RESUMEN
We report the long-term follow-up of the efficacy and safety of islet transplantation in seven type 1 diabetic subjects from the United States enrolled in the multicenter international Edmonton Protocol who had persistent islet function after completion of the Edmonton Protocol. Subjects were followed up to 12 years with serial testing for sustained islet allograft function as measured by C-peptide. All seven subjects demonstrated continued islet function longer than a decade from the time of first islet transplantation. One subject remained insulin independent without the need for diabetic medications or supplemental transplants. One subject who was insulin-independent for over 8 years experienced graft failure 10.9 years after the first islet transplant. The remaining six subjects demonstrated continued islet function upon trial completion, although three had received a supplemental islet transplant each. At trial completion, five subjects were receiving insulin and two remained insulin independent, although one was treated with liraglutide. The median hemoglobin A1c was 6.3% (45 mmol/mol). All subjects experienced progressive decline in the C-peptide/glucose ratio. No patients experienced severe hypoglycemia, opportunistic infection, or lymphoma. Thus, although the rate and duration of insulin independence was low, the Edmonton Protocol was safe in the long term. Alternative approaches to islet transplantation are under investigation.
Asunto(s)
Péptido C/análisis , Diabetes Mellitus Tipo 1/terapia , Hemoglobina Glucada/análisis , Supervivencia de Injerto , Hipoglucemia/prevención & control , Trasplante de Islotes Pancreáticos , Adulto , Glucemia/análisis , Femenino , Estudios de Seguimiento , Humanos , Hipoglucemiantes/uso terapéutico , Insulina/uso terapéutico , Masculino , Persona de Mediana Edad , Pronóstico , Factores de RiesgoRESUMEN
A potential link between arsenic (ATO)-based therapy and delayed hematopoietic recovery after autologous hematopoietic SCT (HSCT) for acute promyelocytic leukemia (APL) has previously been reported. We retrospectively reviewed the clinical histories of 58 patients undergoing autologous HSCT for APL at 21 institutions in the United States and Japan. Thirty-three (56%) of the patients received ATO-based therapy prior to stem cell collection. Delayed neutrophil engraftment occurred in 10 patients (17%): 9 of the 10 patients (90%) received prior ATO (representing 27% of all ATO-treated patients), compared with 1 of the 10 patients (10%) not previously treated with ATO (representing 4% of all ATO-naïve patients; P<0.001). Compared with ATO-naïve patients, ATO-treated patients experienced significantly longer times to ANC recovery (median 12 days vs 9 days, P<0.001). In multivariate analysis, the only significant independent predictor of delayed neutrophil engraftment was prior treatment with ATO (hazard ratio 4.87; P<0.001). Of the available stem cell aliquots from APL patients, the median viable post-thaw CD34+ cell recovery was significantly lower than that of cryopreserved autologous stem cell products from patients with non-APL AML. Our findings suggest that ATO exposure prior to CD34+ cell harvest has deleterious effects on hematopoietic recovery after autologous HSCT.
Asunto(s)
Antineoplásicos , Arsenicales , Supervivencia de Injerto/efectos de los fármacos , Leucemia Promielocítica Aguda/terapia , Óxidos , Trasplante de Células Madre de Sangre Periférica , Adolescente , Adulto , Antineoplásicos/administración & dosificación , Antineoplásicos/efectos adversos , Trióxido de Arsénico , Arsenicales/administración & dosificación , Arsenicales/efectos adversos , Autoinjertos , Femenino , Humanos , Leucemia Promielocítica Aguda/sangre , Masculino , Persona de Mediana Edad , Óxidos/administración & dosificación , Óxidos/efectos adversosRESUMEN
Crystal structures of four pyrrolo(2,3-d)pyrimidine-based antifolate compounds, developed as inhibitors of thymidylate synthase (TS) in a strategy to circumvent drug-resistance, have been determined in complexes with their in vivo target, human thymidylate synthase, and with the structurally best-characterized Escherichia coli enzyme, to resolutions of 2.2-3.0 A. The 2.9 A crystal structure of a complex of human TS with one of the inhibitors, the multi-targeted antifolate LY231514, demonstrates that this compound induces a "closed" enzyme conformation and leads to formation of a covalent bond between enzyme and substrate. This structure is one of the first liganded human TS structures, and its solution was aided by mutation to facilitate crystallization. Structures of three other pyrrolo(2,3-d)pyrimidine-based antifolates in complex with Escherichia coli TS confirm the orientation of this class of inhibitors in the active site. Specific interactions between the polyglutamyl moiety and a positively charged groove on the enzyme surface explain the marked increase in affinity of the pyrrolo(2,3-d)pyrimidine inhibitors once they are polyglutamylated, as mediated in vivo by the cellular enzyme folyl polyglutamate synthetase.
