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1.
Indian J Nephrol ; 24(1): 20-3, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24574626

RESUMO

In this retrospective study, we describe the anesthetic management and its implications in parturients with chronic kidney disease (CKD; n = 9), who underwent elective caesarean delivery. Nine parturients with CKD of various etiologies, who underwent elective Caesarean delivery, were included in this study. Spinal anest-hesia was administered in all parturients with normal coagulation profile through a 25-gauze spinal needle (Quincke) with 0.5% (H) bupivacaine in L2-3 space and T6 level was achieved. Hemodynamics and side effects such as nausea, vomiting, headache, and backache were record. The mean age was 28.22 ± 4.43 years. The mean levels of serum creatinine and serum potassium were 2.78 ± 1.29 mg/dl and 4.11 ± 0.46 meq/l, respectively. Mean baseline values of systolic blood pressure, diastolic blood pressure, and pulse rate were higher which decreased after spinal anesthesia. However, the incidence of hypotension, which required mephentermine treatment, was 11.1%. One patient had symptoms of nausea and vomiting/dizziness at the time of hypotension, which disappeared after treatment with 5 mg of intravenous mephentermine. Baseline value of PR remained high throughout the operation. Parturients with CKD with normal coagulation profile remained hemodynamically stable under spinal anesthesia with minimal side effects. However, a large number of studies are required to determine the safety of spinal anesthesia in this setting.

2.
Saudi J Kidney Dis Transpl ; 24(6): 1280-4, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24231504

RESUMO

In a developing country such as India, deceased donor renal transplantation (DDRTx) accounts for only about 1% of all renal transplants (RTx). Our institute initiated an intercity DDRTx in the year 2006, which significantly increased the number of RTx. We retrieved 74 kidneys from 37 deceased donors from various cities of Gujarat from January 2006 to December 2009. We transplanted the allografts in 66 recipients and a retrospective analysis of the donor profile and management and recipient outcome was performed. The mean age of the donors was 43.3 ± 18.8 years. The causes of death included road traffic accident in 51.35% of the donors and cerebrovascular stroke in 48.65% of the donors; 83.78% of the donors required ionotropes for hemodynamic stability in addition to vigorous intravenous fluid replacement. The average urine output of the donors was 350 ± 150 mL. The organs were perfused and stored in HTK solution. The mean cold ischemia time (CIT) was 9.12 ± 5.25 h. The mean anastomosis time in the recipient was 30.8 ± 8.7 min. 57.6% of the recipients established urine output on the operating table and 42.4% developed delayed graft function. At the end of 1 month after transplantation, the mean serum creatinine was comparable to the Ahmadabad city DDRTx, although the CIT was significantly longer in the intercity patients. Intercity organ harvesting is a viable option to increase the donor pool. Distance may not be an impediment, and good recipient outcome is possible in spite of prolonged CIT in case of proper harvesting and preservation.


Assuntos
Transplante de Rim , Doadores de Tecidos/provisão & distribuição , Obtenção de Tecidos e Órgãos/organização & administração , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Feminino , Humanos , Índia , Masculino , Pessoa de Meia-Idade , Adulto Jovem
3.
Cell Death Dis ; 2: e205, 2011 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-21881607

RESUMO

Apoptosis research has been significantly aided by the generation of antibodies against caspase-cleaved peptide neo-epitopes. However, most of these antibodies recognize the N-terminal fragment and are specific for the protein in question. The aim of this project was to create antibodies, which could identify caspase-cleaved proteins without a priori knowledge of the cleavage sites or even the proteins themselves. We hypothesized that many caspase-cleavage products might have a common antigenic shape, given that they must all fit into the same active site of caspases. Rabbits were immunized with the eight most prevalent exposed C-terminal tetrapeptide sequences following caspase cleavage. After purification of the antibodies we demonstrated (1) their specificity for exposed C-terminal (but not internal) peptides, (2) their ability to detect known caspase-cleaved proteins from apoptotic cell lysates or supernatants from apoptotic cell culture and (3) their ability to detect a caspase-cleaved protein whose tetrapeptide sequence differs from the eight tetrapeptides used to generate the antibodies. These antibodies have the potential to identify novel neo-epitopes produced by caspase cleavage and so can be used to identify pathway-specific caspase cleavage events in a specific cell type. Additionally this methodology may be applied to generate antibodies against products of other proteases, which have a well-defined and non-promiscuous cleavage activity.


