Phenotypic variation in the response to the human immunodeficiency virus among derivatives of the CEM T and WIL-2 B cell lines.

Derivatives of the CEM T and WIL-2 B cell lines showed striking diversity in their responses to the HTLV-IIIB strain of the human immunodeficiency virus (HIV). Several stable phenotypic patterns could be defined, based on whether cells were permissive (P+, P-) for virus production, were sensitive or insensitive to cytopathic effects after infection by free virus (C+, C-), and whether they underwent fusion on contact with virus-infected cells (F+, F-). Although expression of CD4 was essential for infection by HTLV-IIIB, very low levels were sufficient for productive infection of WIL-2 derivatives. Conversely, some CEM T cell lines that expressed ample CD4, and which were able to bind virus gp120 and undergo fusion, did not support productive infection by free virus. One nonpermissive, CD4+ derivative of CEM could bind gp120 but failed to undergo fusion, suggesting an alteration in some membrane protein other than CD4 that is essential for virus entry and HIV-induced cell fusion. The AA2 derivative of the WIL-2 cell line is also described, which is remarkably permissive for HIV replication and exquisitely sensitive to virus cytopathic effect. The panel of related cell lines with different host-virus phenotypes could be useful for more precisely defining steps in the infectious cycle of HIV, and for identifying host cell genes and gene products that determine the outcome of HIV infection.

IgG antibody response to polyethylene glycol-modified adenosine deaminase in patients with adenosine deaminase deficiency.

Polyethylene glycol (PEG)-modified bovine adenosine deaminase (ADA) is used for replacement therapy of severe combined immunodeficiency disease due to inherited ADA deficiency. We monitored IgG anti-ADA antibody in 17 patients treated by intramuscular injections of PEG-ADA for 1 to greater than 5.5 yr. ELISA-detectable anti-ADA IgG appeared in 10 patients, usually between the third and eighth months of treatment. Anti-ADA levels did not correlate with trough plasma ADA activity, which averaged 1.8-5 times normal blood (erythrocyte) ADA activity, depending on dose (15-60 U/kg per wk). ELISA-detectable anti-ADA antibodies were directed primarily at bovine-specific peptide (rather than PEG-containing) epitopes. Enhanced enzyme clearance, mediated by antibody that directly inhibited native and PEG-modified bovine ADA, and native, but not PEG-modified human ADA, occurred in two patients. In one, tolerance was induced; in the second, twice weekly injections of PEG-ADA compensated for accelerated clearance. We speculate that inhibitory antibodies recognize conserved, relatively PEG-free epitope(s) encompassing the active site, and that in human, but not bovine, ADA a PEG-attachment site "shields" the active site from immune recognition. We conclude that PEG-modification largely prevents the development of high affinity, or high levels of clearing antibodies to bovine ADA, and that PEG-modified human ADA should be further investigated as a possible treatment for ADA deficiency.

Paradoxical expression of adenosine deaminase in T cells cultured from a patient with adenosine deaminase deficiency and combine immunodeficiency.

T lymphocytes cultured from a patient (T.D.) with adenosine deaminase (ADA) deficiency expressed ADA activity in the normal range, inconsistent with her severe immunodeficiency, metabolic abnormalities, and with the absence of ADA activity in her B lymphocytes and other nucleated hematopoietic cells. ADA from T.D. T cells had normal Km, heat stability, and sensitivity to ADA inhibitors. Examination of HLA phenotype and polymorphic DNA loci indicated that T.D. was neither chimeric nor a genetic mosaic. Amplified and subcloned ADA cDNA from ADA+ T.D. T cells was shown by allele-specific oligonucleotide hybridization to possess the same mutations (Arg101----Trp, Arg211----His) previously found in the ADA-T.D. B cell line GM 2606 (Akeson, A. L., D. A. Wiginton, M. R. Dusing, J. C. States, and J. J. Hutton. 1988. J. Biol. Chem. 263:16291-16296). Our findings suggest that one of these mutant alleles can be expressed selectively in IL-2-dependent T cells as stable, active enzyme. Cultured T cells from other patients with the Arg211----His mutation did not express significant ADA activity, while some B cell lines from a patient with an Arg101----Gln mutation have been found to express normal ADA activity. We speculate that Arg101 may be at a site that determines degradation of ADA by a protease that is under negative control by IL-2 in T cells, and is variably expressed in B cells. Il-2 might increase ADA expression in T cells of patients who possess mutations of Arg101.

High-dose chemotherapy with autologous stem-cell rescue in patients with recurrent and high-risk pediatric brain tumors.

