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V Najfeld

Publications and source records attributed to V Najfeld.

At least 19 recordsLinked to original sources
PubMed2004

Novel integrase-defective lentiviral episomal vectors for gene transfer.

High levels of circular viral extrachromosomal DNA (E-DNA) are normally produced after infection with integration-competent and -incompetent lentiviruses. Although E-DNA has been shown to be transcriptionally active, it lasts for only a short time in replicating cells. Here, we report an integrase (IN)-defective lentiviral episomal vector in which insertion of the simian virus 40 (SV40) promoter, containing the origin of replication (ori), is associated with long-term expression and persistence of E-DNA in the presence of SV40 large T antigen (TAg) from 293T cells. 293 and 293T cell lines transduced with IN-competent lentiviral vectors expressing green fluorescent protein (GFP) or luciferase from the cytomegalovirus (CMV) or SV40 promoter gave similar levels of transduction and expression. In contrast, only transient reporter expression occurred when using the CMV IN-defective control vector in both 293 and 293T cells. However, reporter gene expression was maintained for more than 8 weeks in 293T, but not 293, cells transduced with the IN-defective lentiviral vector containing the SV40-ori promoter. Polymerase chain reaction for two-long terminal repeat (2LTR) extrachromosomal circular forms, a marker of lentiviral E-DNA, and fluorescence in situ hybridization analysis confirmed the persistence and episomal nature of circular E-DNA up to 60 days after transduction. Taken together, these results indicate that insertion of the SV40-ori promoter in a lentiviral vector contributes to long-term expression by promoting episomal replication when TAg is provided in trans. Lentiviral episomal vectors may serve as specific tools for therapeutic approaches to diseases, particularly those associated with episomal replication of DNA viruses including papillomaviruses, polyomaviruses, and herpesviruses.

Cells, Cultured↗
PubMed2003

Collection of autologous PBSC in patients with polycythemia vera.

BACKGROUND: Allogeneic stem-cell transplantation (SCT) can eradicate myelofibrosis (MF), but is limited by donor availability and toxicity. We previously reported normalization of counts and resolution of MF after ablative, syngeneic SCT in spent phase polycythemia vera (PV). Hence, GvL is not required to eradicate MF. Autologous SCT may advance treatment for spent phase PV by restoring effective hematopoiesis. The influence of organomegaly, myelosuppression and MF on PBSC collection has not been studied in the setting of PV. METHODS: Sixteen patients with PV underwent PBSC collection. Mobilization was with filgrastim alone, with a target cell content of 2.5 x 10(6) CD34(+)/kg. All myelosuppression was discontinued 2 weeks prior to collection. RESULTS: Median ages at diagnosis and collection were 47 and 57 years, respectively. Organomegaly, MF and use of myelosuppressive therapies were present in 10 (63%), 4 (25%) and 7 (44%) patients. Median total nucleated cells (TNC) and CD34(+) counts were 8.3 x 10(8)/kg and 4.98 x 10(8)/kg. MF had an adverse effect on TNC (p=0.05) but not on the CD34(+) content. Time from diagnosis and the use of myelosuppresion had no influence on TNC and CD34(+) contents. Four patients had CD34(+) contents <2.5 x 10(6)/kg. Complete blood count (CBC) parameters were not predictive of CD34(+) content. DISCUSSION: Autologous PBSC collection is feasible in PV several years after diagnosis. Organomegaly and MF are not absolute contraindications for collection. Discontinuing myelosuppresion for 2 weeks before mobilization appears sufficient to collect adequate numbers of CD34(+) progenitors.

Adult↗
PubMed2001

Identification of a proviral structure in human breast cancer.