Assuntos
Anticorpos/metabolismo , Caspases/metabolismo , Oligopeptídeos/química , Animais , Anticorpos/química , Anticorpos/imunologia , Apoptose/efeitos dos fármacos , Caspases/imunologia , Linhagem Celular Tumoral , Epitopos/imunologia , Fluoruracila/toxicidade , Humanos , Imunoprecipitação , Oligopeptídeos/imunologia , Coelhos
4.
Transplant Proc ; 40(10): 3451-4, 2008 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19100411

RESUMO

BACKGROUND: Appropriate anesthesia for pediatric renal transplantation requires stable intraoperative hemodynamics, optimal perfusion of the newly transplanted kidney and good analgesia during recovery. The aim of this study was to assess the preliminary application, success and safety of combined epidural and general anesthesia in pediatric renal transplantation in a small cohort. METHODS: We retrospectively reviewed the anesthesia records of 46 consecutive pediatric patients who received renal transplantation under combined epidural and general anesthesia from January 2003-2007. RESULTS: The mean patient age and weight were 13.2 +/- 2.4 years and 25.7 +/- 5.46 kg, respectively. The infused crystalloids, 20% albumin and red blood cell concentrates were 120 +/- 2 mL/kg to achieve a CVP of 13 to 15 mm Hg. Brisk diuresis was observed in all patients. Epidural tramadol (2 mg/kg) provided good postoperative analgesia in 89% patients. 15% patients developed radiological evidence of pulmonary edema, only one required mechanical ventilation for hypoxemia. Minor adverse effects were nausea and vomiting (17.5%) and convulsions (8.5%). No perioperative mortality or major morbidity was recorded. CONCLUSION: Epidural anesthesia is a useful adjunct to general anesthesia due to stable intraoperative haemodynamics and good postoperative analgesia.


Assuntos
Anestesia Epidural , Anestesia Geral , Transplante de Rim/métodos , Adolescente , Analgésicos/uso terapêutico , Criança , Terapia Combinada , Feminino , Humanos , Período Intraoperatório , Nefropatias/classificação , Nefropatias/cirurgia , Masculino , Metilprednisolona/administração & dosagem , Metilprednisolona/uso terapêutico , Complicações Pós-Operatórias/classificação , Complicações Pós-Operatórias/epidemiologia , Período Pós-Operatório , Estudos Retrospectivos , Vômito/prevenção & controle
5.
Indian J Otolaryngol Head Neck Surg ; 60(1): 69-71, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23120506

RESUMO

A variety of swellings located on or near the gums is clinically included under the heading of epulis. There are various types of epulis. In today's era of super specialization gum swellings more commonly present to the dental surgeon than to the practicing otolaryngologist. We present an interesting case of a fibrous epulis managed in our institute along with a brief review of literature.

6.
Science ; 293(5537): 2111-4, 2001 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-11509691

RESUMO

The B cell activating factor BAFF (BlyS/TALL-1/zTNF4) is a tumor necrosis factor (TNF)-related ligand that promotes B cell survival and binds to three receptors (BCMA, TACI, and the recently described BAFF-R). Here we report an absolute requirement for BAFF in normal B cell development. Examination of secondary lymphoid organs from BAFF-deficient mice revealed an almost complete loss of follicular and marginal zone B lymphocytes. In contrast, mice lacking BCMA had normal-appearing B lymphocyte compartments. BAFF therefore plays a crucial role in B cell development and can function through receptors other than BCMA.


Assuntos
Linfócitos B/fisiologia , Proteínas de Membrana/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Fator de Necrose Tumoral alfa/fisiologia , Animais , Antígenos CD/análise , Fator Ativador de Células B , Antígeno de Maturação de Linfócitos B , Subpopulações de Linfócitos B/citologia , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/fisiologia , Linfócitos B/citologia , Linfócitos B/imunologia , Células da Medula Óssea/citologia , Separação Celular , Sobrevivência Celular , Citometria de Fluxo , Imunoglobulinas/sangue , Leucopoese , Linfonodos/citologia , Linfonodos/imunologia , Contagem de Linfócitos , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos A , Camundongos Knockout , Fenótipo , Receptores do Fator de Necrose Tumoral/deficiência , Receptores do Fator de Necrose Tumoral/genética , Transdução de Sinais , Baço/citologia , Baço/imunologia , Timo/citologia , Timo/imunologia , Fator de Necrose Tumoral alfa/deficiência , Fator de Necrose Tumoral alfa/genética
7.
Science ; 293(5537): 2108-11, 2001 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-11509692

RESUMO

B cell homeostasis has been shown to critically depend on BAFF, the B cell activation factor from the tumor necrosis factor (TNF) family. Although BAFF is already known to bind two receptors, BCMA and TACI, we have identified a third receptor for BAFF that we have termed BAFF-R. BAFF-R binding appears to be highly specific for BAFF, suggesting a unique role for this ligand-receptor interaction. Consistent with this, the BAFF-R locus is disrupted in A/WySnJ mice, which display a B cell phenotype qualitatively similar to that of the BAFF-deficient mice. Thus, BAFF-R appears to be the principal receptor for BAFF-mediated mature B cell survival.


