Renata Barresi

A negative cis-acting G-fer element participates in the regulation of expression of the human H-ferritin-encoding gene (FERH)

Ferritin (Fer) is the major iron storage protein in man. Its synthesis is regulated both at the translational and transcriptional levels. In previous studies on transcriptional regulation of the human H-ferritin-encoding gene (FERH), a 160-bp promoter segment was analyzed [Bevilacqua et al., Gene 111 (1992) 255-260]. In order to obtain a more complete view of the elements involved in the transcriptional regulation of FERH, we have studied, in a further upstream region of the human FERH promoter (pFERH), a sequence between -272 and -291, named G-fer, because it contains a stretch of ten G, which binds a nuclear factor present in different cell types. DNA-binding assays and competition experiments suggest that the factor binding to G-fer has binding properties very similar to inhibitory factor-1 (IF-1), an ubiquitous factor that interacts with G-rich elements in the promoters of the mouse type-I collagen genes. DNA transfection experiments in HeLa cells, using either a wild-type or mutated pFERH fused to a reporter gene, showed that a 3-bp substitution mutation, that abolished the binding of the specific factor to G-fer, increased the promoter activity, thus suggesting an inhibitory role for the G-fer element and its cognate trans-acting factor.

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.

First external quality assurance program of the Italian HLA-B*57:01 Network assessing the performance of clinical virology laboratories in HLA-B*57:01 testing

BACKGROUND Since the HLA-B*57:01 allele is strongly associated with abacavir hypersensitivity reaction, testing for the presence of HLA-B*57:01 is mandatory before administration of abacavir. While HLA-B*57:01 testing is usually provided by pharmacogenetics, genetics or blood transfusion services, clinical virology laboratories can be an optimal opportunity for HLA-B*57:01 testing since they receive blood samples for routine HIV monitoring and have the expertise for convenient and less expensive PCR-based point mutation assays. OBJECTIVES The Italian HLA-B*57:01 Network gathers accredited clinical virology laboratories offering HLA-B*57:01 testing in Italy with the aim to share protocols, test new methods, develop and maintain external quality assurance (EQA) programs. STUDY DESIGN A panel of 9HLA-B*57:01-positive and 16HLA-B*57:01-negative frozen blood samples were blindly distributed to 10 units including 9 clinical virology laboratories and one reference pharmacology laboratory. Each laboratory was free to use its own routine method for DNA extraction and HLA-B*57:01 testing. RESULTS DNA was extracted by automated workstations in 6 units and by manual spin columns in 4. Eight units used the Duplicα Real Time HLA-B*57:01 kit by Euroclone and two units used two different PCR homemade protocols. All the 10 units correctly identified all the 25 samples. CONCLUSIONS The first HLA-B*57:01 EQA program run in Italy showed that clinical virology units are equipped and proficient for providing HLA-B*57:01 testing by inexpensive assays easy to integrate into their routine.

Genotypic determination of HIV tropism in a cohort of patients perinatally infected with HIV-1 and exposed to antiretroviral therapy.

Abstract The aim of this study was to determine the coreceptor tropism by performing genotypic HIV-1 tropism testing in a cohort of patients perinatally infected with HIV-1 and exposed to antiretroviral therapy. Genotypic coreceptor tropism was determined in patients with HIV-1 RNA<100 copies/mL using PBMC samples by gp120 V3 sequencing followed by geno2pheno interpretation (set at a false positive rate [FPR] of 20%) and in patients with >100 copies/mL using plasma samples (set at a FPR of 20%), according to European guidelines. Out of 55 patients, 50 had an HIV-1 subtype B strain, and mean (SD) age was 18.2 (4.6) years. The median duration of antiretroviral therapy was 13 years (range, 3–23). Thirty-three (60%) patients harbored the R5 virus. At the time of the testing, the median CD4+ T lymphocyte cell count and percentage were 705 cells/mm3 (474–905) and 32.5% in group R5 and 626 cells/mm3 (450–755) and 31.7% in group X4/D-M, respectively. The nadir of CD4+ T-cell count in groups R5 and X4/D-M were 322 cells/mm3 (230–427) and 340 cells/mm3 (242–356), respectively. These differences were not statistically significant. Fifteen patients had HIV-1 RNA >50 copies/mL. The median HIV-1 RNA and HIV-1 DNA were comparable in both groups without a statistical difference. The study provides an overview of the prevalence of coreceptor tropism in a cohort of patients who were vertically infected with HIV-1. The high prevalence of X4/D-M-tropic strains may simply reflect the long-term exposure to HIV.

