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Biomedical subjects

Laurence A Boxer

Publications and source records attributed to Laurence A Boxer.

16 recordsLinked to original sources
PubMed2006

The incidence of leukemia and mortality from sepsis in patients with severe congenital neutropenia receiving long-term G-CSF therapy.

In patients with severe congenital neutropenia (SCN), sepsis mortality is reduced by treatment with granulocyte colony-stimulating factor (G-CSF), but myelodsyplastic syndrome and acute myeloid leukemia (MDS/AML) have been reported. We studied 374 patients with SCN and 29 patients with Shwachman-Diamond syndrome (SDS) on long-term G-CSF enrolled in the Severe Chronic Neutropenia International Registry. In SCN, sepsis mortality was stable at 0.9% per year. The hazard of MDS/AML increased significantly over time, from 2.9% per year after 6 years to 8.0% per year after 12 years on G-CSF. After 10 years, the cumulative incidence was 8% for sepsis mortality and 21% for MDS/AML. A subgroup of SCN patients (29%) received more than the median dose of G-CSF (> or = 8 microg/kg/d), but achieved less than the median absolute neutrophil count (ANC) response (ANC < 2.188 x 10(9)/L [2188/microL] at 6-18 months). In these less-responsive patients, the cumulative incidence of adverse events was highest: after 10 years, 40% developed MDS/AML and 14% died of sepsis, compared with 11% and 4%, respectively, of more responsive patients whose ANC was above the median on doses of G-CSF below the median. Risk of MDS/AML may be similar in SDS and SCN. In less-responsive SCN patients, early hematopoietic stem cell transplantation may be a rational option.

Female↗
PubMed2005

Phagocytic signaling molecules in lipid rafts of COS-1 cells transfected with FcgammaRIIA.

COS-1 cells bearing FcgammaRIIA were used as a model to demonstrate co-localization of several enzymes previously shown to regulate neutrophil phagocytosis. In COS-1 cells, phospholipase D (PLD) in the membrane fraction was activated during phagocytosis. PLD was found almost exclusively in lipid rafts, along with RhoA and ARF1. Protein kinase C-delta (PKCdelta) and Raf-1 translocated to lipid rafts. In neutrophils, ceramide levels increase during phagocytosis, indicating that FcgammaRIIA engagement initiates ceramide generation. Applying this model, we transfected COS-1 cells with FcgammaRIIA that had been mutated in the ITAM region, rendering them unable to ingest particles. When the mutant receptors were engaged, ceramide was generated and MAPK was activated normally, thus these processes did not require actual ingestion of particles. These results indicate that signaling proteins for phagocytosis are either constitutively present in, or are recruited to, lipid rafts where they are readily available to activate one another.

Animals↗
PubMed2005

Ceramide 1-phosphate, a mediator of phagocytosis.

The agonist-stimulated metabolism of membrane lipids produces potent second messengers that regulate phagocytosis. We studied whether human ceramide kinase (hCERK) activity and ceramide 1-phosphate formation could lead to enhanced phagocytosis through a mechanism involving modulation of the membrane-structural order parameter. hCERK was stably transfected into COS-1 cells that were stably transfected with the FcgammaRIIA receptor. hCERK-transfected cells displayed a significant increase in phagocytic index in association with increased ceramide kinase activation and translocation to lipid rafts after activation with opsonized erythrocytes. When challenged with opsonized erythrocytes, hCERK-transfected cells increased phagocytosis by 1.5-fold compared with vector control and simultaneously increased ceramide 1-phosphate levels 2-fold compared with vector and unstimulated control cells. Control and hCERK-transfected cells were subjected to cellular fractionation. Utilizing an antibody against hCERK, we observed that CERK translocates during activation from the cytosol to a lipid raft fraction. The plasma membrane-structural order parameter of the transfectants was measured by labeling cells with Laurdan. Cells transfected with hCERK showed a higher liquid crystalline order than control cells with stimulation, conditions that are favorable for the promotion of membrane fusion at the sites of phagocytosis. The change in the structural order parameter of the lipid rafts probably contributes to phagocytosis by promoting phagosome formation.