Asunto(s)
Resistencia a Medicamentos/genética , Escherichia coli/enzimología , Antagonistas del Ácido Fólico/química , Antagonistas del Ácido Fólico/metabolismo , Guanina/análogos & derivados , Mutación/genética , Timidilato Sintasa/química , Timidilato Sintasa/metabolismo , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Nucleótidos de Desoxiuracil/metabolismo , Dimerización , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Antagonistas del Ácido Fólico/farmacología , Glutamatos/química , Glutamatos/metabolismo , Glutamatos/farmacología , Guanina/química , Guanina/metabolismo , Guanina/farmacología , Humanos , Enlace de Hidrógeno , Ligandos , Modelos Moleculares , Pemetrexed , Péptido Sintasas/metabolismo , Conformación Proteica/efectos de los fármacos , Pliegue de Proteína , Electricidad Estática , Timidilato Sintasa/antagonistas & inhibidores , Timidilato Sintasa/genéticaRESUMEN
The T-lymphocyte transmembrane glycoprotein CD2 plays an important physiological role in facilitating adhesion between T-lymphocytes and their cognate cellular partners. This interaction is mediated by binding of CD2 to the broadly distributed surface polypeptide LFA-3 and augments the recognition function of the CD3-Ti antigen-major histocompatibility complex receptor via stabilization of conjugate formation between cells. To define better the structural components of the CD2 extracellular region which are important in contact-mediated cellular adhesion, a single-domain CD2 immunoadhesion protein has been prepared from papain digestion of a soluble two-domain CD2 molecule. This amino-terminal domain fragment binds to LFA-3 on human B-cells with a dissociation constant of 0.4 microM, possesses functional immunoadhesion epitopes as defined by the binding of monoclonal antibodies raised to native CD2, and retains the ability to inhibit sheep erythrocyte rosette formation with human T-cells. Thus, all of the immunoadhesion functions ascribed to CD2 reside within the amino-terminal domain. Circular dichroism analysis of the isolated CD2 adhesion domain suggests the presence of substantial alpha-helical character (22%), consistent with earlier computer modeling analyses that predicted a pattern of alternating alpha-helices and beta-sheets within the extracellular region of CD2. Despite the existence of short stretches of sequence homology between CD2 and immunoglobulin superfamily members, the circular dichroism data provide supporting biophysical evidence for classification of CD2 in an alpha-beta (either alpha/beta or alpha + beta) protein folding class.
Asunto(s)
Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Glicoproteínas de Membrana/inmunología , Receptores Inmunológicos/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Antígenos CD2 , Adhesión Celular , Línea Celular , Dicroismo Circular , Ensayo de Inmunoadsorción Enzimática , Humanos , Datos de Secuencia Molecular , Mapeo Peptídico , Péptidos/síntesis química , Conformación Proteica , Proteínas Recombinantes/inmunología , Formación de RosetaRESUMEN
The CD2 molecule is a 50-55KD transmembrane glycoprotein expressed on the vast majority of thymocytes and virtually all peripheral T lymphocytes. Its functions are two-fold: adhesion and activation. CD2 serves to facilitate conjugate formation between the T-lineage cell and its cognate partner via intermolecular interaction of CD2 and LFA-3 on the former and latter cells, respectively. Perturbation of the CD2 extracellular segment by certain combinations of anti-CD2 MAbs or LFA-3 and a single anti-CD2 MAb activate T-lineage function. These CD2-mediated activation events also synergize with signals mediated through the TCR to augment T-cell response. Based on microchemical analysis of immunoaffinity-purified human CD2 and cDNA and genomic cloning of mouse and human molecules, considerable structural information is now available. The mature surface human CD2 molecule consists of 327 amino acids: a 185 aa extracellular segment; a 25 aa hydrophobic transmembrane segment; and a 117 aa cytoplasmic domain rich in prolines and basic residues. The CD2 gene is comprised of five exons which span approximately 12 Kb on chromosome 1. A similar protein structure and gene exon organization is found for the mouse CD2 homologue. The CD2 adhesion domain is approximately 103 aa in length and is encoded by a single exon (exon 2). This domain is resistant to proteolysis, even though it lacks any intrachain disulfides and, like the entire extracellular segment protein expressed in a