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D Zucker-Franklin

Publications and source records attributed to D Zucker-Franklin.

At least 19 recordsLinked to original sources
PubMed1999

Absence of human T-lymphotropic virus type I tax sequences in a population of normal blood donors in the Baltimore, MD/Washington, DC, area: results from a multicenter study.

BACKGROUND: It was reported recently that sequences corresponding to the human T-lymphotropic virus type I (HTLV-I) tax gene were detected in peripheral blood mononuclear cells from 8 to 11 percent of healthy blood donors without detectable antibodies to HTLV-I. A multicenter blind study was conducted to determine if these results could be independently confirmed. STUDY DESIGN AND METHODS: Specimens were collected from 100 anti-HTLV-I-negative healthy blood donors and from 11 anti-HTLV-I- or anti-HTLV-II-positive individuals. All samples were coded and distributed to each of four independent testing laboratories for polymerase chain reaction analysis to detect sequences of the HTLV-I or HTLV-II tax gene, using detailed procedures specified by the laboratory reporting the original observation. Each laboratory also tested a dilution panel of a plasmid containing HTLV-I tax to determine the analytical sensitivity of the procedure. RESULTS: The analytical sensitivity of the screening methods permitted detection of as few as 1 to 10 copies of the tax gene. However, HTLV-I tax sequences could not be detected in any of the anti-HTLV-I-negative blood donors at more than one test site. CONCLUSION: HTLV-I tax sequences appear not to be present in this population of 100 blood donors negative for anti-HTLV-I.

Adult↗
PubMed1998

Human T-cell lymphotropic virus type 1 tax among American blood donors.

In the United States, all blood used for transfusion is tested for the presence of antibodies to the structural components of the human T-cell lymphotropic viruses types 1 and 2 (HTLV-1 and -2). Based on such serologic tests, the prevalence of HTLV-1 infection is estimated to range from 0.016 to 0.1%. As a consequence of studies of patients with mycosis fungoides and some of their healthy relatives who are antibody negative but were found to carry the tax sequence of HTLV-1 in their lymphocytes and who had antibodies to the p40(tax) protein, a study was undertaken to determine the prevalence of the "tax-only" state in 250 healthy blood donors and other volunteers. Using PCR and Southern analysis for cell lysates and using Western blotting for plasmas, 8.6% of the blood donors proved to be tax sequence positive and antibody positive. Sequence analysis of specimens from 22 individuals proved that 20 of the sequences were homologous with that of HTLV-1 while 2 resembled the HTLV-2 sequence. The latter were obtained from volunteers of Indian origin. The possible clinical significance of the tax-only carrier state is discussed.

Arthritis, Rheumatoid↗
PubMed1998

The effects of Mpl-ligand, interleukin-6 and interleukin-11 on megakaryocyte and platelet alpha-granule proteins.

Thrombopoietin (Mpl ligand), interleukin-6 (IL-6), and interleukin-11 (IL-11) differ in their effects on megakaryocyte maturation and development. In the present study, the impact of these thrombocytopoietic cytokines on biochemical and structural granule and membrane components was examined. Western blotting was performed on equivalent amounts of isolated megakaryocyte or platelet protein and the relative intensities of the enhanced chemiluminescent-visualized bands were quantitated by densitometry. Megakaryocyte growth and development factor (MGDF), a recombinant thrombopoietin-related molecule, significantly increased megakaryocyte fibronectin (72%), thrombospondin (55%), von Willebrand factor (28%) and p-selectin (CD62p) (37%) when compared to equivalent amounts of protein from saline-treated controls (p<0.01). Megakaryocyte fibrinogen was not increased. Ultrastructurally, there was a marked increase in ribosomes and rough endoplasmic reticulum even in mature-appearing megakaryocytes. Platelets from MGDF-treated mice showed small increases in fibronectin (8%), and CD62p (18%), but did not show increases in other measured alpha-granule proteins. Neither IL-6 nor IL-11 increased megakaryocyte or platelet alpha-granule proteins over levels observed in saline controls. IL-11 treated megakaryocytes, while also exhibiting an increase in ribosomes, were characterized by prominent cytoplasmic fragmentation. The study demonstrates the impact of Mpl ligand in increasing synthesized megakaryocyte alpha-granule proteins and of IL-11 in promoting megakaryocyte fragmentation.