Involvement of a virus similar to mouse mammary tumor virus (MMTV) in human breast cancer has long been postulated but never demonstrated. We have detected by PCR a 660-bp sequence similar to the env gene of MMTV but not to the known endogenous viruses, in 38% of human breast cancers examined (Wang et al., Cancer Res., 55: 5173-5179, 1995). This sequence was expressed in 66% of the env-positive tumors as detected by reverse transcription-PCR (Wang et al., Clin. Cancer Res., 4: 2565-2568, 1998). In this article we report the amplification of a whole proviral structure from each of two human breast carcinomas that were env positive. Using nested extra-long PCR and primers from specific MMTV sequences, overlapping env-long terminal repeat (LTR), LTR-gag, gag-pol, and pol-env segments were successfully amplified. The 9.9-kb provirus is 95% homologous to MMTV but only 57% to human endogenous retrovirus K10 in 3.5 kb of the gag and pol genes. The provirus displays typical features of a replication competent virus, plus the open reading frame for the superantigen and the glucocorticoid responsive element. Fluorescence in situ hybridization with a 2.7-kb env-LTR sequence of an env-positive breast cancer cell line revealed that the sequence is inserted in several chromosomes but not in chromosomes from normal breast cells. The origin of the MMTV-like sequences is uncertain. Because they are undetectable in normal tissues, because the similarity between the two isolates is high (96%), and because they maintain open reading frames, they appear to be exogenous.

Breast Neoplasms↗
PubMed2001

Deletions of PURA, at 5q31, and PURB, at 7p13, in myelodysplastic syndrome and progression to acute myelogenous leukemia.

Deletions or monosomy of chromosomes 5 and 7 are frequently observed in myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). In this study two genes, PURA and PURB, encoding functionally cooperative proteins in the Pur family, are localized to chromosome bands 5q31.1 and 7p13, respectively. One or both of these loci are shown to be hemizygously deleted in 60 MDS or AML patients using fluorescence in situ hybridization (FISH). High-resolution mapping of PURA localizes it approximately 1.1 Mb telomeric to the EGR-1 gene. Frequency of PURA deletion and segregation with EGR-1 indicate that PURA is within the most commonly deleted segment in myeloid disorders characterized by del(5)(q31). No mutations have been detected within the coding sequence of PURA. Concurrent deletions of PURA and PURB occur in MDS at a rate nearly 1.5-fold higher than statistically expected and in AML at a rate > 5-fold higher. This novel simultaneous deletion of two closely related gene family members may thus have consequences related to progression to AML. Pur alpha, an Rb-binding protein, has been implicated in cell cycle control and differentiation, and Pur alpha and Pur beta are reported to function as heterodimers. Alterations in these genes could affect a delicate balance critical in myeloid development.

Acute Disease↗
PubMed2001

Functional reconstitution, membrane targeting, genomic structure, and chromosomal localization of a human urate transporter.

Elevated serum levels of uric acid have been associated with an increased risk for gout, hypertension, cardiovascular disease, and renal failure. The molecular mechanisms for the diminished excretion of urate in these disorders, however, remain poorly understood. Human galectin 9, which is highly homologous to the rat urate transporter rUAT, has been reported to be a secreted or cytosolic protein. We provide data that galectin 9 is hUAT, the first identified human urate transporter. hUAT is a highly selective urate ion channel when inserted in lipid bilayers. When expressed in renal epithelial cells it is an integral plasma membrane protein with at least two transmembrane domains. The gene for hUAT consists of 11 exons and is mapped to chromosome 17; a highly homologous gene, hUAT2, maps to a nearby region of chromosome 17 and is also likely to be a urate transporter. hUAT is expressed in a wide variety of tissues and is present in at least three isoforms; hUAT2 is less widely expressed at severalfold lower levels than hUAT. Further knowledge about the functions of hUAT, its isoforms, and hUAT2, as well as mutational analysis of hUAT1 and hUAT2 in individuals or families with hyperuricemia, should significantly improve our understanding of the molecular mechanisms of urate homeostasis.

Amino Acid Sequence↗
PubMed2000

An immunological syndrome featuring transverse myelitis, Evans syndrome and pulmonary infiltrates after unrelated bone marrow transplant in a patient with severe aplastic anemia.