baculovirus system, binds to its cellular ligand, LFA-3. The latter occurs with a micromolar Kd. This relatively low affinity suggests that multivalent interactions among CD2 monomers on the T cells and individual LFA-3 structures on the cognate partner are important in enhancing the avidity of the T-cell interaction with its target or stimulator cell. The affinity of the CD2 extracellular segment for LFA-3 is not affected by truncations in the CD2 cytoplasmic domain, implying that ligand binding is not regulated by intracellular mechanisms. Given that CD2 mRNA expression and surface CD2 copy number are increased by more than one order of magnitude post-TCR stimulation, it is more likely that adhesion via CD2 is modulated by alteration in surface copy number. Analysis of early transduction events occurring via CD3-Ti (TCR) and CD2 including single channel Ca2+ patch-clamp recordings on living human T lymphocytes indicate a virtual identity of signals.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Antígenos de Diferenciación de Linfocitos T , Receptores Inmunológicos , Secuencia de Aminoácidos , Animales , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos de Diferenciación de Linfocitos T/inmunología , Antígenos de Diferenciación de Linfocitos T/fisiología , Antígenos CD2 , Complejo CD3 , Adhesión Celular , Humanos , Activación de Linfocitos , Glicoproteínas de Membrana/inmunología , Datos de Secuencia Molecular , Conformación Proteica , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/fisiología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Timo/citologíaRESUMEN
The 50-kD CD2 (T11) surface glycoprotein on human T lymphocytes and thymocytes plays a critical role in T lineage cell activation and adhesion via its ligand LFA-3. To begin to define structure-function relationships in the extracellular segment of the transmembrane CD2 molecule, we have used a eukaryotic expression system and a CD2 cDNA to produce milligram amounts of recombinant soluble CD2 molecule that corresponds to the two extracellular segment exons. We show that this protein, termed T11ex2, behaves as a monomer in aqueous solution and includes a proteolytically resistant NH2-terminal fragment (domain I) encoded by the first extracellular segment exon. Circular dichroism analysis of T11ex2 demonstrates that its stabilized secondary structure is dependent on the intrachain disulfide bonds present in domain II. The T11ex2 monomer binds directly to the CD2 ligand LFA-3 with a dissociation constant of 0.4 microM. This relatively low affinity implies that cooperative binding resulting from an array of transmembrane CD2 molecules is important to facilitate physiologic T cell adhesion.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T , Receptores Inmunológicos , Linfocitos T/inmunología , Anticuerpos Monoclonales , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos de Superficie , Linfocitos B/inmunología , Antígenos CD2 , Línea Celular , Dicroismo Circular , Codón , ADN/genética , Disulfuros , Electroforesis en Gel de Poliacrilamida , Epítopos/análisis , Exones , Humanos , Técnicas de Inmunoadsorción , Glicoproteínas de Membrana , Papaína/metabolismo , Plásmidos , Conformación Proteica , Receptores Inmunológicos/genética , Proteínas Recombinantes , Formación de Roseta , TransfecciónAsunto(s)
Antígenos CD/inmunología , Antígenos de Diferenciación de Linfocitos T/inmunología , Receptores Inmunológicos/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Antígenos de Diferenciación de Linfocitos T/genética , Antígenos de Superficie/inmunología , Secuencia de Bases , Antígenos CD2 , Humanos , Ratones , Datos de Secuencia Molecular , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Inmunológicos/genéticaRESUMEN
The 50-kDa T11 (CD2) T-lymphocyte surface glycoprotein facilitates physical adhesion between T-lineage cells and their cognate cellular counterparts (cytotoxic T-lymphocytes-target cells, helper T lymphocytes-antigen-presenting cells, or thymocytes-thymic epithelium) as well as signaling through the antigen-specific T3-Ti receptor complex. To examine the relationship between the structure and function of the T11 molecule, we have utilized a baculoviral expression system to produce milligram quantities of the hydrophilic extracellular T11 segment. Enzyme cleavage, microsequencing, and HPLC analyses of the expressed protein in conjunction with genomic cloning information show that the domain involved in cellular adhesion is encoded by a single 321-base-pair exon.
