RESUMO
A combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions was used to identify phage clones capable of binding to the surface of Candida albicans blastoconidia. Single chain antibody variable fragments (scFv) derived from three clones detected C. albicans antigens by indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and Western blotting. The antigens detected were conserved among different strains of C. albicans and several other Candida species. Two scFv clones detected antigens specifically expressed by C. albicans blastoconidia; the third detected antigens in both blastoconidia and filamentous forms of C. albicans. The antigens containing the epitopes recognized by all three scFv could be extracted from blastoconidia by dithiothreitol, suggesting attachment to the cell wall via sulfhydryl bonds. Epitope detection by the scFv was sensitive to treatment of C. albicans blastoconidia with sodium periodate, but not proteinase K, indicating the cognate epitopes were composed of carbohydrate. Antigenic determinants for each of the three scFv were detected by immunohistochemical staining of skin sections from a model of cutaneous candidiasis, demonstrating expression in vivo. Through selection for the ability to bind intact organisms, the phage display system provides a means to rapidly identify monoclonal binding ligands to Candida surface antigens. Being entirely human, mature antibodies generated from the scFv have potential utility in the treatment of candidiasis.
Assuntos
Antígenos de Fungos/imunologia , Antígenos de Superfície/imunologia , Candida albicans/imunologia , Região Variável de Imunoglobulina/genética , Sequência de Aminoácidos , Animais , Antígenos de Fungos/genética , Antígenos de Superfície/genética , Sequência de Bases , Western Blotting , Candida albicans/genética , Candidíase Cutânea/imunologia , Candidíase Cutânea/microbiologia , Candidíase Cutânea/patologia , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Epitopos/genética , Epitopos/imunologia , Escherichia coli/genética , Técnica Indireta de Fluorescência para Anticorpo , Expressão Gênica , Vetores Genéticos , Humanos , Camundongos , Dados de Sequência Molecular , Biblioteca de Peptídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
The study of human B cell tolerance has been hampered by difficulties in identifying a sizable population of autoreactive B lymphocytes whose fate could be readily determined. Hypothesizing that B cells expressing intrinsically autoreactive antibodies encoded by the VH4-34 heavy chain gene (VH4-34 cells) represent such a population, we tracked VH4-34 cells in healthy individuals. Here, we show that naive VH4-34 cells are positively selected and mostly restricted to the follicular mantle zone. Subsequently, these cells are largely excluded from the germinal centers and underrepresented in the memory compartment. In healthy donors but not in patients with systemic lupus erythematosus (SLE), these cells are prevented from differentiating into antibody-producing plasma cells. This blockade can be overcome ex vivo using cultures of naive and memory VH4-34 cells in the presence of CD70, IL-2, and IL-10. VH4-34 cells may therefore represent an experimentally useful surrogate for autoantibody transgenes and should prove valuable in studying human B cell tolerance in a physiological, polyclonal environment. Our initial results suggest that both positive and negative selection processes participate in the maintenance of tolerance in autoreactive human B cells at multiple checkpoints throughout B cell differentiation and that at least some censoring mechanisms are faulty in SLE.
Assuntos
Linfócitos B/imunologia , Tolerância Imunológica/imunologia , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Células da Medula Óssea/imunologia , Diferenciação Celular , Centro Germinativo , Nível de Saúde , Humanos , Memória Imunológica , Lúpus Eritematoso Sistêmico/imunologia , Tonsila Palatina/citologia , Tonsila Palatina/imunologiaRESUMO
To determine the pathogenic role of chromosomes 11 and 17 in the carcinogenesis of human ovarian cancers, neo(R)-tagged chromosome 11 or 17 was transferred from cell lines A9H11 or A9H17, respectively, into the ovarian carcinoma cell line SKOV-3 using microcell-mediated chromosome transfer. The chromosome transfer was verified by polymerase chain reaction detection of the neo(R) gene, fluorescence in situ hybridization detection of an extra chromosome 11, and microsatellite polymorphism detection of an exogeneous chromosome 11. Five SKOV-3/A9H11 hybrids and five SKOV-3/A9H17 hybrid clones were generated. For the chromosome 11 transfer, complete suppression of tumorigenicity was observed in four clones, (11)9-8 and 11(H)7-2, 11(H)8-3, and 11(H)7-2, 100 days post implantation. For the chromosome 17 transfer, no complete suppression of tumorigenicity was observed. However, an increased latency period ranging from 25 to 49 days in contrast to 7 days for the SKOV-3 parental line, and a significant reduction in tumor size was observed. There was no correlation between the in vitro growth rate and the tumorigenicity or length of latency period. Our results demonstrate functionally that chromosome 11 may carry a tumor suppressor gene(s) while chromosome 17 may carry a tumor growth-inhibitor gene(s) for the ovarian carcinoma cell line, SKOV-3.
