Biomedical subjects

J Mirro

Publications and source records attributed to J Mirro.

At least 19 recordsLinked to original sources

PubMed2006

Mutant murine leukemia virus Gag proteins lacking proline at the N-terminus of the capsid domain block infectivity in virions containing wild-type Gag.

We have investigated the properties of murine leukemia virus Gag mutants in which the p12-CA cleavage site is altered. In one mutant, the cleavage is blocked; in the other, the conserved proline at the N-terminus of CA has been replaced with glycine. No infectivity was detected in either mutant. Mutant particles cannot synthesize full-length DNA upon infecting permissive cells. Particles composed of a mixture of wild-type and mutant proteins have severely impaired infectivity. These mixed particles are defective in their ability to synthesize DNA upon infection, but this defect is less severe than the loss of infectivity. Thus, proteins lacking the correct N-terminus of CA inhibit DNA synthesis and also interfere with formation or integration of a full-length, normal provirus. The results imply that CA proteins function as part of a large, highly organized structure in reverse transcription and apparently at a later step as well.

Animals↗

PubMed2001

RNA is a structural element in retrovirus particles.

A single retroviral protein, Gag, is sufficient for virus particle assembly. While Gag is capable of specifically packaging the genomic RNA into the particle, this RNA species is unnecessary for particle assembly in vivo. In vitro, nucleic acids profoundly enhance the efficiency of assembly by recombinant Gag proteins, apparently by acting as "scaffolding" in the particle. To address the participation of RNA in retrovirus assembly in vivo, we analyzed murine leukemia virus particles that lack genomic RNA because of a deletion in the packaging signal of the viral RNA. We found that these particles contain cellular mRNA in place of genomic RNA. This result was particularly evident when Gag was expressed by using a Semliki Forest virus-derived vector: under these conditions, the Semliki Forest virus vector-directed mRNA became very abundant in the cells and was readily identified in the retroviral virus-like particles. Furthermore, we found that the retroviral cores were disrupted by treatment with RNase. Taken together, the data strongly suggest that RNA is a structural element in retrovirus particles.

Animals↗

PubMed2000

Treatment of childhood acute myelogenous leukemia with an intensive regimen (AML-87) that individualizes etoposide and cytarabine dosages: short- and long-term effects.

The purpose of this study was to assess the feasibility and efficacy of a treatment regimen for pediatric acute myelogenous leukemia (AML) that uses four rotating drug pairs and adjusts dosages of etoposide and cytarabine to target specific plasma concentrations. Thirty-one girls and 27 boys (median age, 9.7 years) with de novo AML were treated on the protocol. Six cycles of chemotherapy were planned. Cycles 1 to 4 comprised the drug combinations cytarabine plus etoposide, cytarabine plus daunomycin, etoposide plus amsacrine, and etoposide plus azacitidine, respectively. For cycles 5 and 6, the first two combinations were repeated. Dosages were adjusted to achieve plasma concentrations of 1.0 microM +/- 0.1 microM cytarabine and 30 microM +/- 0.3 microM etoposide. Forty-four patients (76%) entered complete remission. Of those, 24 have had relapses; 23 remain alive in first or subsequent remission. The 5-year event-free survival (EFS) estimate was 31.0% +/- 5.9%; the 5-year survival estimate was 41.4% +/- 6.3%. Six patients (10%) died of the toxic effects of therapy. Severe neutropenia occurred in all cycles. Long-term complications of therapy included hepatitis C, cardiac insufficiency, and hearing loss. Adjustment of cytarabine and etoposide dosage was feasible for achieving targeted plasma drug concentrations; however, the potential clinical efficacy of this approach was offset by substantial acute and long-term toxicity.

Adolescent↗

PubMed1998

Evidence for cooperation between murine leukemia virus Env molecules in mixed oligomers.