Characterization, localization, and biosynthesis of acetylcholinesterase in K 562 cells☆

K 562 cell acetylcholinesterase (AChE), identifiable by active site labeling with radioactive diisopropylfluorophosphate (DFP), showed a Mr around 55,000 in both a crude lysate and a purified sample. The K 562 AChE was reactive with one polyclonal and two monoclonal antibodies produced against human erythrocyte AChE. Subcellular localization, investigated by assay on cell fractions, showed that AChE is membrane bound and that it is located on the cell surface as well as on microsomal and Golgi membranes. Biosynthesis of new enzyme molecules, after inactivation of the constitutive AChE with the irreversible inhibitor DFP, allowed us to follow the kinetics of reappearance in the intracellular compartment and at the cell surface (4 and 8 h, respectively).

Urinary Granulopoietic Activity in Chronic Myelogenous Leukemia: Follow-Up and Correlation with Various Phases of the Disease

Daily urinary colony-stimulating factor (CSF) output was tested in 16 patients with chronic myeloid leukemia, 12 of which were in stable phase and 4 in blast crisis of the disease. The CSF of stable-phase patients varied within normal range, while all patients in blast crisis had values lower than normal. Also the number of CFUC ranged within normal values in stable-phase patients, while no colony growth was detectable in blast-crisis patients. Sequential studies (28 or 11 months) of 2 patients revealed an inverse relation between daily urinary CSF and presence of peripheral immature or blast cells in the patient in stable or blast phase, respectively.

Feasibility and Reproducibility of HIV-1 Genotype Resistance Test in Very-Low-Level Viremia

Bianca Bruzzone, Antonio Di Biagio, Laura Sticchi, Renata Barresi, Francesco Saladini, Giancarlo Icardi, Maurizio Setti Hygiene Unit, IRCCS AOU San Martino-IST, Genoa, Italy; Infectious Diseases Unit, IRCCS AOU San Martino-IST, Genoa, Italy; Department of Health Sciences, University of Genoa, Genoa, Italy; Department of Medical Biotechnologies, University of Siena, Siena, Italy; Internal Medicine and Clinical Immunology Unit, IRCCS AOU San MartinoIST, Genoa, Italy

Electrophoretic pattern of NADPH-dependent oxidoreductive activities in K 562 and HL 60 leukemic cell lines.

Morphological and functional differentiation of hemopoietic cells is accompanied by the expression of lineage-specific protein markers. NADPH-oxidoreductive enzymatic activities in HL 60 and K 562 leukemic cell lines, compared with granulocytes and erythrocytes, show a NADPH-oxidizing and a NADPH-diaphorase activity. The oxidizing activity, absent in erythrocytes, has the same electrophoretic migration in HL 60 cells and granulocytes while it is different in K 562 cells. The diaphorase, absent in HL 60 cells and granulocytes, has the same migration in erythrocytes and K 562 cells, although with a slightly different quantitative expression. K 562 cells induced to differentiation with arabinofuranosylcytosine show the appearance of a band of NADPH-oxidizing activity of granulocytic type, together with the major band found in these cells.

Urinary Colony-Stimulating Factor in Acute Leukemia Follow-Up and Correlation with Various Phases of the Disease

Urinary colony-stimulating factor (CSF) was assayed in 19 patients with various leukemias and was monitored in various phases of acute leukemia in 3 patients. Significantly higher CSF levels were found at the onset of leukemia with a monoblastic component. Continuous monitoring of CSF in a patient with acute myelomonocytic leukemia revealed a decrease in CSF level during the remission phase, followed by a rebound to high levels preceding the clinical and hematological relapse. Concomitantly, a colony-inhibitory factor (CIF) was detected. Both CSF and CIF of this patient were isolated and partially characterized.