Assuntos
Carcinoma/genética , Cromossomos Humanos Par 11/genética , Técnicas de Transferência de Genes , Genes Supressores de Tumor/genética , Neoplasias Ovarianas/genética , Animais , Divisão Celular/genética , Cromossomos Humanos Par 17/genética , Células Clonais , Progressão da Doença , Feminino , Marcadores Genéticos , Humanos , Hibridização in Situ Fluorescente , Camundongos , Camundongos SCID , Transplante de Neoplasias , Reação em Cadeia da Polimerase , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Burkitt's lymphoma cell lines have been important in vitro models for studying the pathogenesis of Burkitt's lymphoma (BL) and for exploring new treatment strategies. A new EBV(-) Burkitt's lymphoma cell line (GA-10) was established from a patient with a clinically aggressive, chemorefractory BL and characterized. Although functional p-glycoprotein could not be demonstrated by dye-efflux assays, both p53 genes were mutated in the GA-10 cells, perhaps contributing to the resistant phenotype of the original neoplasm. Two properties of BL cells which may be useful targets for novel cytotoxic therapeutics are their surface expression of CD77, the receptor for Shiga toxin (Stx), and their high rate of proliferation. Expression of CD77 on the GA-10 cells was heterogeneous in that certain subclones expressed high levels of CD77 and correspondingly exhibited strong growth inhibition by Stx while others showed low levels of CD77 expression and weak Stx-induced growth inhibition. Flavopiridol, a potent inhibitor of cell cycle progression through G1 and G2, induced cytotoxicity of the GA-10 cells with an LC(50) of approximately 40 nM vs 70 nM for HL-60 cells (P < 0.05). The concentrations of flavopiridol at which only 10% of the cells were viable (LC(10)) were approximately 280 nM for the GA-10 cells and 520 nM for the HL-60 cells (P < 0.05). Dose-related induction of apoptosis in response to flavopiridol was demonstrated in the GA-10 cells by morphology, TUNEL assay, and activation of caspase-3. Flavopiridol was also cytotoxic to seven other BL cell lines tested. These data suggest that flavopiridol may have therapeutic value in the treatment of Burkitt's lymphoma.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linfoma de Burkitt/genética , Linfoma de Burkitt/patologia , Caspases/metabolismo , Flavonoides/farmacologia , Genes p53/genética , Piperidinas/farmacologia , Células Tumorais Cultivadas/citologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Adulto , Linfoma de Burkitt/metabolismo , Caspase 3 , Caspases/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Humanos , Masculino , Mutação , Toxina Shiga/farmacologia , Triexosilceramidas/metabolismoRESUMO
Tetracyclines have recently been shown to exert a number of pleiotropic anti-inflammatory and immunomodulatory activities, independent of their antibiotic properties. These include the ability to inhibit metalloproteinases (MP), a class of enzymes involved in crucial cellular functions such as the shedding of soluble mediators and their receptors from the cell surface, as well as interaction with, and remodeling of, the extracellular matrix. Here we report that doxycycline at therapeutic concentrations (1--5 microg/ml) significantly suppresses Ig secretion and class switching by in vitro activated murine B cells. Suppression of Ig secretion correlates with a decrease in levels of mRNA for the terminal B cell differentiation-associated genes Blimp-1 and mad-4, as well as to a reduction in expression of the plasma cell markers Syndecan-1 and J chain. Inhibition of class switching occurs at the recombination stage and is also induced by other MP inhibitors, including tetracycline analogs lacking antibiotic activity and the chemically unrelated hydroxamate KB8301. These novel, direct effects of MP inhibitors on B lymphocytes suggest an intrinsic role for MP in B cell activation and likely explain some of the observed in vivo immunomodulatory properties of tetracyclines. Moreover, these findings have significant implications for tetracycline therapy in Ig-mediated autoimmune or allergic diseases and raise questions about the use of doxycycline-inducible transgenic systems for the study of B cell function.