2-Naphthylamine↗
PubMed2003

Ceramide inhibition of phospholipase D and its relationship to RhoA and ARF1 translocation in GTP gamma S-stimulated polymorphonuclear leukocytes.

Phospholipase D (PLD) regulates the polymorphonuclear leukocyte (PMN) functions of phagocytosis, degranulation, and oxidant production. Ceramide inhibition of PLD suppresses PMN function. In streptolysin O-permeabilized PMNs, PLD was directly activated by guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) stimulation of adenosine diphosphate (ADP)-ribosylation factor (ARF) and Rho, stimulating release of lactoferrin from specific granules of permeabilized PMNs; PLD activation and degranulation were inhibited by C2-ceramide but not dihydro-C2-ceramide. To investigate the mechanism of ceramide's inhibitory effect on PLD, we used a cell-free system to examine PLD activity and translocation from cytosol to plasma membrane of ARF, protein kinase C (PKC)alpha and beta, and RhoA, all of which can activate PLD. GTP gamma S-activated cytosol stimulated PLD activity and translocation of ARF, PKC alpha and beta, and RhoA when recombined with cell membranes. Prior incubation of PMNs with 10 microM C2-ceramide inhibited PLD activity and RhoA translocation, but not ARF1, ARF6, PKC alpha, or PKC beta translocation. However, in intact PMNs stimulated with N-formyl-1-methionyl-1-leucyl-1-phenylalamine (FMLP) or permeabilized PMNs stimulated with GTP gamma S, C2-ceramide did not inhibit RhoA translocation. Exogenous RhoA did not restore ceramide-inhibited PLD activity but bound to membranes despite ceramide treatment. These observations suggest that, although ceramide may affect RhoA in some systems, ceramide inhibits PLD through another mechanism, perhaps related to the ability of ceramide to inhibit phosphatidylinositol-bisphosphate (PIP2) interaction with PLD.

ADP-Ribosylation Factor 1↗
PubMed2003

Severe chronic neutropenia: treatment and follow-up of patients in the Severe Chronic Neutropenia International Registry.

Severe chronic neutropenia (SCN) is defined as an absolute neutrophil (ANC) of less than 0.5 x 10(9)/L, lasting for months or years. Congenital, cyclic, and idiopathic neutropenia are principal categories of SCN. Since 1994, the Severe Chronic Neutropenia International Registry (SCNIR) has collected data to monitor the clinical course, treatments, and disease outcomes for SCN patients. This report summarizes data for 853 patients, almost all treated with daily or alternate-day recombinant human granulocyte colony-stimulating factor (G-CSF or Filgrastim). G-CSF treatment increased the ANC overall from 0.34 x 10(9)/L +/- 0.018 pre-treatment to 3.70 x 10(9)/L +/- 0.18 during the first year of treatment. For most patients, the responses were durable with patients remaining on the same dose of G-CSF for many years. Long-term hematological observations showed stable mean leukocyte and neutrophil counts and gradually increasing hemoglobin levels. Thrombocytopenia developed in 4% of patients. As of January 1, 2000, myelodysplasia (MDS) or acute myelogenous leukemia (AML) has occurred in 35 of 387 patients with congenital neutropenia with a cumulative risk of 13% after 8 years of G-CSF treatment. This event occurred without a predictable relationship to the duration or dose of G-CSF treatment. No patients with cyclic or idiopathic neutropenia developed MDS or AML. Other important adverse events included hepatomegaly, osteoporosis, vasculitis, glomerulonephritis, and deaths in 4 of 14 cases requiring splenectomy. Growth and development and the outcome of pregnancy appeared to be unaffected by G-CSF treatment. These data indicate that congenital, cyclic, and idiopathic neutropenia can be effectively treated with long-term G-CSF. The risk of leukemia, osteoporosis, other potentially adverse events, and pregnancy outcome need to be further evaluated with continuing long-term observations.

Adolescent↗
PubMed2003

Chemotaxis of non-compressed blood polymorphonuclear leukocytes from an adolescent with severe leukocyte adhesion deficiency.

