Ciprofloxacin-induced neutropenia and erythema multiforme.
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Aged↗
Biomedical subjects
A Keating
Publications and source records attributed to A Keating.
Investigation of bcr-abl transcription by Ph-positive chronic myeloid leukemia progenitors.
It is now feasible to investigate bcr-abl transcription by the progeny of Ph-positive (Ph+) early and committed hemopoietic progenitor cells from patients with chronic myeloid leukemia (CML). Cells from individual colonies can be bisected and each half analyzed by cytogenetics or the reverse transcriptase-polymerase chain reaction (RT-PCR) method to detect the bcr-abl transcript using internal nested oligonucleotide primers that flank the chimeric gene junction. We previously showed that some Ph+ colonies have undetectable PCR products for bcr-abl. When colonies are generated in the presence of alpha interferon (IFN-alpha) bcr-abl transcripts are undetectable in the majority of Ph+ colonies. These data suggest a potential mechanism for the action of IFN-alpha in Ph+ CML and indicate the need for a combined approach with cytogenetics and RT-PCR in analyzing the bcr-abl gene.
DNA, Neoplasm↗
CD34 antigen: molecular features and potential clinical applications.
Despite the wide variety of functions exhibited by mature peripheral blood cells, all are derived from a small pool (1-3%) of primitive precursor cells in the bone marrow (BM) that bear a unique surface glycoprotein, CD34. Isolated CD34+ cells are capable of reconstituting all hematopoietic lineages, both in experimental animals and in humans following intensive therapy. CD34+ cells capable of reconstituting hematopoiesis are also found at low frequency in peripheral blood (PB), a frequency which can be dramatically increased by combinations of chemotherapy and recombinant cytokines. In some cases, PB "stem cells" (PBSC) can be used to augment or even replace conventional BM autografts. The availability of CD34 antibodies has greatly aided the development of techniques for the enrichment of primitive progenitor cells, thus allowing studies of the hematopoietic potential of stem cells in vitro. Additionally, the use of CD34 antibodies for the "positive selection" of hematopoietic stem/progenitor cells from tumor-contaminated marrow may possibly represent an alternative "purging" strategy prior to transplantation. The availability of pure populations of the most primitive hematopoietic progenitor cells will also facilitate study of genetic manipulation as a practical therapeutic modality.
Antigens, CD↗
The v-raf oncogene enhances tumorigenicity and suppresses differentiation in vivo in a rat hepatocyte cell line.
raf oncogenes have been implicated in hepatic carcinogenesis. We studied the effects of the v-raf of murine retrovirus 3611-MSV on the growth and differentiation of a simian virus 40 (SV40)-immortalized rat liver cell line (ALB-8) which maintained many of characteristics of differentiated hepatocytes. Cells were co-transfected with v-raf and the neo gene followed by selection with G418 for transfectants. In culture, the expression of v-raf stimulated cell proliferation without altering cell morphology or expression of liver-specific genes: albumin, fibrinogen, alpha-1-antitrypsin and alpha-1-acid glycoprotein. The v-raf-transfected cells induced rapidly growing tumors in 100% of nude mice, while control DNA-transfected cells were only weakly tumorigenic, producing slowly growing tumors in 2/7 mice after a long latency. These slowly growing tumors were histologically moderately to well-differentiated hepatocellular carcinomas in which the liver-specific genes were highly expressed. In contrast, v-raf-induced tumors were histologically poorly differentiated and showed a dramatic decline in the expression of the liver-specific genes. In a tumor cell culture established from a v-raf-induced tumor, however, expression of the liver-specific genes was coordinately recovered. These observations indicate that v-raf is capable of inducing progression of SV40-immortalized hepatocytes into highly malignant cells and the progression is accompanied by loss, in vivo, of the hepatic differentiation.
Albumins↗
Myth smashers, myth makers: (re)visionary techniques in the works of Paula Gunn Allen, Gloria Anzaldúa and Audre Lorde.