Assuntos
Antibacterianos/farmacologia , Linfócitos B/efeitos dos fármacos , Doxiciclina/farmacologia , Animais , Linfócitos B/imunologia , Biomarcadores , Diferenciação Celular , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Rearranjo Gênico do Linfócito B/efeitos dos fármacos , Switching de Imunoglobulina , Imunoglobulina G/biossíntese , Isotipos de Imunoglobulinas , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Baço/citologiaRESUMO
From five mice immunized with Escherichia coli K1 bacteria, we produced 12 immunoglobulin M hybridomas secreting monoclonal antibodies (MAbs) that bind to Neisseria meningitidis group B (NMGB). The 12 MAbs also bound the capsular polysaccharide (PS) of E. coli K1 [which, like NMGB, is alpha(2-8)-linked polysialic acid (PSA)] and bound to EV36, a nonpathogenic E. coli K-12 strain producing alpha(2-8) PSA. Except for HmenB5, which cross-reacted with N. meningitidis group C, none of the MAbs bound to N. meningitidis groups A, C, and Y. Of the 12 MAbs, 6 were autoantibodies as defined by binding to CHP-134, a neuroblastoma cell line expressing short-chain alpha(2-8) PSA; five of these MAbs killed NMGB in the presence of rabbit complement, and two also killed NMGB with human complement. The other six MAbs, however, were nonautoreactive; all killed NMGB with rabbit complement, and five killed NMGB with human complement. To obtain peptide mimotopes of NMGB PS, four of the nonautoreactive MAbs (HmenB2, HmenB3, HmenB13, and HmenB14) were used to screen two types of phage libraries, one with a linear peptide of 7 amino acids and the other with a circular peptide of 7 amino acids inserted between two linked cysteines. We obtained 86 phage clones that bound to the screening MAb in the absence but not in the presence of E. coli K1 PSA in solution. The clones contained 31 linear and 4 circular mimotopes expressing unique sequences. These mimotopes nonrandomly expressed amino acids and were different from previously described mimotopes for NMGB PS. The new mimotopes may be useful in producing a vaccine(s) capable of eliciting anti-NMGB antibodies not reactive with neuronal tissue.
Assuntos
Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Neisseria meningitidis/imunologia , Polissacarídeos Bacterianos/imunologia , Sequência de Aminoácidos , Animais , Vacinas Bacterianas/imunologia , Epitopos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BLRESUMO
Telomerase activity is up-regulated 1000-fold higher in human tonsil germinal center B cells compared to resting naive or memory B cells, and telomerase expression can be re-activated in vitro resting B cells. To understand the mechanism(s) of telomerase regulation, quiescent B cell from peripheral blood or tonsil were activated with different combinations of various stimuli. Cross-linking surface (s)IgD or sIgM of B cells induced marked up-regulation of telomerase enzymatic activity in the absence of cellular proliferation. Low level cross-linkage of surface molecules by soluble anti-IgM did not up-regulate the telomerase activity. However, the inability of soluble anti-IgM to up-regulate the telomerase activity was corrected by additional signals from soluble anti-CD40 antibody engagement or IL-4 / IL-10. Activation of B cell proliferation with Epstein-Barr virus failed to up-regulate telomerase, further suggesting that up-regulation of telomerase is an event independent of B cell proliferation. Telomerase induction occurred in the late G1 phase of the cell cycle and did not require entry into S phase. Up-regulation of telomerase enzymatic activity correlated primarily with the induction of expression of the hTERT gene, the catalytic subunit to telomerase, suggesting that control of telomerase regulation resides at the level of the catalytic subunit of this holoenzyme.
