Fibroblastoid colony-forming cells in myeloproliferative disorders

The properties of human bone marrow fibroblastoid colonies (CFU-F) were studied in normal subjects and in patients with myeloproliferative disorders. Colony incidence was within normal values in all groups of patients analyzed except for myelodisplastic syndromes, with higher mean value. The growth rate of CFU-F is inversely related to the initial colony-forming efficiency both in normal subjects and in patients. Direct correlation between CFU-F and granulocyte-macrophage colony-forming unit (GM-CFU) was detected only in normal subjects, but lacked in patients. Higher number of CFU-F was observed in subjects with increased incidence of bone marrow megakaryocytes or peripheral blood platelets, irrespective of the underlying disorders and the platelet-derived growth-factorenhanced cloning efficiency of bone marrow fibroblasts.     

Comparison between disk diffusion and microdilution methods for determining susceptibility of clinical fungal isolates to caspofungin

We compared the caspofungin (CAS) susceptibility testing results generated by the disk diffusion (DD) assay with the results of the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BD) reference method for 106 yeast isolates. The isolates represented 11 different fungal species, including Candida albicans (n = 50), C. parapsilosis (n = 10), C. glabrata (n = 10), C. tropicalis (n = 10), C. guillermondii (n = 6), C. rugosa (n = 5), C. krusei (n = 5), C. kefyr (n = 2), C. pelliculosa (n = 2), Saccharomyces cerevisiae (n = 3), and Geotrichum candidum (n = 3). The DD assay was performed in supplemented Mueller-Hinton agar with CAS, which was tested at concentrations of 2, 10, and 25 mug per disk. MICs and inhibition zone diameters were evaluated at 24 and 48 h. In general, the results obtained by the DD assay correlated well with those obtained by the BD method. In particular, a significant correlation between methods was observed when CAS was used at concentration of 2 mug/disk at a reading time of either 24 or 48 h.     

Analysis of clinically relevant single-nucleotide polymorphisms by use of microelectronic array technology

BACKGROUND: Microelectronic DNA chip devices represent an emerging technology for genotyping. We developed methods for detection of single-nucleotide polymorphisms (SNPs) in clinically relevant genes. METHODS: Primer pairs, with one containing a 5'-biotin group, were used to PCR-amplify the region encompassing the SNP to be interrogated. After denaturation, the biotinylated strand was electronically targeted to discrete sites on streptavidin-coated gel pads surfaces by use of a Nanogen Molecular Workstation. Allele-specific dye-labeled oligonucleotide reporters were used for detection of wild-type and variant sequences. Methods were developed for SNPs in genes, including factor VII, beta-globin, and the RET protooncogene. We genotyped 331 samples for five DNA variations in the factor VII gene, >600 samples from patients with beta-thalassemia, and 15 samples for mutations within the RET protooncogene. All samples were previously typed by various methods, including DNA sequence analysis, allele-specific PCR, and/or restriction enzyme digestion of PCR products. RESULTS: Analysis of amplified DNA required 4-6 h. After mismatched DNA was removed, signal-to-noise ratios were >5. More than 940 samples were typed with the microelectronic array platform, and results were totally concordant with results obtained previously by other genotyping methods. CONCLUSIONS: The described protocols detect SNPs of clinical interest with results comparable to those of other genotyping methods.     

Neutrophils from patients with TNFRSF1A mutations display resistance to tumor necrosis factor-induced apoptosis: pathogenetic and clinical implications

OBJECTIVE: To explore tumor necrosis factor (TNF)-induced apoptosis in neutrophils from patients with TNF receptor-associated periodic syndrome (TRAPS) and to correlate the results with the different kinds of TNFRSF1A mutations. METHODS: Two hundred sixty-five patients with clinically suspected inherited autoinflammatory syndrome were screened for mutations of the TNFRSF1A gene. Neutrophils were isolated from heparinized blood by dextran sedimentation and incubated with and without cycloheximide (CHX) and TNFalpha. Cell apoptosis was assessed by human annexin V binding, and caspase 8 activation was assessed by flow cytometry. RESULTS: Twenty-one patients were found to carry a variant of the TNFRSF1A gene: 13 patients had an R92Q substitution, and 8 patients presented other missense substitutions, 1 splicing mutation, and 1 in-frame interstitial deletion. Neutrophil stimulation with TNF and CHX was associated with induction of apoptosis in 12 normal controls and in 10 subjects with the R92Q mutation. Conversely, neutrophils from 8 TRAPS patients with mutations of cysteine or threonine residues or interstitial deletion did not show any induction of apoptosis after stimulation. The incidence of the R92Q mutation among patients with recurrent autoinflammatory syndromes was similar to that observed in the normal population. CONCLUSION: Resistance to TNF-mediated apoptosis is a feature in TRAPS patients who have mutations of cysteine residues or interstitial deletion, and may play a pathogenic role. The R92Q mutation does not appear to be significantly associated with TRAPS.     

Isolation and characterization of a K562 cell line resistant to 1-beta-D-arabinofuranosylcytosine-mediated erythroid induction

The isolation of a K562 cell line, K562(S)R, resistant to 1-beta-D-arabinofuranosylcytosine (ara-C)-mediated erythroid induction, is described. Ara-C (10-50 microM) inhibits cell growth of K562(S)R cells but is not able to activate the program of erythroid induction. This failure is associated with the lack in the increase of accumulation of epsilon-globin and gamma-globin mRNA sequences in ara-C-treated K562(S)R cells. This cell line could be of interest for studies focused on molecular mechanisms of activation of globin genes in K562 cells.