Animals↗
PubMed1997

Reexamination of human T cell lymphotropic virus (HTLV-I/II) prevalence.

In the United States, blood donors are being screened for infection with human T cell lymphotropic viruses I and II (HTLV-I/II) by serologic means, which detect antibodies to the structural proteins of these viruses. Because patients with mycosis fungoides (MF) usually do not have such antibodies even though their cells harbor HTLV-I Tax and/or pol proviral sequences, it was questioned whether the prevalence of HTLV infection among healthy blood donors may also be underestimated by current means of testing. To examine this possibility, a study on specimens of relatives of mycosis fungoides patients (MFR) was begun. In addition, to collect data more expeditiously, a cohort of former injection drug users (IDUs) was tested by routine serologic methods, as well as by PCR/Southern blot analysis for Tax, pol, and gag proviral sequences and Western blot analysis for antibodies to the Tax gene product. To date, 6/8 MFRs and 42/81 (51.8%) of HIV-negative IDUs proved to be positive for HTLV, whereas routine serology identified none of the MFR and only 18/81 (22.2%) of the IDUs. Among the latter test subjects, the incidence of HTLV-I also proved to be 10 times higher than expected. Therefore, it is likely that among healthy blood donors infection with HTLV-I/II is more prevalent than is currently assumed. Since Tax is the transforming sequence of HTLV-I/II, testing for Tax sequences and antibodies to its gene product may be desirable in blood transfusion and tissue donor facilities.

Blood Donors↗
PubMed1996

The difficulty of detecting HTLV-1 proviral sequences in patients with mycosis fungoides.

Although most patients with cutaneous T cell lymphomas, including mycosis fungoides (MF) and its leukemic variant, the Sézary syndrome, are seronegative for antibodies to the human T cell lymphotropic viruses (HTLV-I/II), it has recently been shown that > 95% of such patients harbor proviral DNA sequences related to the region of the HTLV genome that encodes the transregulatory/transforming gene, tax. However, the demonstration of HTLV sequences, even after amplification by polymerase chain reaction (PCR), has not been universally successful, and some investigators continue to question this observation. In an effort to resolve this controversy, we have compared published methodologies that have been less successful with techniques currently used in this laboratory. Major differences were found in (a) the nature of the cells used [freshly isolated versus cultured peripheral blood mononuclear cells (PBMC)] and (b) the methods used to prepare samples for PCR (whole cell lysates versus DNA extracts). PBMC from 10 different MF patients and the healthy daughter of 1 of the patients were subjected to comparative analyses. While all of the PBMC lysates were positive, the DNA extract from only one of these individuals revealed HTLV tax sequences. Studies were also conducted comparing cell lysates and DNA extracts of cultured cells derived from tax sequence-positive PBMC from seven different MF patients. The cells from four of the seven were shown to have retained tax sequences after varying times in culture, when whole-cell lysates were used as targets for PCR amplification and Southern analysis, whereas none of the DNA extracts were positive. It appears that the use of whole-cell lysates instead of DNA extracts and the use of fresh instead of cultured cells greatly enhance the ability to detect HTLV-1 tax sequences in specimens from MF patients.

Blotting, Southern↗
PubMed1996

Demonstration of antibodies to human T-cell lymphotropic virus-I tax in patients with the cutaneous T-cell lymphoma, mycosis fungoides, who are seronegative for antibodies to the structural proteins of the virus.