Assuntos
Linfócitos B/enzimologia , Linfócitos B/imunologia , Regulação Enzimológica da Expressão Gênica , RNA , Telomerase/genética , Regulação para Cima , Linfócitos B/citologia , Domínio Catalítico , Divisão Celular , Células Cultivadas , Criança , Proteínas de Ligação a DNA , Fase G1 , Células HeLa , Humanos , Imunoglobulina D/imunologia , Receptores Imunológicos , Telomerase/metabolismoRESUMO
To study inherent properties of somatic hypermutation of human immunoglobulin genes in the absence of antigen selection, mutations of human non-productive VH6 rearrangements enriched by subtractive hybridization were characterized. Ten unique clones arising from nine non-productive rearrangements were isolated. The frequency of mutation was 3.0%. Analysis of these mutations showed intrinsic bias for transitions and cytosine (C) to guanine (G) and G to C transversions. Bias for the strand of DNA targeted by mutation was not evident. Replacement mutations in the complementarity-determining region (CDR) occurred more frequently than expected based on the primary DNA sequence. This targeting of replacement mutations to the CDR may explain the conservation of the VH6 sequence in primates.
Assuntos
Rearranjo Gênico/imunologia , Genes de Imunoglobulinas/imunologia , Mutação Puntual , Sequência de Bases , Humanos , Região Variável de Imunoglobulina/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Baço/imunologiaRESUMO
Little is known of the persistence and pathogenicity of human herpesvirus 6 (HHV-6) after primary infection, including the role of strain variant. Over 2 to 5 years, 2,716 children and 149 families were studied. Peripheral blood mononuclear cell (PBMC), saliva, and cerebrospinal fluid (CSF) specimens were examined for HHV-6 DNA and variant. Ninety-nine percent of isolates causing primary infection were HHV-6 variant B (HHV-6B), which predominated in 95%-98% of the variants persisting in PBMC and saliva specimens from children and adults. Of 668 CSF samples, 13% contained HHV-6 DNA; of 77 children examined after primary infection, 61% had HHV-6 DNA detected only in their CSF and 39% had HHV-6 DNA in both CSF and PBMCs. HHV-6 variant A (HHV-6A) was detected significantly (P = .0001) more frequently in CSF than in PBMCs or saliva. In children for whom HHV-6 was identified in both CSF and PBMCs, PBMCs contained only HHV-6B, while CSF contained HHV-6A or HHV-6B, not both. Thus, in patients with dual infection, only HHV-6A persisted in CSF, which suggests that HHV-6A has greater neurotropism. Findings for adults indicate that dual infection occurs; variant persistence is similar to that for children. The frequency of HHV-6A infection increased little with age, thereby indicating that HHV-6A infection remains uncommon into adulthood. This study suggests that HHV-6 variants have different immunobiologic courses and neurotropism.
Assuntos
Encéfalo/virologia , Infecções por Herpesviridae/virologia , Herpesvirus Humano 6/classificação , Adulto , Pré-Escolar , DNA Viral/análise , Herpesvirus Humano 6/isolamento & purificação , Humanos , Lactente , Recém-Nascido , Leucócitos Mononucleares/virologia , Saliva/virologiaRESUMO
The extensive proliferation that B lymphocytes undergo in germinal centers could compromise generation of long term B cell memory if there occurs shortening of the telomeres of germinal center B cells with cell division. Telomere length, which is thought to act as a "mitotic clock" for somatic cells that dictates cellular senescence, can be preserved by the enzyme telomerase. Human tonsil germinal center B cells consistently expressed 100- to 1000-fold higher levels of telomerase activity than naive or memory B cells, which had no or very low detectable activity, as analyzed by the telomere repeat amplification protocol assay. In vitro stimulation of human memory B cells through surface Ig or CD40 was capable of up-regulating telomerase. The findings suggest that longevity of B cell memory is maintained, despite multiple cell divisions in the generation of a memory B cell, by up-regulation of telomerase in germinal center and activated memory B cells.
