RESUMO
INTRODUCTION: Hemophilia comprises a group of X-linked hemorrhagic disorders that result from a deficiency of coagulation factors. The disorder affects mainly males and leads to chronic pain, joint deformity, reduced mobility, and increased mortality. Current therapies require frequent administration of replacement clotting factors, but the emergence of alloantibodies (inhibitors) diminishes their efficacy. New therapies are being developed to produce the deficient clotting factors and prevent the emergence of inhibitors. AREAS COVERED: This article provides an update on the characteristics and disease pathophysiology of hemophilia A, as well as current treatments, with a special focus on ongoing clinical trials related to gene replacement therapies. EXPERT OPINION: Gene replacement therapies provide safe, durable, and stable transgene expression while avoiding the challenges of clotting factor replacement therapies in patients with hemophilia. Improving the specificity of the viral construct and decreasing the therapeutic dose are critical toward minimizing cellular stress, induction of the unfolded protein response, and the resulting loss of protein production in liver cells. Next-generation gene therapies incorporating chimeric DNA sequences in the transgene can increase clotting factor synthesis and secretion, and advance the efficacy, safety, and durability of gene replacement therapy for hemophilia A as well as other blood clotting disorders.
Assuntos
Hemofilia A , Hemofilia B , Fatores de Coagulação Sanguínea/genética , Fator VIII/uso terapêutico , Terapia Genética/tendências , Hemofilia A/genética , Hemofilia A/terapia , Hemofilia B/tratamento farmacológico , Hemofilia B/terapia , Humanos , Isoanticorpos/genética , Masculino , TransgenesRESUMO
Blood vessels and nerve tissues are critical to the development and functionality of many vital organs. However, little is currently known about their interdependency during development and after injury. In this study, dual fluorescence transgenic reporter mice were utilized to observe blood vessels and nervous tissues in organs postnatally. Thy1-YFP and Flt1-DsRed (TYFD) mice were interbred to achieve dual fluorescence in the offspring, with Thy1-YFP yellow fluorescence expressed primarily in nerves, and Flt1-DsRed fluorescence expressed selectively in blood vessels. Using this dual fluorescent mouse strain, we were able to visualize the networks of nervous and vascular tissue simultaneously in various organ systems both in the physiological state and after injury. Using ex vivo high-resolution imaging in this dual fluorescent strain, we characterized the organizational patterns of both nervous and vascular systems in a diverse set of organs and tissues. In the cornea, we also observed the dynamic patterns of nerve and blood vessel networks following epithelial debridement injury. These findings highlight the versatility of this dual fluorescent strain for characterizing the relationship between nerve and blood vessel growth and organization.
Assuntos
Vasos Sanguíneos , Córnea , Isoanticorpos , Proteínas Luminescentes , Imagem Óptica , Nervos Periféricos , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Animais , Vasos Sanguíneos/diagnóstico por imagem , Vasos Sanguíneos/crescimento & desenvolvimento , Córnea/irrigação sanguínea , Córnea/diagnóstico por imagem , Córnea/inervação , Feminino , Isoanticorpos/biossíntese , Isoanticorpos/genética , Proteínas Luminescentes/biossíntese , Proteínas Luminescentes/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Nervos Periféricos/diagnóstico por imagem , Nervos Periféricos/crescimento & desenvolvimento , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Natural antibodies produced by CD5+ B1 B cells include anti-thymocyte autoantibody (ATA). Transgenic mice bearing the Ig-µ heavy chain of a prototypic ATA, V(H)3609Vκ21c, demonstrated a critical requirement for self-antigen in the accumulation of ATA B cells and production of high levels of serum ATA. Further work with ATA-µκ transgenic mice revealed that, while development of most B cells were blocked at an immature stage in spleen, some mature ATA B cells were present. ATA-µκ transgenic mice with varying levels of Thy-1 autoantigen showed a clear relationship between BCR crosslinking and B cell fate, with low levels generating marginal zone ATA B cells and complete antigen absence allowing maturation to follicular ATA B cells. Finally, different fates of developing ATA B cells encountering high levels self-antigen may be accounted for by variations in the response of newly formed B cells arising from foetal and adult development.