A patient with severe aplastic anemia underwent a matched unrelated bone marrow transplant, following which he developed a complex autoimmune syndrome. This featured transverse myelitis, immune mediated Coombs positive hemolytic anemia and immune thrombocytopenia (Evans syndrome), pulmonary infiltrates, eosinophilia, muscle pains and cramps and lichenoid dermatitis all of which may represent manifestations of graft-versus-host disease as they showed response to immunosuppression. Thus, although immune-mediated cytopenias after an allogeneic bone marrow transplant are rare, they should be considered as a possible cause of cytopenia in post-transplant patients.

Adult↗
PubMed1999

Genomic organization and chromosomal localization of the human histone deacetylase 3 gene.

Reversible acetylation of histone proteins plays a critical role in transcriptional regulation, cell cycle progression, and developmental events. The steady state of histone acetylation is controlled by the enzymatic activities of multiple histone acetyltransferases and histone deacetylases (HDACs). Three distinct human HDACs are homologous to RPD3, a yeast transcriptional regulator. We have isolated and sequenced a genomic clone for the human HDAC3 gene. This is a single-copy gene spanning a region of at least 13 kb. Determination of the intron-exon splice junctions established that the gene is encoded by 15 exons ranging in size from 56 to 657 bp. Fluorescence in situ hybridization studies localized this gene to 5q31. Double-target experiments in which both HDAC3 and the early-growth response 1 gene (EGR1), which is localized in the 5q31.2 region, were used as probes showed that the HDAC3 gene lies in region 5q31.3, immediately distal to EGR1 with respect to the centromere.

Amino Acid Sequence↗
PubMed1999

Identification of novel chromosomal rearrangements in acute myelogenous leukemia involving loci on chromosome 2p23, 15q22 and 17q21.

Chromosomal translocations are frequently linked to multiple hematological malignancies. The study of the resulting abnormal gene products has led to fundamental advances in the understanding of cancer biology. This is the first report of t(2;15)(p23;q22) and t(2;17)(p23;q21) translocations in human malignancy. Patient 1, a 73-year-old male, was diagnosed with myeloblastic (FAB M1 sub-type) AML. Cytogenetic analysis showed a 47,XY,t(2;15)(p23;q22),+13 karyotype. Fluorescent in situ hybridization (FISH) showed that the PML gene was transferred intact to the short arm of chromosome 2 while the ALK gene on chromosome 2p23 was passively transferred to the long arm of chromosome 15. Patient 2 was a 60-year-old male diagnosed with monocytic (FAB M4-type) AML. Cytogenetic analysis showed 46,XY,t(2;17)(p23;q21) karyotype. FISH analysis showed that neither RARalpha nor ALK were disrupted by the translocation. None of the coding region of the three genes studied were translocated in these patients. This raises the possibilities that other neighboring genes could be involved or that noncoding regulatory sequences of the studied genes could be put in contact and deregulate expression of other genes. Alternatively, displacement of ALK, RARalpha and PML to novel positions could lead to loss of their normal regulation

Acute Disease↗
PubMed1998

The value of interphase fluorescence in situ hybridization in the study of patients with lymphoproliferative disorders: further evidence for a higher sensitivity of detecting chromosomes 7 and 8 aneuploidy.

Dual-color interphase fluorescence in situ hybridization (I-FISH) for chromosomes 7 and 8 was studied retrospectively on 32 patients with suspected lymphoid disorders, and the results were compared with standard cytogenetics. One of 29 (3.4%) patients with lymphoid malignancy showed cytogenetically detectable aneuploidy for chromosomes 7 and 8. In an additional 5 patients (17.2%), I-FISH unmasked chromosomal loss and gain that were not detected by standard metaphase analysis. This represents 19% of the 21 studied patients with acute lymphoblastic leukemia (ALL). These findings indicate that aneuploidies for chromosomes 7 and 8 are underreported in ALL and further demonstrate higher sensitivity of I-FISH for detecting numerical chromosomal rearrangements in leukemic cells.