Assuntos
Antígenos CD , Subpopulações de Linfócitos B/enzimologia , Centro Germinativo/enzimologia , Memória Imunológica , Ativação Linfocitária , Telomerase/biossíntese , Regulação para Cima/imunologia , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Antígenos de Diferenciação/análise , Subpopulações de Linfócitos B/imunologia , Células Cultivadas , Criança , Centro Germinativo/citologia , Centro Germinativo/imunologia , Humanos , Imunoglobulina D/análise , Interfase/imunologia , Glicoproteínas de Membrana , N-Glicosil Hidrolases/análise , Tonsila Palatina , Telomerase/genéticaRESUMO
Relatively little is known about the relationship of lymphoid-associated gene expression to the proliferation and differentiation potential of early human bone marrow lymphoid progenitors. Surface expression of interleukin-7 (IL-7) receptor-alpha (IL-7R alpha), a component of the high-affinity receptor for the lymphoid precursor growth factor IL-7, defined a CD34+ progenitor subset lacking the CD19+ pro-B phenotype but demonstrating markedly enhanced lymphoid clonogenic capacity and the ability to differentiate into pro-B cells in short-term culture. These progenitors expressed mRNA for the lymphoid-associated genes Ig beta, RAG-1, and PAX-5, and were uniformly TdT-positive (TdT+). In contrast, IL-7R alpha-/CD19-/ CD34+ progenitors had a 50-fold reduced lymphoid clonogenic capacity and did not differentiate into pro-B cells in short-term culture. Expression of TdT and the lymphoid-associated genes Ig beta and RAG-1, but not PAX-5, was detected in this fraction, although at lower levels than in the IL-7R alpha+ progenitors. In contrast to IL-7R alpha, loss of the stem cell factor receptor c-kit was associated with enhanced lymphoid clonogenic potential and increased B-lineage differentiation potential. These results indicate that IL-7R alpha expression defines entry into a developmental stage characterized by upregulation of multiple lymphoid-associated genes and enhanced fitness for B-lymphoid differentiation. The onset of IL-7R alpha and PAX-5 expression immediately before acquisition of CD19 is consistent with evidence suggesting upregulation of CD19 through pathways involving PAX-5 and IL-7.
Assuntos
Antígenos CD/biossíntese , Subpopulações de Linfócitos B/metabolismo , Medula Óssea/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Interleucina-7/metabolismo , Ativação Linfocitária , Receptores de Interleucina/biossíntese , Adulto , Antígenos CD19/análise , Antígenos CD34/análise , Subpopulações de Linfócitos B/enzimologia , Subpopulações de Linfócitos B/imunologia , Medula Óssea/imunologia , Células da Medula Óssea , Diferenciação Celular/genética , Células Clonais , DNA Nucleotidilexotransferase/análise , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Hematopoéticas/enzimologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Imunofenotipagem , Antígenos Comuns de Leucócito/análise , Ativação Linfocitária/genética , Proteínas Proto-Oncogênicas c-kit/biossíntese , Receptores de Interleucina-7RESUMO
Haemophilus influenzae type b (Hib) is a significant pathogen for young children, and three Hib vaccines (named PRP-OMPC, HbOC, and PRP-T) are currently available for young children. Extensive studies of anti-Hib polysaccharide (PS) antibodies (Abs) have shown that the V regions of Abs against the Hib PS comprise a VH gene in the VH3 gene family and a VL gene from various K kappa and V lambda subgroups. To study immunogenic properties of the three vaccines in young children, we determined the VL subgroups and avidities of anti-Hib-PS Abs induced by the three clinically available conjugate vaccines. Ab avidity was measured by determining the concentration of a Hib-PS oligomer that abrogates half of the binding of immunoglobulin G anti-Hib-PS Abs to microwells. The PRP-OMPC vaccine induced lower-avidity Abs than the prelicensure HbOC vaccine (P = 0.05). When we compared anti-Hib-PS Abs expressing V kappa Ia, V kappa II, and V lambda subgroups, a greater Ab response was induced by the prelicensure HbOC vaccine than other vaccines (P < 0.05). When anti-Hib-PS Abs with the V kappa III subgroup were compared, however, both PRP-T and prelicensure HbOC vaccines induced a comparable response, which in turn was greater than those induced by the PRP-OMPC or the postlicensure HbOC vaccine (P < 0.001). The VL repertoire of Abs induced with the prelicensure HbOC or PRP-T vaccine in young children is dominated (about 80%) by anti-Hib-PS Abs using subgroup V kappa II. However, anti-Hib-PS using V kappa II VL accounts for only about 40% of the total anti-Hib-PS Abs induced with the PRP-OMPC vaccine or the postlicensure HbOC. Our data suggest that immunogenic properties of Hib vaccines in young children vary depending on the vaccine preparations as well as the vaccine types.