Assuntos
Autoanticorpos/imunologia , Autoimunidade , Subpopulações de Linfócitos B/imunologia , Seleção Clonal Mediada por Antígeno , Isoanticorpos/imunologia , Animais , Autoanticorpos/metabolismo , Subpopulações de Linfócitos B/metabolismo , Antígenos CD5/imunologia , Genótipo , Humanos , Imunoglobulina M/imunologia , Imunoglobulina M/metabolismo , Isoanticorpos/genética , Isoanticorpos/metabolismo , Camundongos Transgênicos , Fenótipo , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Antígenos Thy-1/imunologia , Antígenos Thy-1/metabolismoRESUMO
It is known that a large number of both genetic and environmental factors contribute to the risk of inhibitor development, but underlying pathogenetic mechanisms are still under investigation. The clinical research on inhibitors towards factor VIII (FVIII) is challenged by the fact that this is an infrequent event occurring in a rare disease. Therefore, it is widely accepted that complementary studies involving animal models can provide important insights into the pathogenesis and treatment of this complication. In this respect, mouse models have been studied for clues to FVIII immunogenicity, natural history of immunity and for different approaches to primary and secondary tolerance induction. In the clinical setting, the type of FVIII product used and the occurrence of product switching are considered important factors which may have an influence on inhibitor development. The evaluation of data currently available in the literature does not prove unequivocally that a difference in the immunogenicity exists between particular FVIII products (e.g. recombinant vs. plasma-derived, full length vs. B-domainless). In addition, national products switches have occurred and, in this context, switching was not associated with an enhanced inhibitor risk. In contrast with severe haemophilia A, patients with moderate and mild haemophilia A receive FVIII treatment infrequently for bleeds or surgery. In this condition the inhibitor risk is low but remains present lifelong, requiring continuous vigilance, particularly after intensive FVIII exposure.
Assuntos
Fator VIII/genética , Fator VIII/imunologia , Hemofilia A/genética , Hemofilia A/imunologia , Isoanticorpos/genética , Isoanticorpos/imunologia , Animais , Modelos Animais de Doenças , Substituição de Medicamentos/efeitos adversos , Interação Gene-Ambiente , Hemofilia A/tratamento farmacológico , Humanos , Proteínas Recombinantes/imunologia , Fatores de RiscoRESUMO
The JR blood group system (ISBT 032) consists of one antigen,Jra, which is of high prevalence in all populations. The rare Jr(a-) phenotype has been found mostly in Japanese and other Asian populations, but also in people of northern European ancestry, in Bedouin Arabs, and in one Mexican. Anti-Jra has caused transfusion reactions and is involved in hemolytic disease of the fetus and newborn. The Jra antigen is located on ABCG2 transporter, a multipass membrane glycoprotein (also known as the breast cancer resistance protein, BCRP), which is encoded by the ABCG2 gene on chromosome 4q22.1. The Jr(a-) phenotype mostly results from recessive inheritance of ABCG2 null alleles caused by frameshift or nonsense changes.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Alelos , Antígenos de Grupos Sanguíneos , Cromossomos Humanos Par 4 , Isoanticorpos , Proteínas de Neoplasias , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/imunologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Antígenos de Grupos Sanguíneos/sangue , Antígenos de Grupos Sanguíneos/genética , Antígenos de Grupos Sanguíneos/imunologia , Cromossomos Humanos Par 4/genética , Cromossomos Humanos Par 4/metabolismo , Eritroblastose Fetal/sangue , Eritroblastose Fetal/genética , Eritroblastose Fetal/imunologia , Humanos , Isoanticorpos/sangue , Isoanticorpos/genética , Isoanticorpos/imunologia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Proteínas de Neoplasias/metabolismoRESUMO