Although most patients with the cutaneous T-cell lymphoma, mycosis fungoides (MF), are seronegative for human T-cell lymphotropic virus-I or -II (HTLV-I/II) when tested by assays that measure only antibodies to the viral structural proteins, the majority of such patients harbor HTLV-I-related pol and tax proviral sequences that encode proteins not included in routinely used serologic tests. Tax mRNA has also been detected in their peripheral blood mononuclear cells (PBMC). Therefore, it seemed possible that these patients have antibodies to the tax protein. To investigate this, enzyme-linked immunosorbent assays (ELI-SAs) and Western blot assays were set up, using as antigens the full-length HTLV-I tax cloned from the prototypic HTLV-I-infected cell line, C91PL, and from PBMC of a MF patient, as well as a synthetic peptide made to the carboxy-terminal 20 amino acids of tax-I. Of 60 MF patients whose PBMC were shown to be positive for tax proviral DNA and mRNA, 50 (83%) were shown to have tax antibodies. The antigen derived from the MF patient was most useful in detecting such antibodies. These results demonstrate the need for including other HTLV-related antigens in addition to gag and env in serologic tests used to identify HTLV-infected individuals. The findings underscore the fact that individuals considered seronegative on the basis of currently used tests can be infected with HTLV.

Amino Acid Sequence↗
PubMed1996

Effect of thrombopoietin on the development of megakaryocytes and platelets: an ultrastructural analysis.

Megakaryocytopoiesis and platelet production can be assessed with reasonable accuracy by quantitative and functional analyses of circulating platelets. The evaluation of megakaryocytopoiesis in culture has remained unsatisfactory, particularly because platelet production is rarely observed. In mouse culture systems, megakaryocytes have been identified almost entirely by measurements of acetyl cholinesterase, size, and ploidy without concomitant assessment of maturation based on such criteria as the formation of granules, demarcation membranes, and cytoplasmic fragmentation. The availability of various thrombopoietic cytokines, in particular thrombopoietin (TPO), and their imminent clinical use has made a more detailed understanding of their effect on differentiation and maturation of the MK lineage more urgent. Therefore, ultrastructural analyses were performed on megakaryocyte-depleted serum-free mouse bone marrow cultures in the presence of TPO alone, TPO plus other cytokines, or under conditions in which TPO and/or other cytokines were blocked with neutralizing agents. These studies show that, while cytokines that use the gp130 receptor subunit may function synergistically with TPO, in the absence of TPO, such cultures do not yield morphologically recognizable MK. On the other hand, TPO alone is able to drive MK to full maturation as evidenced by the generation of granules, demarcation membranes, and cytoplasmic fragmentation into platelets.

Acetylcholinesterase↗
PubMed1996

Immunophenotypic identification of Sezary cells in peripheral blood.

Despite anecdotal literature that Sezary cells express the CD4+ CD7- immunophenotype, no formal validation has been published establishing the use of this immunophenotype for clinical or experimental purposes. Consequently, the only method presently available for Sezary cell identification is nuclear contour analysis, a labor-intensive procedure not generally available at most major medical centers. In this study, the accuracy of CD4+ CD7- subset quantitation for the identification of Sezary cells was examined. The study found that the percentage of CD4+ CD7- cells is elevated in many Sezary syndrome/MF patients relative to normal, healthy individuals. In addition, CD4+ CD7- enumeration correlates with enumeration by nuclear contour analysis in most patients (11 of 15) with elevated CD4/CD8 ratios. The CD4+ CD7- subset also correlates with the expression of other aberrant immunophenotypes, such as CD3low or CD4low. Lastly, CD4+ CD7- subset quantitation correlates with the number of clonal T lymphocytes, as measured using V beta-specific T-cell receptor monoclonal antibodies. The study found this method to be exceptionally accurate, with two caveats: (1) the absence of an expanded CD4+ CD7- subset in patients with a normal CD4/CD8 ratio is uninformative; and (2) in approximately 25% of patients with an elevated CD4/CD8 ratio, the Sezary cells are CD7+.