A retroviral Env molecule consists of a surface glycoprotein (SU) complexed with a transmembrane protein (TM). In turn, these complexes are grouped into oligomers on the surfaces of the cell and of the virion. In the case of murine leukemia viruses (MuLVs), the SU moieties are polymorphic, with SU proteins of different viral isolates directed towards different cell surface receptors. During maturation of the released virus particle, the 16 C-terminal residues of TM (the R peptide or p2E) are removed from the protein by the viral protease; this cleavage is believed to activate the membrane-fusing potential of MuLV Env. We have tested the possibility that different MuLV Env proteins in the same cell can interact with each other, both physically and functionally, in mixed oligomers. We found that coexpressed Env molecules can be precipitated out of cell lysates by antiserum which reacts with only one of them. Furthermore, they can evidently cooperate with each other: if one Env species lacks the R peptide, then it can apparently induce fusion if the SU protein of the other Env species encounters its cognate receptor on the surface of another cell. This functional interaction between different Env molecules has a number of implications with respect to the mechanism of induction of membrane fusion, for the genetic analysis of Env function, and for the design of targeted retroviral vectors for gene therapy.

3T3 Cells↗

PubMed1997

Delayed resection in the management of non-orbital rhabdomyosarcoma of the head and neck in childhood.

This retrospective study was undertaken to evaluate the effect of delayed resection on outcome of head and neck rms in a single institution which has experience in cranial base surgery. Since 1988, patients with primary non-orbital rms of the head and neck following treatment at the Children's Hospital of Pittsburgh, were evaluated by the Department of Otolaryngology, Eye and Ear Hospital at the University of Pittsburgh Medical Center either at the time of presentation or when response to chemotherapy and/or radiation therapy was thought to have been optimized for the possibility of definitive surgery. Medical records of patients who did or did not have delayed surgery were reviewed and compared with respect to demographics, tumor stage, response to therapy, survival, and cosmetic results. Of 16 children diagnosed with non-orbital head and neck rms from 1988-1994 and treated with chemotherapy according to IRS II-IV, 3 had group I or II disease following extensive surgery at diagnosis. Thirteen had group III or IV disease. Of these, 6 patients had delayed resection and 7 did not. Delayed resection was undertaken 3-12 months (median, 4 months) from diagnosis in 4 children who had a partial response (PR) and 2 children who had stable disease (SD) with chemotherapy and/or radiation. Delayed resection converted all children to complete responses (CR), including one child with clinical SD and one with PR who were found to have no viable tumor at surgery. The overall percentages of CRs for patients with group III or IV disease (documented any time post-diagnosis) were at least as good for patients who had undergone delayed surgery as for those who had not (100% vs. 71%, p = .465). Median survivals for patients with advanced disease were 3 1/2 years and 2 years, respectively (p = .2801). Cosmetic and functional problems attributable to surgery were not severe but included facial asymmetry (n = 4), trismus (n = 1), cranial nerve deficits (n = 1), and abnormal dentition (n = 1). In locally extensive head and neck rms, cranial base surgery should be considered after initial cytoreductive therapy, since it may contribute to achievement of CR and to survival with acceptable morbidity.

Child↗

PubMed1997

The use of counterflow centrifugal elutriation for the depletion of T cells from unrelated donor bone marrow.

Transplantation of marrow from unrelated donors is associated with an increased incidence and severity of graft-versus-host disease (GVHD). In an attempt to minimize GVHD without compromising engraftment, unrelated donor marrow was depleted of lymphocytes by counterflow centrifugal elutriation (CCE), and a fixed dose of 0.5 x 10(6) CD3+ T cells/kg, as measured in real time by flow cytometry, was added back to the graft. Patients received cyclosporine (CYA) and corticosteroids for GVHD prophylaxis and to facilitate engraftment. In the first cohort (study I), 7 patients received busulfan (16 mg/kg) and cyclophosphamide (120 mg/kg) (CY) and one patient received CY (200 mg/kg) + 1260 cGy fractionated TBI. Of 6 who were evaluable for both engraftment and rejection, 4 rejected their graft. The study was terminated, and the protocol was modified (study II) by the addition of antithymocyte globulin (ATG) to the pre-BMT and post-BMT therapy. Twelve patients received CY + TBI as above plus ATG given pre-BMT and post-BMT. Ten of twelve who received ATG engrafted. Twelve patients from studies I and II were evaluable for acute GVHD. Two developed grade I acute GVHD. Two patients developed grade II acute GVHD, 2 patients developed grade III GVHD, and 1 patient developed grade IV acute GVHD. Two of three cases of acute GVHD (> grade II) occurred later than day 100 after BMT concomitant with reduction of immunosuppressive therapy. The rate of engraftment was significantly higher in study II (p = .054). In numbers of CD34+ cells infused, numbers of CFU-GM infused, and numbers of nucleated cells did not correlate with engraftment. We conclude that (a) in contrast to the results seen in recipients of marrow from HLA-matched sibling donors, the depletion of unrelated donor marrow of all but 0.5 x 10(6) CD34+ cells/kg together with CYA + corticosteroids was not sufficient to facilitate engraftment. The use of a more immunosuppressive regimen containing TBI and ATG appeared to improve engraftment. (b) The reduction of the graft T cell dose to 0.5 x 10(6) CD34+ cells/kg resulted in a higher incidence of acute GVHD than that seen in recipients of marrow from genotypically identical donors whose marrow was similarly processed.