Asunto(s)
Antígenos de Diferenciación/genética , Exones , Receptores Inmunológicos/genética , Linfocitos T/inmunología , Secuencia de Aminoácidos , Antígenos de Diferenciación/inmunología , Antígenos CD2 , Adhesión Celular , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , ADN Recombinante , Electroforesis en Gel de Poliacrilamida , Epítopos , Glicosilación , Humanos , Técnicas de Inmunoadsorción , Sustancias Macromoleculares , Datos de Secuencia Molecular , Peso Molecular , Fragmentos de Péptidos , Receptores Inmunológicos/inmunología , Formación de Roseta , Relación Estructura-Actividad , TripsinaRESUMEN
Common acute lymphoblastic leukemia antigen (CALLA) is a 100-kDa cell-surface glycoprotein expressed on most acute lymphoblastic leukemias and certain other immature lymphoid malignancies and on normal lymphoid progenitors. The latter are either uncommitted to B- or T-cell lineage or committed to only the earliest stages of B- or T-lymphocyte maturation. To elucidate to homogeneity, obtained the NH2-terminal sequence from both the intact protein and derived tryptic and V8 protease peptides and isolated CALLA cDNAs from a Nalm-6 cell line lambda gt10 library using redundant oligonucleotide probes. The CALLA cDNA sequence predicts a 750-amino acid integral membrane protein with a single 24-amino acid hydrophobic segment that could function as both a transmembrane region and a signal peptide. The COOH-terminal 700 amino acids, including six potential N-linked glycosylation sites compose the extracellular protein segment, whereas the 25 NH2-terminal amino acids remaining after cleavage of the initiation methionine form the cytoplasmic tail. CALLA+ cells contain CALLA transcripts of 2.7 to 5.7 kilobases with the major 5.7- and 3.7-kilobase mRNAs being preferentially expressed in specific cell types.
Asunto(s)
Antígenos de Diferenciación/genética , Antígenos de Neoplasias/genética , Leucemia Linfoide/inmunología , Proteínas de la Membrana/genética , Proteínas de Neoplasias/genética , Secuencia de Aminoácidos , Secuencia de Bases , ADN/genética , Humanos , Leucemia Linfoide/genética , Datos de Secuencia Molecular , Neprilisina , ARN Neoplásico/análisisRESUMEN
To determine whether a full-length human T 11 (CD 2) cDNA encodes a protein which expresses all three T 11 epitopes (T 11(1), T 11(2), T 11(3] and independently triggers activation in a cell other than a T lymphocyte, a baculovirus expression system was employed. Here we show that a recombinant T 11 cDNA-containing baculovirus can induce high-level expression of T 11(1), T 11(2) and T 11(3) epitopes on the surface of gut epithelial SF9 cells. However, in this environment the T 11 protein cannot be triggered to transduce a signal resulting in an elevation of the cytosolic free Ca2+ as is known to occur in T lymphocytes. These results support the notion that T 11 functions in a coordinate fashion with other intracellular lymphoid components.
Asunto(s)
Calcio/metabolismo , Proteínas Portadoras/genética , Epítopos/genética , Proteínas de la Membrana/genética , Receptores Inmunológicos/genética , Animales , Antígenos CD2 , Proteínas Portadoras/inmunología , Proteínas Portadoras/fisiología , Células Cultivadas , ADN/genética , Células Epiteliales , Epitelio/metabolismo , Vectores Genéticos , Humanos , Virus de Insectos/genética , Mucosa Intestinal/metabolismo , Intestinos/citología , Canales Iónicos/metabolismo , Lepidópteros , Proteínas de la Membrana/fisiología , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/fisiología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/fisiologíaRESUMEN
Genomic DNA clones containing the human and murine genes coding for the 50-kDa T11 (CD2) T-cell surface glycoprotein were characterized. The human T11 gene is approximately equal to 12 kilobases long and comprised of five exons. A leader exon (L) contains the 5'-untranslated region and most of the nucleotides defining the signal peptide [amino acids (aa) -24 to -5]. Two exons encode the extracellular segment; exon Ex1 is 321 base pairs (bp) long and codes for four residues of the leader peptide and aa 1-103 of the mature protein, and exon Ex2 is 231 bp long and encodes aa 104-180. Exon TM is 123 bp long and codes for the single transmembrane region of the molecule (aa 181-221). Exon C is a large 765-bp exon encoding virtually the entire cytoplasmic domain (aa 222-327) and the 3'-untranslated region. The murine T11 gene has a similar organization with exon-intron boundaries essentially identical to the human gene. Substantial conservation of nucleotide sequences between species in both 5'- and 3'-gene flanking regions equivalent to that among homologous exons suggests that murine and human genes may be regulated in a similar fashion. The probable relationship of the individual T11 exons to functional and structural protein domains is discussed.