Immunosuppressive CD11b(+)Gr1(+) myeloid-derived suppressor cells and TGF-ß have been shown to negatively regulate host immunity against allografts. Our results demonstrated that Smad3-deficient mice or mice reconstituted with Smad3-deficient hematopoietic cells rejected allogeneic skin or heart grafts in a significantly slower manner compared with littermates or wild-type (WT) control mice. Transplanted Smad3(-/-) recipients produced markedly less anti-donor IgG Abs, especially IgG1 and IgG2b subclasses. T cells in alloskin-grafted Smad3-deficient mice were more likely to participate in a Th2-type immune response, as evidenced by more Th2-specific transcription factor, GATA3 expression, and increased IL-4 and IL-10 production, as well as less Th1-specific transcription factor, T-bet expression, and decreased IL-2 and IFN-γ production. More CD11b(+)Gr1(+) neutrophil infiltration and less monocyte/macrophage and T cell infiltration in allografts were observed in Smad3(-/-) recipients compared with WT recipients. Increased CXCL1 and CXCL2 as well as decreased CCL3, MCP-1, and RANTES chemokines in allografts of Smad3(-/-) recipients were consistently detected by real-time PCR. Further studies indicated that the increased CD11b(+)Gr1(+) myeloid cells in Smad3-deficient mice were immunosuppressive and responsible for the delayed allograft rejection mainly via an NO-dependent pathway. Thus, this study identifies Smad3 as an intrinsic negative regulator that critically inhibits the differentiation and function of immunosuppressive CD11b(+)Gr1(+) myeloid-derived suppressor cells.
Assuntos
Antígeno CD11b , Rejeição de Enxerto/imunologia , Transplante de Coração/imunologia , Células Mieloides/imunologia , Óxido Nítrico/imunologia , Transplante de Pele/imunologia , Proteína Smad3 , Animais , Quimiocinas/genética , Quimiocinas/imunologia , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/imunologia , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Rejeição de Enxerto/genética , Rejeição de Enxerto/patologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Isoanticorpos/genética , Isoanticorpos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Óxido Nítrico/genética , Células Th2/imunologia , Transplante HomólogoRESUMO
BACKGROUND: Alloantibodies that define some high-prevalence MNS antigens are made by people with glycophorin A (GPA) altered by a single-amino-acid change or replacement of amino acids from part of the Pseudoexon 3 of GYP*B. The finding of a patient whose plasma contained a novel alloanti-En(a)FR prompted this study. RESULTS: The patient's serum contained an alloantibody to a high-prevalence antigen, resistant to papain, ficin, trypsin, alpha-chymotrypsin, or dithiothreitol. The antibody was strongly reactive with all panel red blood cells (RBCs) tested, showed reduced reactivity with ENEP- and ENAV- RBCs, and was nonreactive with M(k)M(k), En(a-), GP.Hil/GP.Hil, and GP.JL/M(k) RBCs. The patient's RBCs typed M+N-S+s-, Wr(a-b+(w)), ENEP-, and ENAV-. These results indicated that the antibody recognized a new high-prevalence antigen in the MNS system. Sequencing of DNA prepared from the patient's white blood cells revealed a GYP*A nucleotide substitution of 242T>G (predicted to change Val62 of GPA to Gly). This change ablates an RsaI restriction enzyme site and polymerase chain reaction-restriction fragment length polymorphism confirmed that the proband was homozygous for Nucleotide 242G. CONCLUSIONS: We describe a novel high-prevalence MNS antigen, characterized by Val62 in GPA and named ENEV. The absence of the antigen is associated with Gly62. The change explains the weakened reactivity of the patient's serum with ENEP- and ENAV- RBCs and nonreactivity with anti-ENEP and anti-ENAV against her RBCs. The ENEV antigen has been assigned the ISBT number MNS45.