Adolescent↗

PubMed1997

A pilot study of continuous infusion Ara-C in combination with rhG-CSF in relapsed childhood acute myeloid leukemia.

Relapse in acute myeloid leukemia (AML) following intensive chemotherapy bears a bad prognosis. We treated 18 children with relapsed AML on two separate protocols that included continuous infusion (CI) of cytosine arabinoside (ara-C) (total dose 4gr-6gr/m2) over 96-120 hours. In an attempt to increase the fraction of blasts in S-phase and render them more sensitive to cell-cycle specific agents such as ara-C, 10 patients received 5mcg/kg rhG-CSF twice daily beginning 48 hours before and continuing through the duration of the CI ara-C (POG #9192 study). The percentage of cells is S phase before and after G-CSF administration was determined. In a second group of patients (n = 8) who received ara-C alone, endogenous concentrations of G-CSF and serial blood counts were measured (St Jude's R4 study). The rationale of the St Jude's R4 was to optimize the schedule of the second course of ara-C at a time when the patient's endogenous G-CSF concentration was increased and thus maximize the percent of cells captured in S phase. Four out of 8 patients receiving CI ara-C alone and 4 out of 10 patients receiving CI ara-C with rhG-CSF achieved a complete remission (CR) after 1 cycle of therapy. Four patients in CR underwent marrow transplantation (2 allogeneic and 2 autologous). Cell cycle analysis of blast cells cultured in vitro with or without G-CSF showed a two fold increase in the percentage of cells in S phase (P = 0.03) whereas cells obtained from patients before and after G-CSF administration showed no difference in cell cycling. Correlation between G-CSF concentrations and ANC showed a negative association indicating that the regulatory mechanisms for G-CSF production remained intact. In our relatively small series, CI ara-C achieved a CR rate of 44% with rhG-CSF having no effect on the remission rate. Although in vitro rhG-CSF increased the percentage of blasts in S phase significantly, in vivo effects were not observed. Larger studies with combinations of different hematopoietic growth factors and cell-cycle active drugs are needed to evaluate the role of these cytokines in the therapy of recurrent AML.

Acute Disease↗

PubMed1997

Suppression of retrovirial replication: inactivation of murine leukemia virus by compounds reacting with the zinc finger in the viral nucleocapsid protein.

All retroviral nucleocapsid (NC) proteins, except those of spumaretroviruses, contain one or two zinc fingers, consisting of the sequence C-X2-C-X4-H-X4-C. Rice et al. (Science 270:1194-1197, 1995) have described a series of compounds which inactivate HIV-1 particles and oxidize the sulfur atoms in the NC zinc finger. We have characterized the effects of three such compounds on Moloney murine leukemia virus (MuLV). We find that, as with HIV-1, the compounds inactivate cell-free MuLV particles and induce disulfide cross-linking of NC in these particles. In contrast, the compounds have no effect on the infectivity of human foamy virus, a spumaretrovirus lacking zinc fingers in its NC protein. The resistance of foamy virus supports the hypothesis that the zinc fingers are the targets for inactivation of MuLV and HIV-1 by the compounds. The absolute conservation of the zinc finger motif among oncoretroviruses and lentiviruses, and the lethality of all known mutations altering the zinc-binding residues, suggest that only the normal, wild-type structure can efficiently perform all of its functions. This possibility would make the zinc finger an ideal target for antiretroviral agents.