Assuntos
Anticorpos Antibacterianos/imunologia , Vacinas Anti-Haemophilus/imunologia , Haemophilus influenzae/imunologia , Afinidade de Anticorpos , Diversidade de Anticorpos , Humanos , Imunoglobulina G/imunologia , Cadeias kappa de Imunoglobulina/imunologia , Cadeias lambda de Imunoglobulina/imunologia , Lactente , Vacinas ConjugadasAssuntos
Genes de Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Mutação , Animais , Anticorpos Antibacterianos/genética , Anticorpos Antibacterianos/imunologia , Cápsulas Bacterianas , Ligante de CD40 , Deleção Clonal , Códon , Rearranjo Gênico do Linfócito B , Vacinas Anti-Haemophilus/imunologia , Haemophilus influenzae/imunologia , Humanos , Isotipos de Imunoglobulinas/genética , Imunoglobulina M/genética , Imunoglobulina M/imunologia , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Glicoproteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Família Multigênica , Polissacarídeos Bacterianos/imunologia , Vacinas Conjugadas/imunologiaAssuntos
Linfócitos B/imunologia , Vacinas Bacterianas/imunologia , Linfócitos T/imunologia , Vacinas Combinadas/imunologia , Vacinas Virais/imunologia , Animais , Reações Antígeno-Anticorpo , Antígenos de Histocompatibilidade/imunologia , Humanos , Imunidade Celular , Polissacarídeos Bacterianos/imunologia , Subpopulações de Linfócitos T/imunologia , Células Th1/imunologia , Células Th2/imunologiaRESUMO
Somatic mutation of Ig variable regions occurs prominently in germinal centers, but it has been debated whether the mutation process initiates in germinal centers or is activated before germinal center entry of B cells. We have analyzed for the presence of somatic mutation in Ig gene rearrangements of the nonpolymorphic human VH6 gene in the X-linked HyperIgM syndrome, which is associated with defective CD40 ligand expression and absence of germinal centers and generation of memory B lymphocytes. IgM and rare IgG VH6 productive rearrangements were isolated from PBL of patients with X-linked HyperIgM syndrome. Although the majority of both the IgM and IgG VH6 rearrangements had a germline VH6 sequence, 7 of 102 VH6 IgM and 1 of 6 IgG rearrangements had a mutated VH6 gene. The mutation frequency (mutations/bp) was 1.4% with a range of 2-9 mutations per clone, a mutation frequency lower, however, than that observed in IgM (3.2%) and IgG (5.4%) VH6 rearrangements of normal individuals. These results suggest that somatic mutation may be initiated in a CD40 ligand-independent pathway before entry of B cells into germinal centers, but fails to achieve the high mutation frequency observed in the presence of germinal centers.
Assuntos
Genes de Imunoglobulinas/genética , Imunoglobulina M/genética , Região Variável de Imunoglobulina/genética , Síndromes de Imunodeficiência/genética , Cromossomo X/genética , Sequência de Aminoácidos , Antígenos CD/análise , Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos B/análise , Antígenos de Diferenciação de Linfócitos B/genética , Doenças Autoimunes/complicações , Doenças Autoimunes/etiologia , Sequência de Bases , Antígenos CD40 , Rearranjo Gênico , Ligação Genética , Humanos , Síndromes de Imunodeficiência/complicações , Linfócitos/imunologia , Dados de Sequência Molecular , Mutação , RNA Mensageiro/genética , Homologia de Sequência de AminoácidosRESUMO
Translationally silent mutations, which are not antigen selected, of human VH6 Ig gene rearrangements isolated from human spleen were analyzed for bias to gain insight into intrinsic features of the mutation process. Sixty-three clones representing 38 VH6DJ rearrangements had an overall mutation frequency of 4.5%, a replacement/silent (R/S) mutation ratio of 2.1 and 167 unique silent mutations. The silent mutations showed bias in: (i) targeting to CDR1 and CDR2, (ii) an increased frequency of mutations of A compared to T nucleotide bases on the coding strand, and (iii) an increased frequency of transitions versus transversions. Bias of C-->G over C-->A, of G-->C over G-->T and of A-->C over A-->T transversions was also present. Hot spots of mutation were observed, some which corresponded to potential sites of stem-loop formation. The results suggest that the somatic mutation process in man may be targeted to the complementarity determining region for some V genes, exhibits specific base substitutions favoring transitions and specific types of transversions, and may be occurring on only one DNA strand.