Assuntos
Ponte de Artéria Coronária , Transfusão de Eritrócitos/métodos , Isoanticorpos/sangue , Isoantígenos/genética , Polimorfismo de Nucleotídeo Único , Idoso , Epitopos/imunologia , Membrana Eritrocítica/imunologia , Eritrócitos/imunologia , Feminino , Glicoforinas/genética , Glicoforinas/imunologia , Hemaglutinação/imunologia , Teste de Histocompatibilidade , Humanos , Período Intraoperatório , Isoanticorpos/genética , Isoanticorpos/imunologia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de RestriçãoRESUMO
BACKGROUND AND OBJECTIVES: In the Rh blood group system, partial D, C, and e antigens are well-known, but a partial c antigen resulting in the production of alloanti-c in a c+ individual is rare. One example of an alloanti-c in a c+ person was an anti-Rh26, which can appear as anti-c, and another was an alloanti-c in a c+ person with a presumed R(1)r phenotype. The finding of an apparent alloanti-c in a transfused c+ patient initiated this investigation. MATERIALS AND METHODS: Haemagglutination tests, DNA extraction, polymerase chain reaction (PCR)-based assays (PCR-restriction fragment length polymorphism, allele-specific PCR), reticulocyte mRNA extraction, reverse transcriptase (RT)-PCR and sequencing were performed by standard procedures. RESULTS: Plasma from a 64-year-old African American woman with a wound infection following a mastectomy contained anti-E, anti-S, anti-K, anti-Fy(a) and anti-Jk(b), reacting by the indirect antiglobulin test. In addition, the patient's plasma gave reactions that were consistent with an anti-c, while her pre-transfusion red blood cells typed c+ with some anti-c reagents. These results are consistent with a partial c antigen. The patient's red blood cells also typed V+(W)VS- and JAL+. Analyses of DNA and Rh-transcripts from this patient showed the presence of the following genes: RHD*D, RHD*DAU0, RHCE*Ce and RHCE*ce(S)(340). CONCLUSION: The nucleotide 340C>T change in RHCE exon 3 (predicted to encode 114Trp) of the RHCE*ce(S)(340) allele is associated with a JAL+ phenotype and the altered expression of the c, V and VS antigens. This alteration in the c antigen allowed the patient to make an alloanti-c. This case reveals that the RHCE*ce(S)(340) allele encodes a partial c antigen.
Assuntos
Éxons , Regulação da Expressão Gênica/genética , Isoanticorpos/sangue , Mutação de Sentido Incorreto , Sistema do Grupo Sanguíneo Rh-Hr/sangue , Sistema do Grupo Sanguíneo Rh-Hr/genética , Transfusão de Eritrócitos , Feminino , Humanos , Isoanticorpos/genética , Pessoa de Meia-Idade , Infecção da Ferida Cirúrgica/sangue , Infecção da Ferida Cirúrgica/genética , Infecção da Ferida Cirúrgica/terapiaAssuntos
Fucosiltransferases , Isoanticorpos , Antígenos do Grupo Sanguíneo de Lewis , Alelos , Bactérias/imunologia , Bactérias/metabolismo , Eritroblastose Fetal/genética , Eritroblastose Fetal/imunologia , Eritroblastose Fetal/metabolismo , Eritrócitos/imunologia , Eritrócitos/metabolismo , Fucosiltransferases/genética , Fucosiltransferases/imunologia , Fucosiltransferases/metabolismo , Rejeição de Enxerto/genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/metabolismo , Humanos , Isoanticorpos/genética , Isoanticorpos/imunologia , Isoanticorpos/metabolismo , Transplante de Rim/imunologia , Antígenos do Grupo Sanguíneo de Lewis/genética , Antígenos do Grupo Sanguíneo de Lewis/imunologia , Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Locos de Características Quantitativas , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
Soluble ABO blood group substance (SAS) in fresh-frozen plasma (FFP) and its cognate alloantibody titer reduction capacity (TRC) are not considered when prescribing this product for plasma exchange (PEX) therapy of ABO incompatible transplant recipients. SAS was quantified in 250 single FFPs using ELISA. Total and IgG class-specific anti-A TRCs of FFPs were measured using a microhemagglutination inhibition assay. SAS level depended not only on the A subtype (p < 0.0001) and the Secretor status (p < 0.0001), but also on the expression of ALe(b) in A1 secretors (p < 0.0001). The variation was as great as 137.6 arbitrary units (aU) for 14 A1 Le(a-b-) secretors and 1.2 aU for 6 A2 non-secretors. Homozygous expression of the A1, A2 and Secretor alleles did not increase SAS levels. Only total anti-A TRC, but not IgG class-specific TRC depended on the detected SAS level (r = 0.566, p = 0.0003).