Assuntos
Linfócitos B/fisiologia , Proteínas de Membrana/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Sequência de Aminoácidos , Animais , Fator Ativador de Células B , Receptor do Fator Ativador de Células B , Antígeno de Maturação de Linfócitos B , Linfócitos B/imunologia , Linfócitos B/metabolismo , Linhagem Celular , Mapeamento Cromossômico , Cromossomos Humanos Par 22 , Clonagem Molecular , Homeostase , Humanos , Ligantes , Tecido Linfoide/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores do Fator de Necrose Tumoral/química , Receptores do Fator de Necrose Tumoral/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Transfecção , Proteína Transmembrana Ativadora e Interagente do CAML
8.
J Immunol ; 166(5): 3226-30, 2001 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-11207276

RESUMO

Expression of the protooncogene A-myb is restricted to the developing CNS, adult testes, breasts in late pregnancy, and germinal centers of secondary B cell follicles. The functional relevance of A-myb expression at three of these sites has been demonstrated previously via the generation and analysis of A-myb-deficient mice, which display behavioral abnormalities, male sterility, and perturbed breast development during pregnancy. In contrast, here we show that the germinal center response driven by T cell-dependent Ag immunization and the associated processes of Ab V gene somatic hypermutation, affinity maturation, and heavy chain class switching are overtly normal in A-myb-deficient mice. Nonetheless, these mice display mild splenic white pulp hypoplasia and blunted primary serum Ab responses, suggesting that although A-myb is not directly involved in the regulation of the memory B cell response, it may play a role in enhancing peripheral B cell survival or proliferative capacity.


Assuntos
Proteínas Aviárias , Linfócitos B/imunologia , Centro Germinativo/imunologia , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Linfócitos T/imunologia , Transativadores/deficiência , Transativadores/genética , Animais , Anticorpos/sangue , Formação de Anticorpos/genética , Linfócitos B/metabolismo , Linfócitos B/patologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Galinhas , Feminino , Centro Germinativo/metabolismo , Centro Germinativo/patologia , Switching de Imunoglobulina/genética , Injeções Intraperitoneais , Ativação Linfocitária/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Proto-Oncogênicas c-myb , Baço/anormalidades , Baço/crescimento & desenvolvimento , Baço/imunologia , Linfócitos T/metabolismo , gama-Globulinas/administração & dosagem , gama-Globulinas/imunologia
9.
Br J Oral Maxillofac Surg ; 39(1): 46-8, 2001 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-11178855

RESUMO

Peritonsillar abscess is a rare presentation of squamous cell carcinoma of the tonsil. We report two cases illustrating the need to be aware of the possibility and reinforcing the need to send all excised tissue for histopathological diagnosis.


Assuntos
Carcinoma de Células Escamosas/diagnóstico , Abscesso Peritonsilar/diagnóstico , Neoplasias Tonsilares/diagnóstico , Adulto , Carcinoma de Células Escamosas/patologia , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Tonsilares/patologia
10.
J Immunol ; 164(12): 6268-75, 2000 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-10843680

RESUMO

Generation of the B cell recall response appears to involve interaction of Ag, in the form of an immune complex (IC) trapped on follicular dendritic cells (FDCs), with germinal center (GC) B cells. Thus, the expression of receptors on FDC and B cells that interact with ICs could be critical to the induction of an optimal recall response. FDCs in GCs, but not in primary follicles, express high levels of the IgG Fc receptor Fc gamma RIIB. This regulated expression of Fc gamma RIIB on FDC and its relation to recall Ab responses were examined both in vitro and in vivo. Trapping of IC in spleen and lymph nodes of Fc gamma RII-/- mice was significantly reduced compared with that in wild-type controls. Addition of ICs to cultures of Ag-specific T and B cells elicited pronounced Ab responses only in the presence of FDCs. However, FDCs derived from Fc gamma RIIB-/- mice supported only low level Ab production in this situation. Similarly, when Fc gamma RIIB-/- mice were transplanted with wild-type Ag-specific T and B cells and challenged with specific Ag, the recall responses were significantly depressed compared with those of controls with wild-type FDC. These results substantiate the hypothesis that FcgammaRIIB expression on FDCs in GCs is important for FDCs to retain ICs and to mediate the conversion of ICs to a highly immunogenic form and for the generation of strong recall responses.