PURPOSE: We treated 49 patients with recurrent or poor-prognosis CNS malignancies with high-dose chemotherapy regimens followed by autologous marrow rescue with or without peripheral-blood stem-cell augmentation to determine the toxicity of and event-free survival after these regimens. PATIENTS AND METHODS: Nineteen patients had medulloblastomas, 12 had glial tumors, seven had pineoblastomas, five had ependymomas, three had primitive neuroectodermal tumors, two had germ cell tumors, and one had fibrosarcoma. Thirty-seven received chemotherapy with cyclophosphamide 1.5 g/m2 daily x 4 and melphalan 25 to 60 mg/m2 daily x 3. Nine received busulfan 37.5 mg/m2 every 6 hours x 16 and melphalan 180 mg/m2 (n = 7) or 140 mg/m2 (n = 2). Three received carboplatin 700 mg/m2/d on days -7, -5, and -3 and etoposide 500 mg/m2/d on days -6, -4, and -2. All patients received standard supportive care. RESULTS: Eighteen of 49 patients survive event-free 22+ to 55+ months (median, 33+) after transplantation, including nine of 16 treated before recurrence and nine of 33 treated after recurrence. There was one transplant-related death from pulmonary aspergillosis. Of five patients assessable for disease response, one had a partial remission (2 months), one has had stable disease (55+ months), and three showed progression 2, 5, and 8 months after transplantation. CONCLUSION: The toxicity of these regimens was tolerable. Certain patients with high-risk CNS malignancies may benefit from such a treatment approach. Subsequent trials should attempt to determine which patients are most likely to benefit from high-dose chemotherapy with autologous stem-cell rescue.

Efficacy and toxicity of 9-beta-D-arabinofuranosylguanine (araG) as an agent to purge malignant T cells from murine bone marrow: application to an in vivo T-leukemia model.

9-beta-D-Arabinofuranosylguanine (araG), an analog of deoxyguanosine which is not degraded by purine nucleoside phosphorylase, has been previously shown in in vitro studies by our laboratory to be effective in purging malignant T cells from human bone marrow (1). We now describe studies in a murine model of T-cell acute lymphoblastic leukemia (ALL) in which we tested whether bone marrow, contaminated with malignant T cells and purged ex vivo with araG, could reconstitute both the lymphoid and myeloerythroid lineages in the absence of leukemic relapse. The model utilized 6C3HED tumor cells, derived from a Thy 1.2+ malignant murine T-cell line, which were shown to cause lethal leukemia in C3H/HeN mice. Intravenous injection of 10(6) 6C3HED cells resulted in 100% mortality within 18 days, with autopsy revealing tumor infiltration of multiple organs. 100% of non-leukemia bearing lethally irradiated C3H/HeN mice transplanted with syngeneic bone marrow, treated ex vivo with 100 microM of araG for 18 hours, survived > 365 days post-transplant with full lymphohematopoietic reconstitution. Evidence of araG's ability to purge bone marrow of malignant tumor cells without causing significant toxicity to normal marrow derived hematopoietic progenitor cells was documented in experiments in which 75% of lethally irradiated mice transplanted with syngeneic bone marrow, contaminated with 6C3HED tumor cells and treated ex vivo with 100 microM araG for 18 hours, survived for > 250 to > 400 days. Death in 25% of mice was secondary to infection which developed before marrow reconstitution occurred. Reconstitution of the lymphoid, myeloid, and erythroid lineages with donor cells in surviving mice was documented. The data presented indicate that araG may effectively purge bone marrow of malignant T cells without irreversible toxicity to hematopoietic stem cells. This purging regimen is recommended for consideration for clinical trials in patients with T-cell malignancies undergoing autologous bone marrow transplantation and may also be a viable option for T-cell depletion as a strategy to prevent graft-versus-host disease.

Treatment of platelet alloimmunization with intravenous immunoglobulin. Two case reports and review of the literature.

Two pediatric patients with severe aplastic anemia, elevated antiplatelet antibody levels, refractoriness to human lymphocyte antigen-matched platelet transfusions, and sustained bleeding problems were treated with intravenous immunoglobulin (IVIG), pH 4.25, for three to over nine months. Improved responses to platelet infusions and improved hemostasis were demonstrated in both patients. A review of the published literature analyzing the role of IVIG in the treatment of platelet alloimmunization is presented.

Efficacy of intravenous gamma globulin in autoimmune-mediated pediatric blood dyscrasias.

Sixteen pediatric patients diagnosed with a variety of autoimmune-mediated hematocytopenias were treated with one to 50 courses of intravenous gamma globulin (IVIG), pH 4.25, over the course of one to 30 months. Thirteen patients had immune thrombocytopenic purpura (ITP), two had autoimmune neutropenia, and one had autoimmune hemolytic anemia. In one patient, chronic ITP was associated with systemic lupus erythematosis, and in a second patient, acute ITP was the presenting manifestation of infection with human immunodeficiency virus. Initial therapy consisted of 400 mg/kg/dose daily for five days for the first seven patients treated, and 1,000 mg/kg/dose daily for two days for the remaining nine patients. In 15 of 16 patients, there was a response to IVIG therapy. In nine of 16 patients, maintenance IVIG therapy for two to more than 30 months was required. Minimal toxicity was experienced in four of 210 separate infusions. Data are presented to support the use of IVIG in the management of childhood autoimmune disorders.