Assuntos
Sistema ABO de Grupos Sanguíneos/química , Fucosiltransferases/análise , Isoanticorpos/análise , Antígenos do Grupo Sanguíneo de Lewis/química , Plasma/química , Sistema ABO de Grupos Sanguíneos/genética , Tipagem e Reações Cruzadas Sanguíneas , Fucosiltransferases/genética , Humanos , Isoanticorpos/genética , Antígenos do Grupo Sanguíneo de Lewis/genética , Troca Plasmática , Galactosídeo 2-alfa-L-FucosiltransferaseAssuntos
Adenoviridae/genética , Hepatócitos/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Animais , Células Cultivadas , Regulação da Expressão Gênica/imunologia , Genes Reporter , Proteínas de Fluorescência Verde , Isoanticorpos/genética , Proteínas Luminescentes/genética , Ratos , Ratos Endogâmicos Lew , Transfecção/métodosRESUMO
BACKGROUND: Natural antibodies that bind the carbohydrate antigen Galalpha1-3Galbeta1-4GlcNAc-R (alphaGal) mediate rigorous rejection of porcine xenografts and represent a major immunological hurdle to successful discordant xenotransplantation. However, little is known about how production of antibodies specific for alphaGal is regulated. METHODS: Transgenic mice expressing an IgM heavy chain isolated from a B-cell hybridoma that produces antibodies specific for alphaGal were constructed. These mice were bred to mutant mice that lack the alphaGal epitope (GT0 mice) or wild-type (GT+) mice to generate animals in which the transgene is expressed in the presence or absence of alphaGal as a "self"-antigen. Development of transgene-expressing B cells and production of alphaGal-specific serum antibodies were then analyzed in transgenic mice on GT0 and GT+ backgrounds. RESULTS: B cells expressing the transgenic heavy chain and transgene-encoded serum antibodies specific for alphaGal were readily detected in mice on the GT0 background. Most alphaGal-reactive antibodies in GT0 mice used the transgene rather than endogenous Ig heavy chains. In contrast, transgene-encoded serum antibodies specific for alphaGal were not detected in GT+ mice. In transgenic mice on the GT+ background, B cells expressing the transgene underwent deletion as a result of encountering alphaGal during their development, indicating that expression of alphaGal as part of self-mediated efficient negative selection of B cells expressing transgene-encoded alphaGal-specific antibodies. CONCLUSIONS: The development of transgenic mice expressing a B cell receptor specific for alphaGal provides a novel system to study developmental regulation of B cells making carbohydrate-specific antibodies. In addition, these mice may be useful for examining methods to prevent production of alphaGal-reactive antibodies.