Amino Acid Sequence↗

PubMed1996

Pain palliation with strontium-89 in children with metastatic disease.

We report two cases of children with metastatic bone disease who received strontium-89 intravenously. An 11-year-old boy with stage IV neuroblastoma received 50 microCi/kg of strontium-89. He had a good response, and his pain abated to the point that he could be taken off IV Dilaudid and was discharged from the hospital. A 7-year-old girl with the diagnosis of squamous cell carcinoma of the lung disclosed minimal increased uptake on a bone scan. Following the strontium-89 therapy, she did not have any significant improvement in pain, probably due to the minimal osteoblastic activity evidenced by the minimal abnormalities on the bone scan. Until this report there has been no reported case of using strontium-89 in the treatment of children with metastatic disease.

Analgesics, Opioid↗

PubMed1996

Inactivation of murine leukemia virus by compounds that react with the zinc finger in the viral nucleocapsid protein.

All retroviral nucleocapsid (NC) proteins, except those of spumaretroviruses, contain one or two copies of the conserved sequence motif C-X2-C-X4-H-X4-C. The conserved cysteine and histidine residues coordinate a zinc ion in each such motif. Rice et al. (W. G. Rice, J. G. Supko, L. Malspeis, R. W. Buckheit, Jr., D. Clanton, M. Bu, L. Graham, C. A. Schaeffer, J. A. Turpin, J. Domagala, R. Gogliotti, J. P. Bader, S. M. Halliday, L. Coren, R. C. Sowder II, L. 0. Arthur, and L. E. Henderson, Science 270:1194-1197, 1995) have described a series of compounds which inactivate human immunodeficiency virus type 1 (HIV-1) particles and oxidize the cysteine thiolates in the NC zinc finger. We have characterized the effects of three such compounds on Moloney murine leukemia virus (MuLV). We find that, as with HIV-1, the compounds inactivate cell-free MuLV particles and induce disulfide cross-linking of NC in these particles. The killed MuLV particles were found to be incapable of synthesizing full-length viral DNA upon infection of a new host cell. When MuLV particles are synthesized in the presence of one of these compounds, the normal maturational cleavage of the Gag polyprotein does not occur. The compounds have no effect on the infectivity of human foamy virus, a spumaretrovirus lacking zinc fingers in its NC protein. The resistance of foamy virus supports the hypothesis that the zinc fingers are the targets for inactivation of MuLV and HIV- I by the compounds. The absolute conservation of the zinc finger motif among oncoretroviruses and lentiviruses and the lethality of all known mutations altering the zinc-binding residues suggest that only the normal, wild-type structure can efficiently perform all of its functions. This possibility would make the zinc finger an ideal target for antiretroviral agents.

3T3 Cells↗

PubMed1995

FK506 (tacrolimus) in the treatment of steroid-resistant acute graft-versus-host disease in children undergoing bone marrow transplantation.

Steroid-resistant graft-versus-host disease (GVHD) is an often lethal complication of bone marrow transplantation (BMT). FK506 (tacrolimus) is a new potent immunosuppressant which has been shown to be superior to conventional immunosuppression in the prevention and treatment of graft rejection in recipients of solid organ transplants. To determine whether FK506 is effective in the treatment of steroid-resistant acute GVHD, 6 children with biopsy-proven severe GVHD were studied. FK506 was administered as intravenous or oral therapy and the dose was adjusted to achieve serum levels between 0.5 and 1.0 microgram/ml by ELISA. Steroid doses were tapered based on clinical grading in each organ. Within 1-2 days, improvement occurred in skin and gut in all patients, and in liver in 3 patients. Toxicity attributable to FK506 was similar to that described in solid organ transplant patients and included neurotoxicity, nephrotoxicity and gastrointestinal effects. While FK506 is effective in the treatment of steroid-resistant acute GVHD, toxicity may limit its use. Further studies evaluating FK506 as GVHD prophylaxis and treatment of less advanced GVHD are needed.