Antigens, CD7↗
PubMed1996

Localization of human T-cell lymphotropic virus-1 tax proviral sequences in skin biopsies of patients with mycosis fungoides by in situ polymerase chain reaction.

The histopathologic diagnosis of mycosis fungoides (MF), even when clinical manifestations of the disease seem convincing, is often tenuous. The observation that practically all patients with MF harbor human T cell lymphotropic virus type I (HTLV-I) proviral sequences in their circulating lymphocytes raised the possibility that such viral footprints could be detected in their cutaneous infiltrates. Application of in situ polymerase chain reaction (PCR) to skin biopsies of 11 of 12 patients demonstrated this assumption to be correct. In addition, cells suspected to be keratinocytes were also positive. None of 10 skin biopsies from a variety of sources used as controls, nor 3 lymph node biopsies from patients with B-cell lymphomas, showed any HTLV proviral sequences on in situ PCR. On the basis of these observations, it is concluded that in situ PCR carried out on skin biopsies of patients with presumptive MF may help to established the diagnosis.

Genes, pX↗
PubMed1996

Determination of the true prevalence of infection with the human T-cell lymphotropic viruses (HTLV-I/II) may require a combination of biomolecular and serological analyses.

Infection with the human T-cell lymphotropic virus types I and II (HTLV-I/II) usually is determined by tests that detect antibodies to the viral structural proteins. However, recent studies revealed that patients with mycosis fungoides have proviral DNA sequences related to the HTLV transactivating-transforming gene tax, without having antibodies to the virus. These results raised the possibility that the prevalence of HTLV infection in the general population of the United States also may be underestimated. To reassess the prevalence of HTLV-I/II infection effectively, a population at increased risk for infection (i.e., a cohort of injection drug users [IDUs]) was studied. Paired sera and peripheral blood mononuclear cells (PBMCs) from 81 IDUs were subjected to testing by Western blot analysis for antibodies to the viral structural proteins gag and env and by polymerase chain reaction (PCR) Southern analysis to detect gag, pol and tax proviral DNA sequences. Western blot assays showed 1 of 81 IDUs to be positive for HTLV-I, 14 to be positive for antibodies to HTLV-II, and 3 to be HTLV-serotype indeterminate. When whole-cell lysates of PBMCs from these individuals were subjected to PCR and Southern analysis. 39 of 81 were found to have HTLV-related sequences. A total of nine IDUs were found to be infected with HTLV-I, a figure nearly 10 times higher than that estimated by serology alone. Bio-molecular analysis showed HTLV-II-specific proviral sequences in 21 IDUs. Three individuals were seropositive for HTLV-II but lacked PCR evidence of gag, pol and tax sequences. Thus, the overall prevalence of HTLV infection among this cohort was 59% (43 of 81) (i.e., more than twice the frequency predicted by serology, 18 of 81 or 22%). These results indicate that it may be necessary to incorporate biomolecular as well as serological methodologies to identify all persons infected with these retroviruses.

Blotting, Southern↗
PubMed1995

Transcription factor NF-E2 is required for platelet formation independent of the actions of thrombopoietin/MGDF in megakaryocyte development.

Despite the importance of blood platelets in health and disease, the mechanisms regulating their formation within megakaryocytes are unknown. We generated mice lacking the hematopoietic subunit (p45) of the heterodimeric erythroid transcription factor NF-E2. Unexpectedly, NF-E2-/- mice lack circulating platelets and die of hemorrhage; their megakaryocytes show no cytoplasmic platelet formation. Though platelets are absent, serum levels of the growth factor thrombopoietin/MGDF are not elevated above controls. Nonetheless, NF-E2-/- megakaryocytes proliferate in vivo in response to thrombopoietin administration. Thus, as an essential factor for megakaryocyte maturation and platelet production, NF-E2 must regulate critical target genes independent of the action of thrombopoietin. These findings provide insight into the genetic analysis of megakaryocyte maturation and thrombopoiesis.