Assuntos
Linfócitos B/imunologia , Dissacarídeos/genética , Cadeias Pesadas de Imunoglobulinas/genética , Isoanticorpos/genética , Animais , Células da Medula Óssea/imunologia , Linfócitos T CD4-Positivos/imunologia , Cruzamentos Genéticos , Dissacarídeos/imunologia , Células-Tronco Hematopoéticas/imunologia , Isoanticorpos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Mapeamento por Restrição , Linfócitos T/imunologia , Transplante Heterólogo/imunologiaRESUMO
The heavy and light chain immunoglobulin variable region nucleotide sequences for 219 mAbs to human red blood cells were collected from workshop participants, published reports, and Genbank. Information regarding antigen specificity, species of origin, method of cloning, and other relevant serological properties was correlated with the sequence data. Immunoglobulin sequences were analyzed to determine the heavy- and light-chain immunoglobulin genes used and the overall extent of somatic mutation from germline configuration. Approximately 50% of the sequences encoded antibodies with Rh(D) specificity with the remaining sequences encoding mAbs to other Rh-related antigens, antigens of the ABO, MNS, and Kell blood group systems, and several others. Surprisingly, no sequence data were available for mAbs with specificity for a number of common Rh antigens, common Kell antigens, or antigens of the Lewis, Kidd, or Duffy blood group systems. The majority of mAbs were of human origin but included a significant number of macaque mAbs, murine mAbs, and a small number of synthetically-designed recombinant antibodies. Both cellular (EBV-transformation, cell fusion) and molecular (phage display) approaches were used for antibody cloning. Analysis of certain groups of sequences demonstrated patterns of immunoglobulin gene restriction, repertoire shift, and somatic mutation. Analysis of other mAbs demonstrated the value of antibody sequence data for the design and production of novel reagents useful in blood group serology.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Grupos Sanguíneos/imunologia , Genes de Imunoglobulinas , Isoanticorpos/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Tipagem e Reações Cruzadas Sanguíneas/normas , Fusão Celular , Linhagem Celular Transformada , Clonagem Molecular , Bases de Dados de Ácidos Nucleicos , Desenho de Fármacos , Epitopos/imunologia , Genes Sintéticos , Haplorrinos , Herpesvirus Humano 4/fisiologia , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Isotipos de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/genética , Imunoglobulina M/química , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Isoanticorpos/química , Isoanticorpos/genética , Camundongos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/imunologia , Hipermutação Somática de ImunoglobulinaRESUMO
BACKGROUND: Anti-At(a) is a rare red cell (RBC) alloantibody found in the black population. It has been described as causing one case of mild hemolytic disease of the newborn, but its ability to cause hemolytic transfusion reactions is uncertain. CASE REPORT: The patient was a 60-year-old black female with a history of three uneventful pregnancies but no transfusions. On admission, her direct and indirect antiglobulin tests were negative, total bilirubin was 0.5 mg per dL, and lactate dehydrogenase was 224 IU per L. She received nine units of compatible RBCs in the perioperative period of a hemicolectomy. Her hemoglobin rose appropriately and stabilized at 12.6 g per dL by the 6th postoperative day. By Day 10 after surgery her hemoglobin had dropped to 6.8 g per dL, and her total bilirubin and lactate dehydrogenase had risen to 1.4 mg per dL and 783 IU per L, respectively. The direct and indirect antiglobulin tests were now newly positive with strengths of 3+. A warm hemolytic autoantibody was suspected. She was transfused two units of incompatible RBCs for a rapidly falling hemoglobin and symptomatic anemia. On Day 11, the total bilirubin rose to 3.5 mg per dL, and the lactate dehydrogenase was 1154 IU per L with a hemoglobin of 7.6 g per dL. Corticosteroids were begun. Studies of serum and an acid eluate revealed anti-At(a), but no other RBC antibodies. The patient stabilized, and further transfusion was avoided. CONCLUSION: Although anti-At(a) was previously described as being of uncertain clinical significance, this patient demonstrated the ability of the antibody to cause a severe delayed hemolytic transfusion reaction.
Assuntos
Antígenos de Grupos Sanguíneos/imunologia , Hemólise/imunologia , Isoanticorpos/imunologia , Reação Transfusional , População Negra/genética , Teste de Coombs , Feminino , Humanos , Isoanticorpos/genética , Pessoa de Meia-Idade , Fenótipo , Fatores de TempoRESUMO
Antibodies to the D antigen of the Rh system use a restricted set of immunoglobulin V and J gene segments, especially VH DP50 and DP63, JH6, Vlambda DPL16 and Jlambda 2/3. These gene segments may confer a natural affinity on the antibodies for the D antigen and this hypothesis has been tested by constructing two single-chain Fv phage-antibody libraries based on the germline gene segments DP50 and DP63; structural variability was obtained by insertion of 11 amino acids in random sequence in the VHCDR3. 10 anti-D antibodies were selected from these libraries, each with a unique VHCDR3. In contrast, selections with the CcEe antigens produced antibodies reacting with the Rh polypeptide molecules but without strict blood group specificity. One of these latter DP50-based antibodies was converted into 12 different antibodies with specificity for E by replacing the original germline light chain with chains from a rearranged L chain library. The CDR1 and CDR2 sequences of the DP50-based antibodies were common to both anti-D and anti-E molecules; differentiation between D and E specificity was dependent on VHCDR3 sequences and their correct pairing with an appropriate L chain.