Adolescent↗

PubMed1994

Pulmonary insufficiency complicating therapy with high dose cytosine arabinoside in five pediatric patients with relapsed acute myelogenous leukemia.

BACKGROUND: The occurrence of fatal or nearly fatal pulmonary insufficiency in 5 of 22 pediatric patients with relapsed acute myelogenous leukemia (AML) treated with high dose cytosine arabinoside (Ara-C) at St. Jude Children's Research Hospital, Memphis, Tennessee, and institutions affiliated with the Pediatric Oncology Group (POG) is reported. METHODS: Cytosine arabinoside (1.0-1.5 g/m2/day) was given as a 5-day continuous infusion to all patients. Four patients with persistent leukemia received a second 3- or 5-day course. The POG protocol included the administration of granulocyte colony stimulating factor for the priming of myeloblasts. Diagnostic criteria for pulmonary insufficiency included noncardiogenic pulmonary edema with exclusion of underlying cardiorespiratory, infectious, or metabolic conditions. Autopsy material also was reviewed. RESULTS: Of the 22 patients 5 died (23%), including 2 who received a second course of Ara-C as a result of pulmonary insufficiency that developed at a median of 8 days (range, 3-38 days) after the first course. Three patients died despite intubation and pressor support. Two patients were managed successfully with colloids, diuresis, and oxygen by face mask; remission was achieved in both. The postmortem examination of one patient disclosed airless lungs, profound pulmonary edema, and subpleural nodules, but no evidence of leukemia. CONCLUSION: Pulmonary insufficiency from high dose Ara-C varies in severity and may be fatal. It may occur during or after treatment. Awareness of this potential complication, careful attention to fluid status, and aggressive supportive care may optimize outcome.

Acute Disease↗

PubMed1994

Gene marking and autologous bone marrow transplantation.

If residual cancer cells in harvested bone marrow could be marked and subsequently detected in patients at relapse, valuable information would be obtained about the source of recurrent disease after autologous marrow transplantation. If normal progenitor cells were also marked, the study would provide useful data on the susceptibility of these human cells to gene transfer and their capacity to express newly introduced genes. We transferred the neomycin-resistance gene (NeoR) into bone marrow cells harvested from 20 children with acute myeloid leukemia (n = 12) or neuroblastoma (n = 8) in clinical and cytological remission using a retrovirus vector. The cells were then returned to the patients as part of an autologous bone marrow transplantation protocol. Two AML and three neuroblastoma patients have relapsed. In all, the resurgent cells contained the NeoR marker by analysis with PCR. These results prove that so-called remission marrow can contribute to relapse in patients who receive autologous transplants. The gene marking technique is now being used to evaluate techniques of pretransplant purging.

Bone Marrow Transplantation↗

PubMed1994

Acute myeloblastic leukemia with a pericentric inversion of chromosome 6 in a child with Down syndrome.

Cytogenetic analysis of bone marrow cells demonstrated a pericentric inversion of chromosome 6 when acute myeloblastic leukemia (AML) was diagnosed in a 15-month-old child with Down syndrome. The abnormal clone with inv(6)(p24q16) was associated with FAB-M1 AML and disappeared when a complete remission was achieved. This is a unique cytogenetic abnormality for FAB-M1 AML in a child with Down syndrome and, to our knowledge, is only the second case of a pericentric inversion of chromosome 6 reported in AML.

Chromosome Inversion↗

PubMed1994

Function of the cytoplasmic domain of a retroviral transmembrane protein: p15E-p2E cleavage activates the membrane fusion capability of the murine leukemia virus Env protein.