This paper examines the revisionist mythmaking strategies employed by three lesbian-feminist writers of color: Paula Gunn Allen (Laguna/Sioux/Lebanese/Scottish), Gloria Anzaldúa (Chicana tejana), and Audre Lorde (Caribbean/African/American). By incorporating creatrix figures such as the West African Mawulisa, the pre-Aztec Coatlicue, and the Laguna Pueblo Spider Old Woman/Thought Woman into their works, they challenge the cultural stereotypes that silence women of color by denying their access to language. Their use of nonwestern mythic material destabilizes monolithic definitions of (white heterosexual) female identity, yet their mythmaking goes beyond this challenge to hegemonic concepts of (white) womanhood. As they replace the Judeo-Christian world-view with modes of perception drawn from Native American, Chicana, and African mythic traditions, they offer a far-reaching critique of western culture's binary structures. By displacing the boundaries between inner/outer, subject/object, spirit/matter, and other dichotomous terms, the new myths they create provide radical alternatives to the existing social structures.
Female↗
Photodynamic elimination of clonogenic Ph+ chronic myeloid leukemia cells.
Benzoporphyrin derivative (BPD) and light is a potent photosensitizer. We investigated this modality as a means to selectively eliminate clonogenic Ph(+) chronic myeloid leukemia (CML) cells. BPD at 10 ng/ml and 10.8 J/cm2 broad spectrum light eliminates from 5 to 6 logs of Ph(+) EM-2 cells. Long-term marrow culture studies of treated mixtures of normal and CML cells indicate that multipotent progenitor cell viability is retained while cells transcribing BCR-ABL are not detected. We conclude that BPD and light may offer a means of providing CML autografts potentially free of Ph(+) clonogenic cells.
Adult↗
Detection and direct sequence identification of BCR-ABL mRNA in Ph+ chronic myeloid leukemia.
The reverse transcriptase-polymerase chain reaction (RT-PCR) for BCR-ABL mRNA is increasingly used to diagnose and monitor patients with Ph+ chronic myeloid leukemia (CML). We investigated an alternative approach to detect BCR-ABL mRNA in CML in order to overcome some of the potential drawbacks of RT-PCR. Nucleic acid sequence based amplification (NASBA) is a homogeneous, isothermal, in vitro process that provides the direct amplification of RNA. Peripheral blood from seven patients with Ph+ CML and Ph+ EM-2 cells were investigated by NASBA and RT-PCR. A nested set of four primers flanking the BCR-ABL junction was used in two serial NASBA reactions performed for 2 hours. The two methods were fully concordant for detection of transcripts with bcr3-abl2 and bcr2-abl2 junctions. Ethidium bromide fluorescence with NASBA indicated in repeated experiments that similar quantities of total RNA from patient material contained different amounts of BCR-ABL mRNA. The data suggest that direct amplification of RNA is suitable for identifying and monitoring patients with Ph+ CML and may provide a means to quantify BCR-ABL mRNA levels.
Base Sequence↗
Hematopoietic stem cells engraft in untreated transplant recipients.
We investigated the engraftment of hematopoietic stem cells in completely untreated transplant recipients to further study hematopoietic cell regulation and for possible inclusion in gene therapy protocols. Untreated female Balb/c recipients received a single infusion of male Balb/c marrow cells. Donor origin of the hematopoietic cells was determined by polymerase chain reaction (PCR), Southern and in situ hybridization analyses with Y-chromosome-specific probes. We found that up to 47% day 12 CFU-S (26.2 +/- 12.6%, mean +/- SD, range 13.3 to 46.7%), 7.3 +/- 5.5% CFU-GM and from 2.5 to approximately 10% nucleated marrow cells were of donor-origin at 8 weeks after marrow infusion. Our results indicate that hematopoietic stem cells can stably engraft in completely unconditioned recipients but, during the interval analyzed, have a low tendency to differentiate. Moreover, the data suggest that under steady-state conditions, niches for primitive hematopoietic cells present in the marrow microenvironment are not saturated, and are readily available. We conclude that the untreated-recipient transplant model, in conjunction with sensitive techniques for the detection of donor cells, provides a valuable means for studying hematopoietic stem cell regulation and indicates a need to reassess our understanding of the interactions between stem cells and the hematopoietic microenvironment.
