obtained and EWS-WT1 fusion transcript detection was positive in the peripheral blood during follow-up in all patients. High-dose chemotherapy did not seem to change the results in desmoplastic small round-cell tumour.

Deregulated FGFR3 mutants in multiple myeloma cell lines with t(4;14): comparative analysis of Y373C, K650E and the novel G384D mutations.

The t(4;14)(p16.3;q32) chromosomal translocation occurs in approximately 20% of multiple myelomas (MM) and leads to the apparent deregulation of two genes located on 4p16.3: the fibroblast growth factor receptor 3 (FGFR3) and the putative transcription factor WHSC1/MMSET. Interestingly, FGFR3 mutations known to be associated with autosomal dominant human skeletal disorders have also been found in some MM cell lines with t(4;14) but their pathogenetic role in MM is still controversial. Since cell lines may represent useful models for investigating the effects of deregulated FGFR3 mutants in MM, we analysed the expression, activation, signaling pathways and oncogenic potential of three mutants identified so far: the Y373C and K650E in the KMS-11 and OPM-2 cell lines respectively, and the novel G384D mutation here identified in the KMS-18 cell line. All of the cell lines present a heterozygous FGFR3 gene mutation and transcribe the mutated allele; unlike KMS-11 and OPM-2 (which express the IIIc isoform), the KMS-18 cell line expresses prevalently the isoform IIIb. We demonstrated that, under serum-starved conditions, KMS-11 and OPM-2 cells express appreciable levels of phosphorylated FGFR3 mutants indicating a constitutive activation of the Y373C and K650E receptors; the addition of the aFGF ligand further increased the level of receptor phosphorylation. Conversely, the FGFR3 mutant in KMS-18 does not seem to be constitutively activated since it was phosphorylated only in the presence of the ligand. In all three MM cell lines, ligand-stimulated FGFR3 mutants activated the MAP kinase signaling pathway but did not apparently involve either the STAT1 or STAT3 cascades. However, when transfected in 293T cells, G384D, like Y373C and K650E, was capable of activating MAPK, STAT1 and STAT3 under serum-starved condition. Finally, a focus formation assay of NIH3T3 cells transfected with FGFR3-expressing plasmid vectors showed that Y373C and K650E (albeit at different levels) but not G384D or the wild-type receptor, can induce transformed foci. Overall, our results support the idea that FGFR3 mutations are graded in terms of their activation capability, thus suggesting that they may play a critical role in the tumor progression of MM patients with t(4;14).

Detection of t(4;14)(p16.3;q32) chromosomal translocation in multiple myeloma by reverse transcription-polymerase chain reaction analysis of IGH-MMSET fusion transcripts.

We and others have recently identified a novel recurring t(4;14)(p16.3; q32) translocation in multiple myeloma (MM) that leads to an apparent deregulation of the FGFR3 and WHSC1/MMSET genes. Because the presence of IGH-MMSET hybrid transcripts has been found in MM cell lines with t(4;14), they may represent a specific tumor-associated marker in MM. In this study, we developed a reverse transcription-PCR (RTPCR) assay for detecting chimeric transcripts from all of the 4p16.3 breakpoints identified thus far, and we used it to investigate a representative panel of 53 MM patients and 16 patients with monoclonal gammopathy of uncertain significance; in addition, t(4;14) was investigated in all of the MM patients by means of two-color fluorescence in situ hybridization. IGH-MMSET transcripts were found in 11 of the 53 (20%) MM cases and 1 of 16 (6%) cases of monoclonal gammopathy of uncertain significance. There was complete concordance between the RT-PCR and fluorescence in situ hybridization analyses of the MM cases. The results of this study indicate that RT-PCR is a sensitive and reliable method of detecting t(4;14) and suggest that it may be useful for monitoring the disease in a significant proportion of patients.

DNA typing of maternal HLA in congenital complete heart block: comparison with systemic lupus erythematosus and primary Sjögren's syndrome.

OBJECTIVE: To investigate which maternal HLA allele or haplotype is primarily associated with isolated congenital complete heart block (CCHB) in offspring. METHODS: HLA class II typings were assessed by line probe assay and polymerase chain reaction-sequence-specific oligonucleotide probe methods, and HLA class I by the microlymphocytotoxicity test, in 13 Italian anti-Ro-positive mothers of children with CCHB and 41 anti-Ro-positive mothers with healthy children (20 mothers with systemic lupus erythematosus [SLE] and 21 with Sjögren's syndrome [SS]). Anti-Ro antibodies were studied by immunoblot. RESULTS: HLA-DRB1*03011 and DRB1*03011; DQA1*0501;DQB1*0201 were more frequent in mothers of infants with CCHB than in mothers who had SLE, but not in mothers who had SS and whose children were healthy. Mothers of infants with CCHB were either HLA-B5/35, B17, or B44 positive and had a higher prevalence of B44;DRB11;DQA1*0501;DQB1*0301 and isolated anti-52-kd antibodies, which were absent in SS and SLE controls. CONCLUSION: Mothers of infants with CCHB presented a strong genetic similarity to mothers who had SS, except for HLA class I phenotype. HLA-DRB1*03011;DQA1*0501;DQB1*0201 seemed not to be primary CCHB-associated genes, but were involved in an SS-like anti-Ro/La response. The combined presence of HLA-DRB1*03011 and anti-52-kd SSA/Ro antibodies conveyed the highest risk of giving birth to an affected child.