Asunto(s)
Antígenos de Diferenciación de Linfocitos T/genética , Proteínas Portadoras/genética , Receptores Inmunológicos/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Antígenos CD2 , Clonación Molecular , Exones , Regulación de la Expresión Génica , Genes , Humanos , Ratones , Datos de Secuencia Molecular , Secuencias Reguladoras de Ácidos Nucleicos , Linfocitos T/fisiologíaRESUMEN
The human CD2 molecule is a 50kd surface glycoprotein expressed on greater than 95% of thymocytes and all peripheral T lymphocytes which mediates both adhesion between T cells and their targets, and subsequent T cell activation events. Molecular cloning of human CD2 cDNAs predicts a mature CD2 protein of 327 amino acids, with an extracellular segment of 185 amino acids, a transmembrane domain of 24 amino acids and an intracytoplasmic region of 117 amino acids. Genomic cloning shows that the extracellular segment is encoded by two exons, the transmembrane segment by a single exon and the intracytoplasmic region by a single exon. Expression and biochemical analysis of a soluble extracellular domain CD2 molecule reveal that it expresses native CD2 epitopes and contains a stable 15kd NH2-terminal fragment corresponding to a single exon. Binding analyses of the soluble CD2 molecule indicate that it binds specifically to a known cell-surface ligand for CD2 at a relatively low affinity, thus suggesting that T cell-target adhesion mediated by CD2 and its ligand depends on multimeric attachment between an array of CD2 molecules and their cognate ligands.
Asunto(s)
Eritrocitos/metabolismo , Receptores de Superficie Celular/metabolismo , Linfocitos T/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/inmunología , Antígenos de Superficie/metabolismo , Antígenos CD58 , ADN , Exones , Humanos , Ligandos/metabolismo , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Mapeo Peptídico , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/fisiología , Ovinos/sangre , Solubilidad , Relación Estructura-Actividad , Linfocitos T/fisiología , Células Tumorales CultivadasRESUMEN
The murine equivalent of the cDNA encoding the human T11 (CD2) sheep erythrocyte-binding protein has been cloned. It codes for a putative transmembrane protein which is homologous to human T11. In contrast to immunoglobulins whose domains consist of anti-parallel beta sheets, we predict that mouse and human T11 external domains probably belong to the alpha/beta protein folding class. The cytoplasmic region of T11 is a lengthy, proline-rich segment; secondary structural analysis predicts it to have a nonglobular conformation. This elongated tail could allow for interaction with multiple other intracellular proteins and may contain a cation-binding site involved in T lineage activation.
Asunto(s)
Antígenos de Superficie/genética , Ratones/genética , Secuencia de Aminoácidos , Animales , Antígenos de Superficie/fisiología , Secuencia de Bases , ADN/genética , Genes , Humanos , Datos de Secuencia Molecular , Conformación Proteica , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis TumoralRESUMEN
The T11 (CD2) sheep-erythrocyte-binding protein is a T-cell surface molecule involved in activation of T lymphocytes and thymocytes, including those lacking the T3-Ti antigen-receptor complex. The primary structure of T11 was deduced from protein microsequencing and cDNA cloning. The mature human protein appears to be divided into three domains: a hydrophilic 185 amino acid external domain bearing only limited homology to the T-cell surface protein T4 and the immunoglobulin kappa light chain variable region, a 25 amino acid hydrophobic transmembrane segment, and a 126 amino acid cytoplasmic domain rich in prolines and basic residues. Transfection of cDNAs encoding either the 1.7- or the 1.3-kilobase T11 mRNA into COS-1 cells resulted in expression of surface T11 epitopes as well as sheep-erythrocyte-binding capacity. The predicted structure is consistent with the possibility that T11 functions in signal transduction.
Asunto(s)
Proteínas Portadoras/genética , Clonación Molecular , ADN/metabolismo , Genes , Receptores de Antígenos de Linfocitos T/genética , Receptores Inmunológicos/genética , Linfocitos T/inmunología , Transcripción Genética , Secuencia de Aminoácidos , Animales , Antígenos de Superficie/genética , Secuencia de Bases , Antígenos CD2 , Línea Celular , Humanos , ARN Mensajero/genéticaRESUMEN
Recent studies have suggested that the T4 molecule may bind to class II major histocompatibility complex (MHC) gene products on target cells. Such an interaction could be with either a polymorphic or monomorphic determinant on class II MHC genes. The first would imply that T4 is structurally variable and represents a component of a dual receptor, whereas the latter would suggest that T4 is structurally invariant and serves as an accessory binding molecule. To resolve this question we studied the structural variability of T4 molecules isolated from four clones of differing antigen/MHC specificities. By one- and two-dimensional electrophoretic analysis and by peptide mapping, we detected no differences among the 55-kDa T4 molecules from these clones. We conclude that T4 serves as an invariant accessory binding structure and that T4 does not confer MHC specificity on T cells.