Animals↗
Interleukin-3 followed by GM-CSF for delayed engraftment after autologous bone marrow transplantation.
Recombinant human interleukin-3 (IL-3) is well-tolerated according to phase I studies, and produces trilineage hematologic responses in patients with normal bone marrow. In addition, promising results have been obtained in a variety of bone marrow failure states. We studied IL-3 in 7 patients with markedly delayed engraftment after autologous bone marrow transplantation (ABMT) for hematologic malignancies (acute myeloid leukemia 4, chronic myeloid leukemia 1, myeloma 1, non-Hodgkin's lymphoma 1). All patients were red blood cell- and platelet transfusion-dependent, had an absolute neutrophil count (ANC) < 0.7 x 10(9)/L and failed to achieve a sustained ANC > 1.0 x 10(9)/L after receiving granulocyte-macrophage colony stimulating factor (GM-CSF) for 28 days. IL-3 was given daily for 21 days at 2 micrograms/kg/d (2 patients) and 5 micrograms/kg/d (5 patients). Toxicity was mild and consisted mostly of low-grade fever and malaise. No changes in platelet, hemoglobin or reticulocyte levels were observed. Four patients had at least a 2-fold increase in ANC at the end of IL-3 treatment. Five patients received GM-CSF 10 micrograms/kg/d subcutaneously for 7 to 10 days immediately after IL-3 and 4 had a further increase in ANC (median 1.7-fold, range 1.6- to 5.8-fold), but no change in platelet transfusion requirements. Hematopoietic colony assays of bone marrow cells obtained before and after treatment showed that granulocyte-macrophage colony-forming cell (CFU-GM) and erythroid blast-forming cell (BFU-E) levels were severely reduced and multilineage progenitors (CFU-GEMM) absent in all patients, and remained low after IL-3 treatment for 21 days. Sequential IL-3 and GM-CSF produced a significant but transient increase in the neutrophil counts of some patients. IL-3 appears to be of limited benefit in patients who are severely aplastic after ABMT and have very low levels of bone marrow progenitors.
Acute Disease↗
Bead transfection: rapid and efficient gene transfer into marrow stromal and other adherent mammalian cells.
We report a simple, rapid, efficient and cost-effective method of gene transfer into bone marrow stromal and other adherent mammalian cells. Our approach involves brief incubation of cells with glass beads in a solution containing the DNA to be transferred. We optimized the technique using COS cells (SV40 transformed kidney cell line from African green monkey) and a transient expression assay for chloramphenicol acetyl transferase (CAT). Factors affecting gene transfer include size and condition of the beads and DNA concentration, but not DNA conformation. Gene transfer efficiency, assessed in a transient expression assay for beta-galactosidase activity, was 5 and 3% in nontransformed human bone marrow stromal cells and COS cells, respectively. Long-term stable expression with the selectable marker, neomycin phosphotransferase, was demonstrated in clonogenic COS cells at a frequency of 27%. Southern analysis of resistant clones revealed the transferred DNA to be integrated in low copy number at one or two sites in the host cell genome. Comparison with electroporation and DEAE-dextran indicates that bead transfection is more efficient than the latter and less costly than either of these methods. In view of its simplicity and because the use of retroviral sequences can be avoided, bead transfection may be an attractive means of gene insertion for gene therapy.
Animals↗
ras transformation of simian virus 40-immortalized rat hepatocytes: an in vitro model of hepatocarcinogenesis.