Assuntos
Genes de Imunoglobulinas , Região Variável de Imunoglobulina/genética , Isoanticorpos/biossíntese , Especificidade de Anticorpos , Humanos , Cadeias Leves de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/genética , Isoanticorpos/genética , Isoanticorpos/imunologia , Imunoglobulina rho(D)RESUMO
A point mutation has been postulated as cause of the phenotype D category VII, based on data of 3 probands only. Repeatedly, D protein variants have been found to be due to heterogenous molecular events. Therefore, the aforementioned cause was to be tested with more probands. In a systematic study, 68 nonrelated probands with D category VII were found. 33 were selected by chance, and the nucleic acid region 280-329 was sequenced after PCR amplification. All examined probands showed the postulated Leu(110)Pro substitution. No further polymorphisms were detected. Our data show that in Southern Germany D category VII is homogenously due to the amino acid substitution Leu(110)Pro. This allows the exploitation of this polymorphism for the prenatal detection of D category VII and the related Tar antigen.
Assuntos
Substituição de Aminoácidos , Isoanticorpos/genética , Doadores de Sangue , Triagem de Portadores Genéticos , Humanos , Leucina , Programas de Rastreamento , Mutação Puntual , ProlinaRESUMO
As a part of our continuing effort to study the antigenic structure of class I molecules, we have undertaken two types of studies. First, we have studied the capacity of five different Kd-reactive mAbs to recognize a panel of 25 site-directed mutants of the H-2Kd molecule. Both the gain and the decrease in Ab binding resulted from a single amino acid substitution at different positions. All mutations that increase the binding of the tested mAbs are located on the alpha-helices, indicating that the replacement of an Ig-contacting surface residue with a charged or polar side chain by a short one generally favors Ab binding. Mutation of two alpha-helix-situated residues, 58 and 166, completely abolished the binding of one mAb (Tu191.7.1), indicating that these two residues contribute to the antigenic determinant defined by this mAb. The overwhelming majority of mutations that diminished Ab binding concerns residues buried within the Ag binding groove, suggesting the possibility of peptide contribution to serologic epitopes defined by alloreactive Abs. We have addressed this issue by comparison of the repertoire of peptides eluted from Kd molecules precipitated by different Kd-reactive mAbs. The results reveal that the two-dimensional profile obtained with one (F35.119.18) of the alloreactive mAbs is clearly different. The use of 21 single amino acid variants of a Kd-restricted 10-mer peptide allowed us to identify the residue of the bound peptide contributing to the epitope recognized by this mAb. Thus, we have shown that at least in some instances, changes induced in the MHC molecules by the binding of distinct peptides can be recognized as alterations in serologic determinants expressed on the class I molecules.
