RESUMEN
The optimal conditioning regimen for adults undergoing transplantation for acute lymphoblastic leukemia (ALL) remains undetermined. Cyclophosphamide and total body irradiation (Cy/TBI) has emerged as a standard myeloablative regimen but is associated with significant toxicity. We compared outcomes between patients undergoing transplant for ALL at centers using Cy/TBI as standard of care and another center using fludarabine, busulfan, and low-dose TBI (400 cGy) in combination with anti-thymocyte globulin as its standard. Among 146 patients (74 Cy/TBI and 72 Flu/Bu/TBI) there were no significant differences in overall or progression-free survival between groups. Non-relapse mortality was similar (12% vs. 16.7% for Cy/TBI and Flu/Bu/TBI, respectively, p = .62) despite the Flu/Bu/TBI group having significantly worse performance status. Flu/Bu/TBI resulted in significantly lower cumulative incidence of relapse compared with Cy/TBI (2-year point estimate 18.5% vs. 31.5%, p = .05). These results demonstrate similar outcomes for patients receiving Flu/Bu/TBI versus Cy/TBI. Flu/Bu/TBI may allow the possibility of providing myeloablative conditioning to patients with poor performance status.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Trasplante de Células Madre Hematopoyéticas , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Acondicionamiento Pretrasplante , Irradiación Corporal Total , Adolescente , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Busulfano/administración & dosificación , Terapia Combinada , Femenino , Enfermedad Injerto contra Huésped/etiología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Masculino , Persona de Mediana Edad , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/mortalidad , Pronóstico , Recurrencia , Análisis de Supervivencia , Acondicionamiento Pretrasplante/efectos adversos , Acondicionamiento Pretrasplante/métodos , Trasplante Homólogo , Resultado del Tratamiento , Vidarabina/administración & dosificación , Vidarabina/análogos & derivados , Adulto JovenRESUMEN
The majority of adults with acute lymphoblastic leukemia will achieve a first complete remission (CR). However relapse is the most common cause of treatment failure. Outcomes after relapse remain poor, with long-term survival in the order of 10 %. Treatment decisions made at the time of first complete remission are thus critical to ensuring long-term survival. Allogeneic hematopoietic cell transplant (HCT) is effective at preventing relapse in many transplant recipients but is also associated with significant treatment related morbidity and mortality. Alternatively, ongoing systemic chemotherapy offers lower toxicity at the expense of increased relapse rates. Over the past decades, both the safety of transplant and the efficacy of non-transplant chemotherapy have improved. Emerging data show substantially improved outcomes for young adults treated with pediatric-inspired chemotherapy regimens that question the role of HCT in the upfront setting. In this review, we review the data supporting the role of allogeneic transplantation in adult acute lymphoblastic leukemia (ALL), and we propose a therapeutic algorithm for upfront therapy of adults with ALL.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Anticuerpos Monoclonales/uso terapéutico , Humanos , Inmunoterapia , Neoplasia Residual , Cromosoma Filadelfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Inducción de Remisión , Trasplante HomólogoRESUMEN
The features that govern the interaction of ligand binding proteins with membrane permeases of cognate ABC transporters are largely unknown. Using sequence alignments and structural modeling based on the structure of the Escherichia coli BtuCD vitamin B12 transporter, we identified six conserved basic residues in the permease, comprised of FhuB and FhuG proteins, in the ferrichrome transporter of Staphylococcus aureus. Using alanine-scanning mutagenesis we demonstrate that two of these residues, FhuB Arg-71 and FhuG Arg-61, play a more dominant role in transporter function than FhuB Arg-74 and Arg-311, and FhuG Arg-64 and Lys-306. Moreover, we show that at positions 71 and 61 in FhuB and FhuG, respectively, arginine cannot be substituted for lysine without loss of transporter function. Previously, our laboratory demonstrated the importance of conserved acidic residues in the ferrichrome binding protein, FhuD2. Taken together, these results support the hypothesis that Glu-Arg salt bridges are critical for the interaction of the ligand binding protein with the transmembrane domains FhuB and FhuG. This hypothesis was further studied by "charge swapping" experiments whereby we constructed a S. aureus strain expressing FhuD2 with conserved residues Glu-97 and Glu-231 replaced by Arg and FhuB and FhuG with conserved basic residues Arg-71 and Arg-61, respectively, replaced by Glu. A strain containing this combination of substitutions restored partial function to the ferrichrome transporter. The results provide a direct demonstration of the functional importance of conserved basic residues on the extracellular surface of the ferrichrome permease in the Gram-positive bacterium S. aureus.