We have defined the defect in a child with severe leukocyte adhesion deficiency-1 (LAD) as resulting from a single amino acid shift in CD18 (from a C to T mutation at position 533) that prevents heterodimerization with the CD11 antigens to produce beta(2) integrins-the first reported patient homozygous for this defect. Although beset by frequent infections, the patient has survived to adolescence despite the lack of these important adhesion molecules. Consistent with his clinical course is the ability of his PMN to respond chemotactically in slide preparations, albeit with difficulty because of their poor purchase on substrate. The operant adhesins are unknown; his polymorphonuclear leukocytes (PMN) remain chemotactically responsive in the presence of antibodies to alphavbeta(3) and beta(1) integrins and to integrin-associated protein (IAP). These findings indicate that not all patients with severe LAD are candidates for early bone marrow transplantation.

Amino Acid Substitution↗
PubMed2003

Cellular and molecular abnormalities in severe congenital neutropenia predisposing to leukemia.

Severe congenital neutropenia (SCN) is a rare hematological disease characterized by a selective decrease in the level of circulating neutrophils in peripheral blood, maturation arrest at the promyelocyte stage of differentiation in the bone marrow, recurrent severe infections, and evolution to acute myelogenous leukemia (AML). Cellular and molecular studies of 12 SCN patients, including 5 patients that evolved to develop AML, revealed impaired proliferative characteristics and accelerated apoptosis of bone marrow progenitor cells in SCN compared with 11 healthy controls as demonstrated by flow cytometry analysis. Sequencing analysis revealed heterozygous deletion or substitution mutations in the neutrophil elastase (NE) gene in 9 of 12 patients but not in healthy controls. Expression of various NE mutants, but not normal NE, resulted in accelerated apoptosis of human promyelocytic HL-60 progenitor cells, similar to impaired survival observed in patients' cells. Bone marrow-derived primitive CD34(+) and CD33(+)/CD34(-) progenitor cells from SCN patients evolving to AML, all with mutations in the granulocyte colony-stimulating factor receptor (G-CSFR) gene, demonstrated normal cell survival, whereas more differentiated CD15(+)/CD33(-)/CD34(-) cells negative for mutant G-CSFR gene, continue to exhibit accelerated apoptosis. These data demonstrate that impaired survival of bone marrow myeloid progenitor cells, probably driven by expression of mutant NE, is the cellular mechanism responsible for neutropenia in SCN. Furthermore, our results suggest that acquired G-CSFR mutations may initiate signaling events that override the pro-apoptotic effect of mutant NE in primitive progenitor cells, resulting in an expansion of the abnormal AML clone.

Antigens, CD34↗
PubMed2003

Combination therapy for refractory idiopathic thrombocytopenic purpura in adolescents.

PURPOSE: To investigate combined immunosuppressive therapy with vincristine, methylprednisolone, and prolonged cyclosporine in adolescents with refractory idiopathic thrombocytopenic purpura (ITP). PATIENTS AND METHODS: Ten adolescent patients with ITP refractory to previous medical management, including gluco-corticosteroid, intravenous immunoglobulin or anti-Rh (D) IgG, or splenectomy, were treated with combination immunosuppressive therapy at the University of Michigan between 1997 and 2001. Therapy consisted of weekly doses of vincristine 1.5 mg/m intravenous push (IVP) (maximum dose 2 mg), weekly methylprednisolone 100 mg/m IVP, and cyclosporine (CSA) 5 mg/kg orally twice daily (goal: CSA trough of 100-200 mg/mL). Vincristine and methylprednisolone were given weekly until the platelet count was greater than 50,000/mm for a minimum of 2 doses and a maximum of 4 doses. CSA was continued until the platelet count was normal for 3 to 6 months. RESULTS: Seven patients had continuous complete responses (platelet count normal after cessation of CSA), a median of 13 months (9-37 months) since completion of therapy. One patient had a partial response (platelet count 80-120 x 10 /L off CSA for 3 months). Two patients were nonresponders (platelet count <40 x 10 /L), one of whom had all therapy discontinued after 2 weeks due to peripheral neuropathy. The median time to response was 7 days (range 7-67 days). CSA was administered for a median of 4 months (range 0.5-19 months). CONCLUSIONS: A combination immunosuppressive approach that includes prolonged cyclosporine therapy may be promising for refractory ITP and is associated with sustained disease remissions in some patients.