Pharmacologic purging of malignant T cells from human bone marrow using 9-beta-D-arabinofuranosylguanine.

Arabinosylguanine (araG) is a nucleoside analog that is rapidly converted by cells of the T lymphoid lineage to its corresponding arabinosylguanine nucleotide triphosphate, resulting in inhibition of DNA synthesis and selective in vitro toxicity to T lymphoblastoid cell lines as well as to freshly isolated leukemia cells from patients with T cell acute lymphoblastic leukemia. In this report, we demonstrate that araG is an effective agent to use for chemoseparation of malignant T lymphoblasts from human bone marrow. When freshly isolated human T leukemia cells or T lymphoblastoid cells were treated with 100 microM araG for 18 hr, up to 6 logs of clonogenic T cells could be eliminated without appreciable toxicity to the normal myeloid, erythroid, and megakaryocytoid clonal progenitor cells. We discuss the use of this agent in ex vivo elimination of residual malignant T cells from marrow of patients requiring myeloablative chemotherapy with autologous bone marrow rescue.

Detection of HIV-1 neutralizing antibodies by a simple, rapid, colorimetric assay.

A rapid, simple, reproducible and semi-quantitative assay to measure neutralizing antibodies has been developed. It employs a unique cell line which is exquisitively sensitive to infection with all HIV isolates tested. The assay is amenable to microtiter formulation as well as analysis by automation.

Toxicity, pharmacology and feasibility of administration of PEG-L-asparaginase as consolidation therapy in patients undergoing bone marrow transplantation for acute lymphoblastic leukemia.

We attempted to administer PEG-L-asparaginase (PEG-L-A) following hematologic recovery to 38 patients undergoing autologous or allogeneic marrow transplantation for acute lymphoblastic leukemia (ALL). Twenty-four patients (12 of 22 receiving allogeneic and 12 of 16 receiving autologous transplants) received between one and 12 doses of PEG-L-A, including nine who completed the planned 12 doses of therapy. The toxicities encountered were similar to those observed in non-transplanted patients undergoing therapy with PEG-L-A and included allergic reactions, pancreatitis, weight loss, hypoalbuminemia, and low levels of anti-thrombin III. Of the 24 who received the drug, eight remain in remission. Of 12 patients in second remission at the time of transplantation who received PEG-L-A, five of seven who received allogeneic and two of five who received autologous transplants remain in remission, 16+ to 46+ months from transplant. While PEG-L-A could be administered to most of the patients undergoing marrow transplantation for ALL, most patients either relapsed while receiving the drug or developed toxicities which resulted in abbreviated courses. At this time, we cannot recommend PEG-L-A as single agent, post-BMT chemotherapy.

Constitutional de novo t(1;22)(p22;q11.2) and ependymoma.

There is a body of evidence suggesting the presence of a tumor suppressor gene on chromosome 22 which plays a role in the pathogenesis of ependymomas. We report a patient with a de novo constitutional t(1;22)(p22;q11.2) who developed a malignant ependymoma at age 5. The patient is otherwise phenotypically normal. By fluorescence in situ hybridization (FISH) analysis, the chromosome 22 breakpoint has been localized to the region between the DiGeorge locus and BCR. Since NF2 and EWS are both distal to BCR, the are presumable not involved in this rearrangement. This patient may offer a unique opportunity to identify the chromosome 22 ependymoma tumor suppressor gene by cloning the translocation breakpoint.

9p- in a girl with acute lymphocytic leukemia and sickle cell disease.

A 4 year old girl with sickle cell disease developed acute lymphocytic leukemia null cell type. The bone marrow karyotype was 46,XX,del(9)(p13). This girl is among the few patients with acute lymphocytic leukemia and abnormalities of #9 to have an isolated 9p-.

Treatment of trilateral retinoblastoma with vincristine and cyclophosphamide.

Current therapy for patients with trilateral retinoblastoma, consisting primarily of surgical intervention and radiotherapy, has resulted in no long-term survivors. The use of adjuvant chemotherapy has not improved this outcome. After observing a tumor response to cyclophosphamide in a patient with suprasellar retinoblastoma, we treated a subsequent patient with trilateral retinoblastoma with both vincristine and cyclophosphamide. Objective tumor regression resulted. Although the tumor ultimately progressed in both patients, these findings suggest that vincristine and cyclophosphamide are active in patients with intracranial (trilateral) retinoblastoma.