Assuntos
Linfócitos B/imunologia , Células Dendríticas Foliculares/imunologia , Células Dendríticas Foliculares/metabolismo , Memória Imunológica/imunologia , Receptores de IgG/fisiologia , Animais , Complexo Antígeno-Anticorpo/imunologia , Complexo Antígeno-Anticorpo/metabolismo , Complexo Antígeno-Anticorpo/fisiologia , Antígenos/imunologia , Linfócitos B/transplante , Células Cultivadas , Feminino , Memória Imunológica/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ovalbumina/imunologia , Receptores de IgG/biossíntese , Receptores de IgG/deficiência , Receptores de IgG/genética , Baço/citologia , Baço/imunologia , Baço/metabolismo , Linfócitos T/transplante , Regulação para Cima/genética , Regulação para Cima/imunologia
11.
J Immunol ; 163(8): 4315-27, 1999 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-10510371

RESUMO

Whether memory B cells possess altered differentiative potentials and respond in a qualitatively distinct fashion to extrinsic signals as compared with their naive precursors is a current subject of debate. We have investigated this issue by examining the participation of a predominant anti-arsonate clonotype in the primary and secondary responses in the spleens of A/J mice. While this clonotype gives rise to few Ab-forming cells (AFC) in the primary response, shortly after secondary immunization its memory cell progeny produce a massive splenic IgG AFC response, largely in the red pulp. Extensive clonal expansion and migration take place during the secondary AFC response but Ab V region somatic hypermutation is not reinduced. The primary and secondary germinal center (GC) responses of this clonotype are both characterized by ongoing V gene hypermutation and phenotypic selection, little or no inter-GC migration, and derivation of multiple, spatially distinct GCs from a single progenitor. However, the kinetics of these responses differ, with V genes containing a high frequency of total as well as affinity-enhancing mutations appearing rapidly in secondary GCs, suggesting either recruitment of memory cells into this response, or accelerated rates of hypermutation and selection. In contrast, the frequency of mutation observed per V gene does not increase monotonically during the primary GC response of this clonotype, suggesting ongoing emigration of B cells that have sustained affinity- and specificity-enhancing mutations.


Assuntos
Linfócitos B/imunologia , Imunização Secundária , Imunização , Memória Imunológica , Animais , Células Produtoras de Anticorpos/imunologia , Células Produtoras de Anticorpos/metabolismo , Arsenicais/imunologia , Linfócitos B/citologia , Linfócitos B/metabolismo , Sequência de Bases , Divisão Celular/imunologia , Movimento Celular/imunologia , Células Clonais , Genes de Imunoglobulinas/genética , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Haptenos/imunologia , Hemocianinas/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Imunofenotipagem , Camundongos , Camundongos Endogâmicos A , Dados de Sequência Molecular , Moluscos/imunologia , Mutação , Transdução de Sinais/imunologia
12.
J Exp Med ; 189(3): 471-82, 1999 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-9927509

RESUMO

Recently, results obtained from mice with targeted inactivations of postreplication DNA mismatch repair (MMR) genes have been interpreted to demonstrate a direct role for MMR in antibody variable (V) gene hypermutation. Here we show that mice that do not express the MMR factor Msh2 have wide-ranging defects in antigen-driven B cell responses. These include lack of progression of the germinal center (GC) reaction associated with increased intra-GC apoptosis, severely diminished antigen-specific immunoglobulin G responses, and near absence of anamnestic responses. Mice heterozygous for the Msh2 deficiency display an "intermediate" phenotype in these regards, suggesting that normal levels of Msh2 expression are critical for the B cell response. Interpretation of the impact of an MMR deficiency on the mechanism of V gene somatic hypermutation could be easily confounded by these perturbations.