Adult↗
PubMed1998

Clonal development of myeloproliferative disorders: clues to hematopoietic differentiation and multistep pathogenesis of cancer.

In November 1996, word reached the University of Washington that Philip Fialkow and his wife, Helen, had died while trekking in Nepal. Over a 30-year period, Dr Fialkow and his colleagues used the cellular mosaicism resulting from X-chromosome inactivation in females as a marker system to investigate the clonal development of human hematopoietic disorders. This review discusses the impact that these studies have had on our understanding of hematopoietic stem cell relationships and the pathogenesis of human neoplasia in general. To appreciate the special role played by studies on clonality, it is necessary to consider how little was known about the origin of leukemias and myeloproliferative disorders and the limited techniques available for their study in the early to mid 1960s. Dr Fialkow and his coworkers were the first to show that myeloproliferative disorders and acute myelogenous leukemias (AML) are clonal diseases at the time of diagnosis and to elucidate the level of differentiation manifested by the originating cell type. Although the myelodysplastic disorders were found to involve a pluripotent stem cell, heterogeneity was found in the level of stem cell involvement in AML. Evidence was obtained to support a multistep pathogenesis of these diseases as well as a clonal but cytogenetically normal stage in some cases of Ph-positive chronic myelogenous leukemia, AML, acute lymphoblastic leukemia and myelodysplasia.

Cell Differentiation↗
PubMed1998

Acquisition of the Ph chromosome and BCR-ABL fusion product in AML-M2 and t(8;21) leukemia: cytogenetic and FISH evidence for a late event.

A patient with the M2 subtype of AML who had a 45,X,-X,t(8;21) karyotype at diagnosis was found to have the Ph chromosome in one out of 37 evaluated cells 18 months after the initial diagnosis. Interphase FISH studies utilizing a BCR-ABL dual-color probe did not detect a fusion product 4 months prior to the appearance of one Ph-positive cell. Nineteen months post diagnosis and 5 months after clinical relapse all evaluated cells had the Ph chromosome in a clone characterized by t(8;21). These observations suggest that late appearing Ph is a secondary event which may be either therapy-related or consistent with one of the later events in a multistep pathogenesis of AML.

Adult↗
PubMed1997

A murine model for human cord blood transplantation: near-term fetal and neonatal peripheral blood cells can achieve long-term bone marrow engraftment in sublethally irradiated adult recipients.

The purposes of the research reported here were first to explore a murine model for human placental and umbilical cord blood transplantation and second to evaluate the engraftment ability of ex vivo cultured hematopoietic cells. Murine near-term fetal and neonatal peripheral blood (FNPB) cells, genetically marked with the human multiple drug resistance transgene (MDR1) were used for syngeneic transplants into sublethally irradiated adult mice. Donor cells were transplanted either fresh and untreated, or after ex vivo culture in the presence of the hematopoietic growth factors recombinant murine stem cell factor, recombinant human interleukin-3 (rHu IL-3), and rHu IL-6, in a liquid culture system. To evaluate, count, and characterize FNPB progenitor cell-derived colonies, neonatal mouse mononuclear cells were cultured directly in methylcellulose with growth factors. To assess their ex vivo expansion ability, FNPB mononuclear cells were first cultured in liquid medium for 3 to 8 days and then transferred to semisolid assay plates. Evaluation of the cell counts after liquid culture showed a 1.4- to 11.6-fold increase, and the numbers of colonies observed in methylcellulose were similar to those produced by fresh FNPB cells. Donor-type engraftment was demonstrated by polymerase chain reaction (PCR) amplification of the human MDR1 transgene in the peripheral blood of all surviving animals (5 of 7 recipients of the fresh, and 3 of 8 recipients of the ex vivo-cultured cells) 2 to 4 months after transplantation. The proportion of donor leukocytes in the peripheral blood of the recipients (chimerism) was evaluated using fluorescence in situ hybridization (FISH) analysis 4 to 6 months after transplantation and ranged from 2% to 26%. In addition, bone marrow cultures were obtained from two recipient animals: one had received fresh-untreated cells and was evaluated 8 months after transplant, the other had received ex vivo cultured cells and was tested 14 months after grafting. The derived hematopoietic colonies were tested by PCR and the transgene was detected, conclusively proving long-term engraftment of donor cells. These results indicate that FNPB transplants can be successfully performed in sublethally irradiated mice with and without ex vivo culture. Long-term donor-type engraftment with sustained chimerism has been demonstrated. Thus, murine neonatal blood grafts can be used as an animal model for cord blood transplantation for gene therapy studies where complete myeloablation is not desirable and partial replacement of defective marrow may be sufficient. Furthermore, the possibility of numerically expanding hematopoietic progenitor cells contained in neonatal blood without affecting their engraftment ability could facilitate use of cord blood grafts in adult recipients.