Animals↗
PubMed1995

Thrombopoietin, the Mp1 ligand, is essential for full megakaryocyte development.

The development of megakaryocytes (MKs) from their marrow precursors is one of the least understood aspects of hematopoiesis. Current models suggest that early-acting MK colony-stimulating factors, such as interleukin (IL) 3 or c-kit ligand, are required for expansion of hematopoietic progenitors into cells capable of responding to late-acting MK potentiators, including IL-6 and IL-11. Recently, the Mp1 ligand, or thrombopoietin (Tpo), has been shown to display both MK colony-stimulating factor and potentiator activities, at potencies far greater than that of other cytokines. In light of these findings, we tested the hypothesis that Tpo is absolutely necessary for MK development. In this report we demonstrate that neutralizing the biological activity of Tpo eliminates MK formation in response to c-kit ligand, IL-6, and IL-11, alone and in combination, but that these reagents only partially reduce MK formation in the presence of combinations of cytokines including IL-3. However, despite the capacity of IL-3 to support the proliferation and initial stages of MK differentiation, elimination of Tpo prevents the full maturation of IL-3-induced MK. These data indicate that two populations of MK progenitors can be identified: one that is responsive to IL-3 but can fully develop only in the presence of Tpo and a second that is dependent on Tpo for both proliferation and differentiation. Thus, our results strongly suggest that Tpo is the primary regulator of MK development and platelet production.

Animals↗
PubMed1995

Interaction of human immunodeficiency virus type 1, human T-cell leukemia/lymphoma virus type I (HTLV-I), and HTLV-II with in vitro-generated dendritic cells.

Although it is known that impairment of dendritic cells (DC) plays a role in the pathogenesis and immunosuppression of retrovirus-associated diseases, it is not clear whether, or to what extent, these antigen-presenting cells themselves become infected. The realization that the cells can be generated in vitro in larger numbers than can be isolated from circulating blood or bone marrow raised the possibility that they could be used for therapeutic purposes. Therefore, we investigated whether DC generated in vitro from CD34 precursors are susceptible to infection when cocultured with human immunodeficiency virus type 1- or human T-cell leukemia/lymphoma virus-infected cell lines. While there appears to be a remarkable affinity of the viruses for the plasma membranes of the DC, interiorization or budding was not observed in 30 experiments carried out under a variety of conditions.

Acquired Immunodeficiency Syndrome↗
PubMed1995

The cutaneous T cell lymphoma, mycosis fungoides, is a human T cell lymphotropic virus-associated disease. A study of 50 patients.

For nearly two decades it has been suspected that the cutaneous T cell lymphoma, mycosis fungoides (MF), and its leukemic variant, the Sézary syndrome, are caused by the human T lymphotropic virus (HTLV-I/II). Arguments against this concept included the finding that only a small number of MF patients have antibodies to HTLV-I/II and that attempts to detect proviral sequences by mere Southern hybridization of extracted DNA usually met with failure. However, we have reported repeatedly that HTLV-like particles emerge in blood mononuclear cell (PBMC) cultures of practically all patients with this disease. In several instances, the particles were identified as HTLV by immunoelectron microscopy as well as biomolecular analysis. With the assumptions that the virus in MF patients may have become detection by Southern hybridization alone, the extracts of freshly isolated PBMC of 50 consecutive patients were subjected to combined PCR/Southern analysis. Here we report the presence of HTLV pol and/or tax proviral sequences in 46 out of 50 (92%) of the patients tested. In addition, five of the patients, who lacked antibodies to HTLV-I/II structural proteins, were found to be seropositive for tax. It thus seems reasonable to conclude that MF/Sézary syndrome is an HTLV-associated disease and that lack of an immune response does not preclude infection with this type of virus.

Adult↗