Pediatric palliative care.

This article presents a model of integrated palliative care for children with life-limiting illnesses, with emphasis on collaboration of care over time among family, primary care providers, and several other groups of providers. Some of the unique aspects of caring for children related to normal developmental changes and the family unit are considered. Issues related to pain and to specific diseases are also reviewed.

Basis for resistance to 3-deazaaristeromycin, an inhibitor of S-adenosylhomocysteine hydrolase, in human B-lymphoblasts.

Clones resistant to 3-deazaaristeromycin, a potent inhibitor of S-adenosylhomocysteine hydrolase, were selected from a nucleoside kinase-deficient derivative of the WIL-2 human B-lymphoblastoid cell line. The resistant clones took up 3-deazaaristeromycin and showed no alteration in the level of S-adenosylhomocysteine hydrolase activity or in the sensitivity of the enzyme to inhibition by 3-deazaaristeromycin. However, they displayed markedly elevated S-adenosylmethionine content during growth in 3-deazaaristeromycin and, following prolonged selection, enhanced export of S-adenosylhomocysteine. As a result they maintained a high ratio of S-adenosylmethionine to S-adenosylhomocysteine and thus were resistant to the inhibition of S-adenosylmethionine turnover and transmethylation caused by 3-deazaaristeromycin. Expanded S-adenosylmethionine pools declined over several weeks of nonselective growth, suggesting a metabolic adaptation rather than a mutational mechanism. No alterations in S-adenosylmethionine synthetase activity were found in the 3-deazaaristeromycin-resistant clones. S-Adenosylhomocysteine export appeared to be carrier-mediated and largely unidirectional. The resistant clones showed a 5-fold increased rate of S-adenosylhomocysteine export compared with parental cells, but a similar Km for intracellular S-adenosylhomocysteine, estimated to be approximately 1 mM. Our results highlight the opposing effects of S-adenosylmethionine and S-adenosylhomocysteine on transmethylation and suggest that the ability to elevate S-adenosylmethionine pools and to export S-adenosylhomocysteine may provide for homeostatic control of transmethylation in lymphoid cells when S-adenosylhomocysteine hydrolase activity is limited.

Enzyme replacement therapy with polyethylene glycol-adenosine deaminase in adenosine deaminase deficiency: overview and case reports of three patients, including two now receiving gene therapy.

During the past 6 y, 29 adenosine deaminase (ADA)-deficient patients with combined immunodeficiency have been treated with polyethylene glycol (PEG)-modified bovine ADA (PEG-ADA). We have monitored plasma ADA activity, metabolic effects of treatment, and the evolution of antibody to PEG-ADA in these patients, in collaboration with immunologists and clinicians in North America, Europe, and Australia, who have monitored immune function and clinical response to treatment. This article summarizes the current status of PEG-ADA therapy and provides recommendations for its use. Recovery of specific immune function during treatment with PEG-ADA is illustrated for three patients, who represent early, delayed, these patients have entered a trial of gene therapy, but continue to receive enzyme replacement.

epsilon-Aminocaproic acid-associated myopathy in a child.

PURPOSE: A 12-year-old girl developed severe autoimmune thrombocytopenia after a bone marrow transplant for acute lymphoblastic leukemia. RESULTS: Although epsilon-aminocaproic acid helped to control her bleeding, it eventually caused a rare myopathy previously undescribed in a pediatric patient. CONCLUSION: The myopathy resolved when the drug was discontinued and a different antifibrinolytic agent was used.

Health insurance access to young adult survivors of childhood cancer in North Carolina.

Historically, there has been evidence to support the hypothesis that survivors of childhood cancer have been discriminated against in the private health insurance market in some areas of the United States. Results of previous studies have been inconsistent and have generally focused on a limited number of outcome variables. A retrospective cohort study of young adult survivors of childhood cancer and their siblings was performed to determine the risk of health insurance access problems of childhood cancer survivors in North Carolina. Mailed questionnaires were completed by 182 cancer survivors from three institutions who were diagnosed between 1976 and 1988, and by 101 of their siblings for a response of 62.1%. Using logistic regression in SAS, cancer survivors were found to be more likely to be denied health insurance than their siblings, with an adjusted odds ratio of 15.1. Childhood cancer survivors also had health insurance policies that excluded care for pre-existing medical conditions more often than their siblings (OR = 5.5). In addition, cancer survivors reported problems obtaining health insurance coverage more frequently than their siblings with an adjusted odds ratio of 22.8. In general, survivors of childhood cancer who were diagnosed in North Carolina have had decreased access to health insurance coverage when compared to their siblings of similar age. North Carolina health insurance regulations permit health insurance firms to discriminate against cancer survivors because of their history of illness, often decreasing their access to needed follow-up care.