Animals↗
PubMed1997

Interphase FISH analysis of sex-mismatched BMT utilizing dual color XY probes.

Interphase FISH analysis, utilizing dual color XY probes, was performed on 27 patients following allogeneic sex-mismatched bone marrow transplantation and on 31 controls. Of the 123 167 examined interphase nuclei, 63 318 were from 19 of the 21 patients (54 specimens) who engrafted, 31 827 from five of the six patients (29 specimens) who relapsed (four) or failed to engraft (one) and 24 703 from the 31 control specimens. In patients who engrafted, the mean percentage of host cells was 0.26% between day 29 and 5 years following BMT. Microchimerism of 0.7% or less than 1-5 years following BMT was not predictive of relapse. Interphase FISH analysis predicted relapse or failure of engraftment in five of the six evaluable patients. In three of five patients both conventional cytogenetics and interphase FISH of bone marrow cells provided important information regarding engraftment status and degree of chimerism.

Bone Marrow Transplantation↗
PubMed1997

A pilot study of allogeneic bone marrow transplantation using related donors stimulated with G-CSF.

Growth factor administration to donors prior to bone marrow (BM) harvesting results in an enrichment of the graft for myeloid precursors. In animals, growth factor-primed BM has a higher repopulating ability than untreated BM. Ten patients received an HLA-identical sibling, allogeneic transplant using granulocyte colony-stimulating factor (G-CSF)-stimulated BM. Stimulation consisted of G-CSF at 10 microg/kg/day for 2 days prior to harvest. Patients were transplanted for various benign and malignant hematological conditions. The GVHD prophylaxis consisted of cyclosporine, methotrexate and/or prednisone. Compared to untreated historical control BM, stimulated BM infusions contained similar number of nucleated cells (mean +/- s.d.: 3.5 +/- 1.5 vs 4.0 +/- 0.9 x 10(8)/kg), CD34+ cells (mean +/- s.d.: 7.5 +/- 3.0 vs 9.4 +/- 6.7 x 10(6)/kg), and CD3+ cells (mean +/- s.d.: 129 +/- 30 vs 190 +/- 59 x 10(6)/kg) but higher numbers of granulocyte-macrophage colony-forming units (mean +/- s.d.: 20 +/- 12 vs 96 +/- 34 x 10(4)/kg). Patients receiving stimulated BM had prompt and stable engraftment of white cells and platelets. On average they attained an ANC of > or = 1 x 10(9)/l 9 days earlier and a platelet count of > or = 20 x 10(9)/l 6 days earlier than historical controls receiving unstimulated HLA-identical sibling BM. Hospitalization was shortened by a mean of 10 days and transfusion requirements were modest. None of the patients developed severe GVHD or disease relapse. Two patients died of severe VOD post-BMT and thus were unevaluable for platelet engraftment. A third patient died of TTP on day 76 post-BMT. Seven patients are alive and well 49-585 days post-BMT. Stimulated BM may provide a valuable alternative to allogeneic BM and PBSC transplants. Ideal stimulation regimens need to be investigated.

Adolescent↗