Assuntos
Anticorpos Monoclonais/química , Antígenos H-2/imunologia , Isoanticorpos/química , Peptídeos/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Sítios de Ligação de Anticorpos/genética , Epitopos/genética , Epitopos/imunologia , Epitopos/metabolismo , Antígenos H-2/genética , Antígenos H-2/metabolismo , Isoanticorpos/genética , Isoanticorpos/metabolismo , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Mutação/imunologia , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica/genética , Ligação Proteica/imunologiaRESUMO
Inhibitor formation is a serious complication, occurring in 24-52% of patients with haemophilia A and in 1.5-3% of patients with haemophilia B. Low-titre inhibitors can easily be overcome and do not represent a treatment challenge. They are treated with increased doses of factor concentrate. However, high-titre inhibitors may still cause death from haemorrhage in this population. Optimal treatment of acute, life-threatening haemorrhage in patients with factor VIII inhibitors includes either porcine factor VIII or human factor VIII in high doses since, with both modalities, circulating factor levels are achievable. If the inhibitor titres are too high, plasmapheresis may be warranted. For routine joint bleeds, PCC or aPCC can be administered at home with variable success. For patients with factor IX inhibitors treatment options are more limited. If the haemorrhage is life-threatening, plasmapheresis should be done to decrease the inhibitor and high doses of pure factor IX administered either by continuous infusion or bolus. Attainment of levels of > 20% should be attempted. New clinical options, such as rFVIIa, may emerge. For both groups of patients, IT should be started as soon as the inhibitor is discovered and its behaviour characterized. Unfortunately, no standard regimen for IT exists and the treater will have to choose whichever schedule he/she feels is the most appropriate.
Assuntos
Fator VIII/imunologia , Hemofilia A/imunologia , Isoanticorpos/imunologia , Animais , Contraindicações , Dessensibilização Imunológica , Fator IX/imunologia , Fator IX/uso terapêutico , Fator VIII/isolamento & purificação , Fator VIII/uso terapêutico , Hemofilia A/complicações , Hemofilia A/tratamento farmacológico , Hemofilia B/tratamento farmacológico , Hemofilia B/imunologia , Hemorragia/tratamento farmacológico , Hemorragia/etiologia , Humanos , Imunização , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Isoanticorpos/biossíntese , Isoanticorpos/genética , Masculino , Especificidade da Espécie , Procedimentos Cirúrgicos Operatórios , Suínos/sangueAssuntos
Autoanticorpos/isolamento & purificação , Clonagem Molecular/métodos , Eritrócitos/imunologia , Biblioteca Gênica , Genes de Imunoglobulinas , Técnicas de Imunoadsorção , Isoanticorpos/isolamento & purificação , Autoanticorpos/genética , Autoanticorpos/imunologia , Subpopulações de Linfócitos B/imunologia , Bacteriófago M13/genética , Bacteriófago M13/imunologia , DNA Complementar/genética , DNA Recombinante/isolamento & purificação , Escherichia coli/genética , Rearranjo Gênico do Linfócito B , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Isoanticorpos/genética , Isoanticorpos/imunologia , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , Imunoglobulina rho(D)/genética , Imunoglobulina rho(D)/isolamento & purificaçãoRESUMO
Many residues involved in polymorphic antibody-binding epitopes on class II molecules are located on the alpha-helix of DR beta chains. Although they have received less attention, residues in the peptide-binding groove and second domain of the DR beta chain may also be critical for polymorphic anti-DR antibody epitopes. In this study, we used transfectants expressing site-directed mutations at positions in the HLA-DR beta 1 and beta 2 domains and flow cytometry to define the epitopes of several polymorphic anti-DR antibodies. Both DR(beta 1*0403) residues 14 and 25 were shown to be involved in the epitopes of mAbs DA6. 164, HU-20, Q5/6, and 50D6, and DR(beta 1*0701) residue 14 was shown to be critical for the epitopes of two DR7-specific mAbs, SFR 16-DR7M and TAL13.1. Unlike most other residues shown to be important in antibody-binding epitopes, residue 14 is located in the floor of the peptide-binding groove and residue 25 is in an outer loop, each with their side chains pointing down, such that antibodies may directly contact these residues from below the binding groove. Two residues in the beta 2 domain, beta 180 and beta 181, were also shown to be involved in the epitopes of three polymorphic anti-DR mAbs, NFLD.D1, NFLD.M1, and LY9. Although these two residues are close to the transmembrane domain in the linear sequence, their solvent accessibility in the DR1 structures is quite impressive. Our data provide new evidence that residues accessible under the peptide-binding groove contribute to polymorphic antibody-binding epitopes.