Asunto(s)
Dipéptidos/metabolismo , Ferricromo/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Staphylococcus aureus/metabolismo , Transporte Biológico , Modelos Moleculares , Staphylococcus aureus/crecimiento & desarrolloRESUMEN
In Staphylococcus aureus, fhuCBG encodes an ATP-binding cassette (ABC) transporter that is required for the transport of iron(III)-hydroxamates; mutation of either fhuB or fhuG eliminates transport. In this paper, we describe construction and characterization of an S. aureus fhuCBG deletion strain. The delta fhuCBG::ermC mutation not only resulted in a strain that was incapable of growth on iron(III)-hydroxamates as a sole source of iron but also resulted in a strain which had a profound growth defect in iron-restricted laboratory media. The growth defect was not a result of the inability to transport iron(III)-hydroxamates since S. aureus fhuG::Tn917 and S. aureus fhuD1::Km fhuD2::Tet mutants, which are also unable to transport iron(III)-hydroxamates, do not have similar iron-restricted growth defects. Complementation experiments demonstrated that the growth defect of the delta fhuCBG::ermC mutant was the result of the inability to express FhuC and that this was the result of an inability to transport iron complexed to the S. aureus siderophore staphylobactin. Transport of iron(III)-staphylobactin is dependent upon SirA (binding protein), SirB (permease), and SirC (permease). S. aureus expressing FhuC with a Walker A K42N mutation could not utilize iron(III)-hydroxamates or iron(III)-staphylobactin as a sole source of iron, supporting the conclusion that FhuC, as expected, functions with FhuB, FhuG, and FhuD1 or FhuD2 to transport iron(III)-hydroxamates and is the "genetically unlinked" ABC-ATPase that functions with SirA, SirB, and SirC to transport iron(III)-staphylobactin. Finally, we demonstrated that the delta fhuCBG::ermC strain had decreased virulence in a murine kidney abscess model.
Asunto(s)
Adenosina Trifosfatasas/fisiología , Proteínas Portadoras/fisiología , Hierro/metabolismo , Staphylococcus aureus/fisiología , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/fisiología , Absceso/microbiología , Adenosina Trifosfatasas/genética , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Transporte Biológico/genética , Transporte Biológico/fisiología , Proteínas Portadoras/genética , Elementos Transponibles de ADN , Modelos Animales de Enfermedad , Eliminación de Gen , Prueba de Complementación Genética , Riñón/microbiología , Ratones , Mutagénesis Insercional , Sideróforos/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/genética , Staphylococcus aureus/patogenicidad , Virulencia/genéticaRESUMEN
Mutagenesis of group B streptococcus (GBS) with TnphoZ, a transposon designed to identify secreted protein genes, identified the gene homologues fhuD and fhuG. The encoded proteins participate in siderophore (hydroxamate)-dependent iron(III) transport in other bacterial species. Sequence analysis of the genome determined that fhuD and fhuG are members of a polycistronic operon comprised of four genes, fhuCDBG, that encode a putative ATPase, cell surface receptor and two transmembrane proteins respectively. We hypothesized that FhuD was a siderophore receptor. Western analysis of cell extracts localized FhuD to the bacterial cell membrane. Fluorescence quenching experiments determined that purified FhuD bound hydroxamate-type siderophores. FhuD displayed highest affinity for iron(III)-desferroxamine, with a K(D) (microM) = 0.05, identical to that described for FhuD2 from Staphylococcus aureus. The role of Fhu in siderophore-iron transport was also characterized. A fhu mutant, ACFhu1, was equally sensitive to the iron-dependent antibiotic streptonigrin as the wild-type strain, suggesting that ACFhu1 was not reduced for intracellular iron concentrations in the absence of exogenous siderophore. However, ACFhu1 transported significantly less siderophore-bound iron in (55)Fe accumulation assays. These data provide the first evidence of siderophore-mediated iron acquisition by GBS.