Increased interferon-gamma (IFN-gamma) levels produced in vitro by alloactivated T lymphocytes in systemic sclerosis and Raynaud's phenomenon.

The aim of the present study was to analyse the in vitro proliferation and cytokine production by alloantigen-stimulated peripheral blood mononuclear cells (PBMC) obtained from patients affected by systemic sclerosis (SSc) and patients with Raynaud's phenomenon (RP). In SSc patients the proliferation of PBMC stimulated in vitro with alloantigens was significantly increased compared with healthy subjects, while no differences were observed for RP patients. Lymphocytes from SSc patients also produced larger amounts of IFN-gamma compared with healthy controls. However, patients with clinically active disease had lower IFN-gamma levels than those found in clinically stable patients. Patients affected by RP showed significantly higher levels of IFN-gamma than healthy subjects. Analysis at the clonal level of the lymphocyte subsets involved in alloantigen stimulation in one patient affected by active SSc, and one subject with RP confirmed the results obtained using PBMC. In particular, in the RP patient but not in the SSc patient, we observed a population of CD4+ T cells which proliferated to alloantigens in vitro and produced high levels of IFN-gamma. We suggest that T lymphocytes producing high levels of IFN-gamma might play a protective role in RP patients and in established scleroderma.

Cytokine production in scleroderma patients: effects of therapy with either iloprost or nifedipine.

OBJECTIVE: To compare the long-term effects of intermittent infusion of iloprost with those of oral nifedipine on the in vitro production of cytokines in patients with systemic sclerosis (SSc), and to evaluate their relationship with the effects of the two treatments on clinical parameters. METHODS: The production of cytokines by alloactivated circulating mononucleated cells was assessed before and after one year of treatment in a subset of 31 patients enrolled in a 12-month randomized clinical trial. Nineteen patients were treated with a 5-day (8 hr per day), 2.0 ng/kg per minute infusion followed by a 1-day infusion every 6 weeks; 12 patients were treated with an oral slow-release formulation of nifedipine, 20 mg twice daily. Quantitative determinations of interleukin-1 beta (IL1-beta) and interleukin-6 (IL6) in the culture supernatants were performed with a commercial ELISA; the levels of tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) were measured by specific radioimmunometric assays. RESULTS: The production of IL1-beta was significantly lower in the iloprost group than in the nifedipine group. Both the cutaneous fibrosis and the capillaroscopic patterns were better in patients treated with iloprost than in patients treated with nifedipine. There was a significant positive covariance between IL1-beta changes and the changes in both the skin score and the capillaroscopic score. CONCLUSION: There are several mechanisms by which iloprost could exert its clinical efficacy. Vasodilatation and inhibition of platelet aggregation are certainly important, but they are transient. We suggest that the long-lasting modulation of the cytokine network observed in the present study could be another potential mechanism responsible for the persistent efficacy of iloprost despite its intermittent administration.

Differential effects of cyclo-oxygenase pathway metabolites on cytokine production by T lymphocytes.

Cyclo-oxygenase pathway metabolites released in the microenvironment by activated platelets and endothelial cells are potential local modulators of the immune response. In the present study, we have investigated the modulatory role of PGE2, iloprost (prostacyclin analogue), U-46619 (thromboxane analogue) on the release of IL-2, IFN-gamma, TNF-alpha and IL-6 by T lymphocytes. Our results show that PGE2 and prostacyclin differ in the regulation of cytokine production. PGE2 inhibited the release of IL-2 and IFN-gamma, while iloprost did not affect their production. The addition of PGE2 or iloprost greatly decreased the amount of TNF-alpha measured in the supernatants, although the rates of inhibition differed according to the kind of stimulation. Unlike that of PGE2, inhibition by iloprost was stronger in alloactivated cultures than in PHA-stimulated ones. In vitro IL-6 production was stimulated by PGE2 in alloactivated cultures and by iloprost, whatever the stimulus. These results are probably due to other cellular subsets contaminating the T-lymphocyte preparations. After complete removal of monocytes from cell cultures, there were inhibitory effects of lloprost and PGE2 on IL-6 released in the supernatants. We did not observe any significant effect of thromboxane analogue on the production of either cytokine.

Functional analysis of a new polymorphism in the human TNF alpha gene promoter.

In this paper the functional relevance of a TNFA promoter polymorphism, a G/A polymorphic sequence at position -238, was tested by analysing its influence on TNF alpha production upon in vitro stimulation of monocytes from 78 healthy, unrelated individuals by lipopolysaccharide (LPS) or after allogenic stimulation in a panel of 32 healthy individuals. All TNFA-A positive individuals were either DR3 or DR7 positive, confirming the previously reported strong linkage disequilibrium of the TNFA-A allele with the two extended haplotypes (B18, F1C30, DR3) and (B57, SC61, DR7). No individuals homozygous for the TNFA-A allele were present in the panel. The mean level of TNF alpha production was not significantly different in TNFA-G/G homozygous and in TNFA-A/G heterozygous individuals after LPS stimulation of monocytes (P = 0.35) or after allogenic stimulation (P = 0.7). After LPS and allogenic stimulation DR3 positive individuals had a higher mean TNF production. This could not be further differentiated by typing for TNF -283.