Adolescent↗
PubMed2002

Regulation of polymorphonuclear leukocyte degranulation and oxidant production by ceramide through inhibition of phospholipase D.

Exogenous C(2)-ceramide has been shown to inhibit polymorphonuclear leukocyte (PMN) phagocytosis through inhibition of phospholipase D (PLD) and downstream events, including activation of extracellular signal-regulated kinases 1 and 2, leading to the hyphothesis that the sphingomyelinase pathway is involved in termination of phagocytosis. Here it is postulated that increased PLD activity generating phosphatidic acid and diacylglycerol (DAG) is essential for superoxide release and degranulation and that ceramide, previously shown to be generated during PMN activation, inhibits PLD activation, thereby leading to inhibition of PMN function. When PMNs were primed with granulocyte colony-stimulating factor (G-CSF) and then activated with N-formyl-methionyl-leucyl-phenylalanine (FMLP), C(2)-ceramide (10 microM) completely inhibited release of superoxide, lactoferrin, and gelatinase; the DAG analog sn-1,2-didecanoylglycerol (DiC10) (10 microM) restored oxidase activation and degranulation in the ceramide-treated cells. Similarly, C(2)-ceramide inhibited oxidase activity and degranulation of PMNs treated with cytochalasin B followed by FMLP, and DiC10 restored function. In contrast, C(2)-ceramide did not inhibit phosphorylation of p47phox or p38 mitogen-activated protein kinase, or translocation of p47phox, PLD-containing organelles, adenosine diphosphate-ribosylation factor 1, RhoA, protein kinase C (PKC)-beta or PKC-alpha to the plasma membrane in G-CSF or cytochalasin B-treated, FMLP-activated PMNs. PLD activity increased by 3-fold in G-CSF-primed PMNs stimulated by FMLP and by 30-fold in cytochalasin B-treated PMNs stimulated by FMLP. Both PLD activities were completely inhibited by 10 microM C(2)-ceramide. In conclusion, superoxide, gelatinase, and lactoferrin release require activation of the PLD pathway in primed PMNs and cytochalasin B-treated PMNs. Ceramide may affect protein interactions with PLD in the plasma membrane, thereby attenuating PMN activation.

Cell Degranulation↗
PubMed2002

Granulocyte colony-stimulating factor primes NADPH oxidase in neutrophils through translocation of cytochrome b(558) by gelatinase-granule release.

Granulocyte colony-stimulating factor (GCSF) primes reduced neutrophil nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in response to formyl peptide but does not increase oxidase activity when used alone. Both oxidase activity and degranulation require phospholipase D (PLD) activation, and exogenous C(2)-ceramide inhibits both functions through inhibition of PLD activity. We extended these observations to investigate neutrophil responses to GCSF. GCSF at a dosage of 30 to 100 ng/mL, a concentration range that primes superoxide release, stimulated a 60% to 100% increase in gelatinase release from tertiary granules but did not stimulate lactoferrin release from secondary granules. A 75% to 100% dose-dependent increase in PLD activity in GCSF-treated neutrophils was also observed. Gelatinase release and PLD activity were inhibited by 10 micromol/L C(2)-ceramide. The increase in gelatinase release in response to priming concentrations of GCSF suggests that tertiary granules contribute a component of the NADPH oxidase to the plasma membrane. Neutrophils treated with 50 ng/mL GCSF were found to contain 20% more cytochrome b(558) in the plasma membrane fraction than unstimulated cells, consistent with degranulation of only tertiary granules. Correspondingly, in the presence of 10 micromol/L C(2)-ceramide, cytochrome b(558) content in the plasma membrane did not increase after neutrophil activation. In contrast, GCSF did not lead to p47phox translocation to the plasma membrane or phosphorylation. Because phosphorylation and translocation of p47phox are required for oxidase activity, these findings account for the inability of GCSF alone to generate the respiratory burst. We conclude that translocation of cytochrome b(558) was responsible for GCSF priming of NADPH oxidase in neutrophils.