Primary rat hepatocytes were transfected with simian virus 40 DNA and cultured in a chemically defined medium. Proliferating colonies developed after 2-3 weeks. Three cell lines were established by cloning albumin-secreting colonies, as identified by an immunooverlay assay. Two of the cell lines, ALB-6 and ALB-8, expressed all five liver-specific mRNAs studied, albumin, alpha-1-antitrypsin, fibrinogen, alpha-1-acid glycoprotein, and histidase. ALB-6 cells were nontumorigenic in nude mice while ALB-8 cells were weakly tumorigenic with only one of four injected nude mice developing a slowly growing tumor. Further transfection of ALB-6 and ALB-8 cells with an activated c-Ha-ras or N-ras oncogene resulted in strongly tumorigenic cells. The tumors induced by ras-transformed ALB-6 cells were moderately differentiated hepatocellular carcinomas. The tumors derived from ras-transformed ALB-8 cells were poorly differentiated, while the slowly growing tumors induced by untransfected or control DNA-transfected ALB-8 cells were well-differentiated trabecular hepatocellular carcinomas, suggesting histological dedifferentiation of cells following ras transformation. However, the synthetic capabilities of the cells were not lost in that the ras-transfected cultures and the tumors induced by ras-transformed cells retained the ability to synthesize the five liver-specific mRNAs. Thus we have developed an in vitro model of carcinogenesis in which, by sequential exposure to SV40 DNA and a ras oncogene, primary rat hepatocytes are transformed.
Animals↗
Successful pregnancy and delivery during alpha-interferon therapy for chronic myeloid leukemia.
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Adult↗
The CD34 antigen: structure, biology, and potential clinical applications.
The diversity of function of mature circulating blood cells is reflected in their respective complements of cell-surface molecules and receptors. Although monoclonal antibodies have been instrumental in the identification and characterization of many cell-surface molecules on mature hematopoietic cells, the CD34 antigen represents to date, the only molecule, similarly identified, whose expression within the blood system is restricted to a small number of primitive progenitor cells in the bone marrow. Although its precise function remains unknown, the pattern of expression of the CD34 structure suggests that it plays an important role in early hematopoiesis. The availability of CD34 antibodies has greatly aided the development of techniques for the enrichment of primitive progenitor cells for studies of hematopoiesis in vitro. Additionally, the use of CD34 antibodies for the 'positive selection' of hematopoietic stem/progenitor cells represents and alternative strategy to 'negative selection' or purging for the large-scale manipulation of bone marrow cells prior to transplantation. The availability of pure populations of the most primitive hematopoietic progenitor cells may also facilitate the development of genetic techniques for the repair of specific blood cell disorders. In this article, we review the biology of the CD34 molecule and assess some of the roles for CD34 antibodies in immunopathology and for progenitor/stem cell purification in clinical applications.
Animals↗
Prolonged disease-free survival in an adult presenting with Burkitt-type acute lymphoblastic leukaemia and CNS disease.
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Adult↗
Differential sensitivity of CD34 epitopes to cleavage by Pasteurella haemolytica glycoprotease: implications for purification of CD34-positive progenitor cells.
Our previous studies have shown that a unique glycoprotease from Pasteurella haemolytica specifically cleaves only proteins containing sialylated O-linked glycans. The hematopoietic progenitor cell antigen, CD34, which is heavily glycosylated with both N- and O-linked glycans, is readily cleaved by this protease. In this study, we demonstrate that the epitopes detected by five of the seven CD34 monoclonal antibodies are removed by the glycoprotease. The differential sensitivity of the CD34 epitopes to cleavage with either neuraminidase and/or glycoprotease establishes three classes of epitopes: 1) (class I) those identified by MY10, B1.3C5, 12.8, and ICH3 that are differentially affected by neuraminidase and removed by the glycoprotease; 2) (class II) the epitope detected by QBEND 10 that is removed only by the glycoprotease; and 3) (class III) those identified by TUK3 and 115.2 that are not removed by either enzyme. Cleavage of the 110-kd CD34 structure by the glycoprotease generates a major cell-bound fragment of about 75 kd, identified by the class III antibodies. We have also used the enzyme to improve the rapid recovery of CD34+ cells selected by immunomagnetic affinity techniques. In a preclinical model, we separated CD34+ KG1 cells with high yield (90%-95%) and high purity (94%-98%) from sham mixtures containing 50% CD34- cells. We also separated CD34+ blast cells from a patient in megakaryoblastic crisis of chronic myelogenous leukemia. In this case, the purity and yield were 93% and 94%, respectively. Enzyme treatment had no detrimental effect on cell viability, and the treated cells showed a normal quantitative expression and distribution of CD34 antigen as assessed with class III antibodies. We conclude that the P. haemolytica glycoprotease has potential to improve the isolation, from human bone marrow, of primitive hematopoietic cells that carry the CD34 antigen.