Asunto(s)
Proteínas Bacterianas/metabolismo , Hierro/metabolismo , Sideróforos/metabolismo , Streptococcus agalactiae/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Cartilla de ADN , Datos de Secuencia Molecular , Operón , Homología de Secuencia de Aminoácido , Streptococcus agalactiae/genética , Streptococcus agalactiae/crecimiento & desarrollo , EstreptonigrinaRESUMEN
Staphylococcus aureus can utilize ferric hydroxamates as a source of iron under iron-restricted growth conditions. Proteins involved in this transport process are: FhuCBG, which encodes a traffic ATPase; FhuD2, a post-translationally modified lipoprotein that acts as a high affinity receptor at the cytoplasmic membrane for the efficient capture of ferric hydroxamates; and FhuD1, a protein with similarity to FhuD2. Gene duplication likely gave rise to fhuD1 and fhuD2. While the genomic locations of fhuCBG and fhuD2 in S. aureus strains are conserved, both the presence and the location of fhuD1 are variable. The apparent redundancy of FhuD1 led us to examine the role of this protein. We demonstrate that FhuD1 is expressed only under conditions of iron limitation through the regulatory activity of Fur. FhuD1 fractions with the cell membrane and binds hydroxamate siderophores but with lower affinity than FhuD2. Using small angle x-ray scattering, the solution structure of FhuD1 resembles that of FhuD2, and only a small conformational change is associated with ferrichrome binding. FhuD1, therefore, appears to be a receptor for ferric hydroxamates, like FhuD2. Our data to date suggest, however, that FhuD1 is redundant to FhuD2 and plays a minor role in hydroxamate transport. However, given the very real possibility that we have not yet identified the proper conditions where FhuD1 does provide an advantage over FhuD2, we anticipate that FhuD1 serves an enhanced role in the transport of untested hydroxamate siderophores and that it may play a prominent role during the growth of S. aureus in its natural environments.
Asunto(s)
Duplicación de Gen , Ácidos Hidroxámicos/química , Hierro/química , Lipoproteínas/química , Lipoproteínas/genética , Proteínas de Transporte de Membrana/genética , Staphylococcus aureus/genética , Bioensayo , Membrana Celular/metabolismo , Proliferación Celular , Citoplasma/metabolismo , Detergentes/farmacología , Endopeptidasa K/farmacología , Evolución Molecular , Técnicas de Transferencia de Gen , Membranas Intracelulares/metabolismo , Hierro/metabolismo , Cinética , Proteínas de Transporte de Membrana/química , Modelos Genéticos , Filogenia , Unión Proteica , Conformación Proteica , Procesamiento Proteico-Postraduccional , Dispersión de Radiación , Sideróforos/metabolismo , Staphylococcus aureus/metabolismo , Rayos XRESUMEN
The fhuD2 gene encodes a lipoprotein that has previously been shown to be important for the utilization of iron(III)-hydroxamates by Staphylococcus aureus. We have studied the function of the FhuD2 protein in greater detail, and demonstrate here that the protein binds several iron(III)-hydroxamates. Mutagenesis of FhuD2 identified several residues that were important for the ability of the protein to function in iron(III)-hydroxamate transport. Several residues, notably Tyr-191, Trp-197, and Glu-202, were found to be critical for ligand binding. Moreover, mutation of two highly conserved glutamate residues, Glu-97 and Glu-231, had no affect on ligand binding, but did impair iron(III)-hydroxamate transport. Interestingly, the transport defect was not equivalent for all iron(III)-hydroxamates. We modeled FhuD2 against the high resolution structures of Escherichia coli FhuD and BtuF, two structurally related proteins, and showed that the three proteins share a similar overall structure. FhuD2 Glu-97 and Glu-231 were positioned on the surface of the N and C domains, respectively. Characterization of E97A, E231A, or E97A/E231A mutants suggests that these residues, along with the ligand itself, play a cumulative role in recognition by the ABC transporter FhuBGC2. In addition, small angle x-ray scattering was used to demonstrate that, in solution, FhuD2 does not undergo a detectable change in conformation upon binding iron(III)-hydroxamates. Therefore, the mechanism of binding and transport of ligands for binding proteins within this family is significantly different from that of other well studied binding protein families, such as that represented by maltose-binding protein.