In the murine leukemia viruses (MuLVs), the Env complex is initially cleaved by a cellular protease into gp70SU and pre15ETM. After the virus particle is released from the cell, the C-terminal 16 residues are removed from the cytoplasmic domain of pre15E by the viral protease, yielding the mature p15ETM and p2E. We have investigated the function of this cleavage by generating a Moloney MuLV mutant, termed p2E-, in which the Env coding region terminates at the cleavage site. This mutant synthesizes only the truncated, mature form of TM rather than its extended precursor. When cells expressing this truncated Env protein are cocultivated with NIH 3T3 cells, they induce rapid cell-cell fusion. Thus, the truncated form, which is normally found in virions but not in virus-producing cells, is capable of causing membrane fusion. We conclude that the 16-residue p2E tail inhibits this activity of Env until the virus has left the cell. p2E- virions were found to be infectious, though with a lower specific infectivity than that of the wild type, showing that p2E does not play an essential role in the process of infection. Fusion was also observed with a chimeric p2E- virus in which gp70SU and nearly all of p15ETM are derived from amphotropic, rather than Moloney, MuLV. In a second mutant, an amino acid at the cleavage site was changed. The pre15E protein in this mutant is not cleaved. While the mutant Env complex is incorporated into virions, these particles have a very low specific infectivity. This result suggests that the cleavage event is essential for infectivity, in agreement with the idea that removal of p2E activates the membrane fusion capability of the Env complex.

Amino Acid Sequence↗

PubMed1994

Evidence that a central domain of nucleocapsid protein is required for RNA packaging in murine leukemia virus.

We have analyzed RNA packaging by a series of mutants altered in the nucleocapsid (NC) protein of Moloney murine leukemia virus (Mo-MuLV). We found that mutants lacking residues 8 through 11 or 44 through 60 of NC package Mo-MuLV RNA with virtually the same efficiency as wild-type Mo-MuLV. In contrast, point mutants altered at the conserved cysteines in the cysteine array (residues 26 and 29) and a mutant lacking residues 16 through 23 packaged Mo-MuLV RNA with approximately 1% of the efficiency of wild-type Mo-MuLV. The deficiency in packaged RNA was observed not only in Northern (RNA) analysis but also in an RNA-PCR assay, which would detect degraded as well as intact RNA. One of the cysteine array mutants was also shown to be defective with respect to encapsidation of hygromycin phosphotransferase mRNA containing a Mo-MuLV packaging signal. We suggest that a central region of NC, consisting of the cysteine array and flanking basic residues, is required for RNA packaging in Mo-MuLV.

Amino Acid Sequence↗

PubMed1994

Molecular evidence for minimal residual bone marrow disease in children with 'isolated' extra-medullary relapse of T-cell acute lymphoblastic leukemia.

Central nervous system (CNS) relapse confers a poor prognosis in children with acute lymphoblastic leukemia (ALL). It is uncertain whether morphologically undetectable leukemia is present in the bone marrow at the time of CNS relapse, or whether the CNS acts as a 'sanctuary' site to allow reseeding of the marrow at a later time. We examined DNA from bone marrow samples from six patients with T-cell ALL with isolated CNS relapse using sensitive polymerase chain reaction (PCR) assays to detect minimal residual disease. One of these PCR assays was based on amplification of leukemia-specific TCR-delta gene rearrangements, while the other assay relied upon detection of the c-tal deletion. In four patients, where bone marrow samples were taken at the time of CNS relapse, residual disease was detectable in every sample at a level below morphological detection. In addition, three patients had residual disease detected in their subsequent bone marrow when CNS disease was not evident. Our findings, although preliminary, suggest that relapse of leukemia in the CNS reflects resurgence of the disease in the bone marrow that is first detected clinically in the CNS. The concomitant molecular detection of bone marrow leukemia at time of 'isolated' CNS relapse in children with T-cell ALL explains subsequent bone marrow relapse in some of these children, and argues for intensive systemic therapy of these patients.

Adolescent↗

PubMed1994

Clinical applications of the hematopoietic growth factors.

Hematopoiesis occurs in a microenvironment containing a variety of growth factors. Normal hematopoietic cells require these growth factors for viability, proliferation, and differentiation. The purification and molecular cloning of human growth factors has permitted a detailed analysis of hematopoiesis. Furthermore, the commercial production of growth factors resulted in their widespread introduction into clinical practice to limit chemotherapy-induced neutropenia. Recently, clinical trials of growth factors have expanded to test their direct anticancer effects, to improve the efficacy of chemotherapy by stimulating cell division, and to use them as biological modulators to inhibit tumor growth. This review focuses on a few clinical uses of recombinant growth factors.

Animals↗