Antigens, CD↗
Maintenance of Philadelphia-chromosome-positive progenitors in long-term marrow cultures from patients with advanced chronic myeloid leukemia.
We investigated the marrows of 19 patients with advanced Philadelphia chromosome positive (Ph+) chronic myeloid leukemia (CML) in long-term marrow culture (LTMC) to determine the frequency of loss of clonogenic leukemic cells in vitro. Sixteen patients were in first chronic phase at a median of 24 months from diagnosis and had received prior therapy with busulphan and/or hydroxyurea. The effect of interferon therapy on loss of Ph+ clonogenic cells in LTMC was also investigated. Of 16 patients who had not previously received interferon, complete loss of Ph+ progenitors was documented by 4-5 weeks in the LTMCs from two (12.5%). Ph+ progenitors persisted at 4-5 weeks in the LTMC derived from 12 patients. Marrows from nine patients treated with interferon were also established in LTMC. Cultures from four patients did not yield colonies with detectable metaphases at 3-5 weeks, while Ph+ clones were present in the cultures initiated with marrows from five patients. Mean hematopoietic colony yields from the adherent layer at 2-4 weeks, and from the supernatant layer at 1-3 weeks, of cultures derived from interferon-treated patients were significantly lower than in LTMCs of patients not treated with interferon (p less than 0.05). The results indicate that in previously treated patients with late chronic phase CML there is a low frequency of conversion of Ph-negative hematopoiesis in long-term culture. Interferon therapy is associated with impaired progenitor yields in LTMC and does not improve selective loss of Ph+ progenitors.
Bone Marrow↗
Retention of progenitor cell function in CD34+ cells purified using a novel O-sialoglycoprotease.
We previously showed that the sialoglycoprotein, CD34, which is expressed on primitive human hematopoietic progenitor cells, is cleaved by a unique glycoprotease from Pasteurella haemolytica (P.h. glycoprotease). This proteolytic enzyme specifically cleaves glycoproteins rich in O-sialoglycans. Glycoproteins containing only N-linked glycans are not cleaved. Cleavage of the CD34 antigen results in the loss of epitopes detected by five of seven CD34-designated antibodies. In this study, we investigated the role of the P.h. glycoprotease in isolating CD34+ cells from unfractionated normal human bone marrow mononuclear cells (MNCs), and determined the effect of the glycoprotease on the proliferative capacity of the progenitor-enriched fraction. CD34+ cells were isolated from MNCs using immunomagnetic beads attached via a CD34 antibody whose epitope is susceptible to removal by the cleavage with the glycoprotease. Subsequent cleavage with P.h. glycoprotease for 30 min at 37 degrees C released the CD34+ cells from the beads with a recovery of up to 78%. Using a CD34 antibody whose epitope was not removed by the glycoprotease, up to 95% of the recovered cells expressed CD34. Compared to unseparated MNCs, the CD34+ cells showed the following enrichment of committed hematopoietic progenitors, as assayed in semi-solid media: CFU-GM, 45-fold; CFU-M, 13-fold; BFU-E, 26-fold and CFU-GEMM, 81-fold. Hematopoiesis was also studied in two-stage long-term bone marrow cultures in which the CD34+ cells were co-cultured over irradiated, allogeneic adherent layers. Output of CFU-GM over a seven week period from these cultures was similar to that from control cultures with autologous adherent-cell-depleted marrow MNCs. These data suggest that the loss of O-sialo-glycosylated peptide moieties from P.h. glycoprotease-released CD34+ cells neither affects the functional capacity of committed progenitors, nor impairs the proliferation of long-term culture-generating cells. The P.h. glycoprotease can be used to facilitate the isolation and recovery of functionally competent CD34+ cells at high yield and purity, without prior removal of other adherent cells. The ability to rapidly purify CD34+ cells using this non-cytotoxic enzyme has important implications for bone marrow transplantation as well as for gene transfer studies in vitro.
Antigens, CD↗
High dose etoposide melphalan, total body irradiation and ABMT for acute myeloid leukemia in first remission.
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Adolescent↗