Cell Membrane↗
PubMed2002

Adolescents with cancer: access to clinical trials and age-appropriate care.

Many adolescents with cancer are denied the benefits of protocol inclusion and optimal supportive care. More than 70% of older adolescent patients are not treated at institutions representing pediatric cooperative groups and are not enrolled in clinical trials, despite a demonstrated survival advantage for patients registered in protocols. Failure to refer adolescent cancer patients to specialized pediatric oncology treatment centers also has an impact on their quality of care. The challenge before both the medical and pediatric health care communities is to assure adolescent patients the benefits of inclusion in clinical trials and treatment in age-appropriate settings.

Adolescent↗
PubMed2002

Mutant elastase in pathogenesis of cyclic and severe congenital neutropenia.

Severe neutropenia is characterized by maturation arrest of myeloid cells at the promyelocyte stage of hematopoiesis. We reported that accelerated apoptosis of bone marrow myeloid progenitor cells was observed in both cyclic (CN) and severe congenital neutropenia (SCN). Short and long-term cultures of bone marrow CD34+ cells revealed reduced production of multipotent progenitors in SCN. In contrast, production of these cells was slightly elevated in CN compared with CD34+ cells from healthy volunteers. Production of myeloid-committed progenitor cells was significantly reduced in both CN and SCN. FACS analysis of CD34+ cells revealed G /G cell cycle arrest in SCN but not in CN.(0) (1) All CN patients and more than 90% of SCN patients have mutation in the neutrophil elastase (NE) gene. Molecular modeling of NE tertiary structure indicates that mutations observed in SCN are primarily located around the glycosylation sites, whereas CN mutations affect predominantly the active site. Transient expression of CN- or SCN-specific mutant NE cDNA results in impaired survival of human myeloid progenitor cells compared with control cells transfected with intact NE cDNA. We hypothesize that abnormal processing and subcellular localization of mutant NE might predetermine the etiology of cyclic or severe congenital neutropenia.

Hematopoiesis↗
PubMed2002

Clinical applications of hematopoietic growth factors in pediatric oncology.

Myeloid growth factors are widely used in both pediatric and adult oncology. Although the literature supporting the use of growth factors in pediatric oncology is less extensive than the adult literature, some uses are clearly established. Both granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor shorten the duration of febrile neutropenia after myelosuppressive chemotherapy, effectively mobilize hematopoietic stem cells for transplantation, and enhance neutrophil engraftment after hematopoietic stem cell transplantation. Although some open-label, uncontrolled trials and retrospective analyses support growth factor use to ameliorate the number of infections, duration of hospitalizations, and duration of intravenous antibiotic use after myelosuppressive chemotherapy or to enhance dose intensity, randomized controlled trials supporting these practices are lacking.

Child↗
PubMed2002

Rituximab for refractory childhood autoimmune hemolytic anemia.

BACKGROUND: Chronic childhood autoimmune hemolytic anemia is an uncommon disorder that is associated with significant morbidity. Treatment with high dose steroids, splenectomy and frequent blood transfusions results in a myriad of complications including growth failure, bone demineralization, Cushing's syndrome, immunosuppression, and transfusional hemosiderosis. OBJECTIVES: To investigate the efficacy of the monoclonal anti-CD20 antibody, rituximab, in treating children with AIHA. METHODS: Four children with chronic AIHA, including two with prior splenectomy, who were dependent on high dose steroids and refractory to other immunosuppressive regimens were treated with four to six weekly doses of rituximab at a dose of 375 mg/m2. RESULTS: All four patients became transfusion-independent and were taken off prednisone completely. Adverse effects included infusion-related reactions that were mild, and infectious complications of Pneumocystis carinii pneumonia and varicella pneumonia. CONCLUSIONS: Treatment with rituximab appears promising for refractory AIHA; if may obviate the need for prednisone and may result in sustained disease remissions in some patients.

Adolescent↗