Assuntos
Linfócitos B/imunologia , Reparo do DNA , Proteínas de Ligação a DNA , Ativação Linfocitária , Proteínas Proto-Oncogênicas/deficiência , Animais , Anticorpos/sangue , Apoptose , Células da Medula Óssea/imunologia , Centro Germinativo/imunologia , Heterozigoto , Homozigoto , Sistema Imunitário/anormalidades , Switching de Imunoglobulina , Isotipos de Imunoglobulinas , Camundongos , Camundongos Mutantes , Proteína 2 Homóloga a MutS , Proteínas Proto-Oncogênicas/genética , Baço/imunologia , Linfócitos T/imunologia
14.
Immunol Rev ; 162: 183-96, 1998 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9602364

RESUMO

Somatic hypermutation and selection of immunoglobulin (Ig) variable (V)-region genes, working in concert, appear to be essential for memory B-cell development in mammals. There has been substantial progress on the nature of the cis-acting DNA elements that regulate hypermutation. The data obtained suggest that the mechanisms of Ig gene hypermutation and transcription are intimately intertwined. While it has long been appreciated that stringent phenotypic selection forces are imposed on the somatically mutated Ig V regions generated during a T-cell dependent B-cell response, the mechanisms involved in this selection have remained enigmatic. Our studies have questioned the role of foreign antigen deposited on follicular dendritic cells in affinity-based positive selection of V regions, and have shown that this selection takes place in a "clone-autonomous" fashion. In addition, our data strongly suggest that affinity for antigen alone is not the driving force for selection of B-cell clones into the memory compartment. In contrast, we suggest that a combination of positive selection for increased foreign antigen binding, and negative selection of antibody V regions that are autoreactive at the onset of the response, or have acquired autoreactivity via hypermutation, results in the "specificity maturation" of the memory B-cell response.


Assuntos
Diversidade de Anticorpos/genética , Linfócitos B/imunologia , Região Variável de Imunoglobulina/genética , Memória Imunológica/genética , Mutação , Seleção Genética , Animais , Células Dendríticas/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética
15.
J Immunol ; 160(2): 728-33, 1998 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-9551908

RESUMO

The behavior of p-azophenylarsonate (Ars)-specific B cell clones during the primary T cell-dependent splenic response of A/J mice was investigated using an immunohistochemical approach. The earliest Ars-specific B cells were observed as isolated cells in the red pulp by day 3 after immunization with Ars-keyhole limpet hemocyanin, (KLH) and at day 6, large clusters of Ars-specific B cells were first detected in germinal centers, which continued to be observed for an additional 8 to 15 days. Surprisingly, no Ars-specific B cell foci were observed in or near the CD4 T cell-rich periarteriolar lymphoid sheath (PALS) during the entire primary response. Nevertheless, A/J mice immunized with (4-hydroxy-3-nitrophenyl)acetyl-chicken gamma globulin (NP-CGG) or Ars-CGG mounted robust splenic (4-hydroxy-3-nitrophenyl)acetyl or CGG-specific PALS-associated focus reactions, respectively. In contrast, no Ars-specific PALS B cell foci were detected in A/J mice immunized with Ars-CGG. These data add to a growing body of evidence indicating that B cell proliferation and differentiation in CD4 T cell-rich microenvironments are not prerequisites for the GC reaction. Taken together with previous results obtained using other model Ags, the data suggest that the specificity of the B cell Ag receptor may strongly influence the lymphoid microenvironment in which a B cell clone first undergoes Ag-driven clonal expansion and differentiation.


Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Tecido Linfoide/irrigação sanguínea , Tecido Linfoide/imunologia , p-Azobenzenoarsonato/imunologia , Animais , Arteríolas , Linfócitos B/citologia , Diferenciação Celular/imunologia , Galinhas , Epitopos/imunologia , Centro Germinativo/citologia , Tecido Linfoide/citologia , Camundongos , Camundongos Endogâmicos A , Nitrofenóis/imunologia , Fenilacetatos , gama-Globulinas/imunologia
16.
J Immunol ; 159(5): 2116-24, 1997 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-9278297

RESUMO

The role of Ag-Ab complexes (or immune complexes; ICs) in the regulation of the maturation of the B cell immune response was investigated in mice perturbed in the deposition and retention of such complexes. Loss of surface expression of Fc gammaRI and Fc gammaRIII due to targeted disruption of the common FcR gamma-chain gene results in dramatically increased deposition of ICs on follicular dendritic cells (FDCs) in germinal centers (GCs), attributed to altered clearance of circulating ICs. Despite these changes in the trapping of ICs by FDCs, serum Ab production, V gene hypermutation, isotype class switching and Ab affinity maturation are overtly unaltered. Thus, substantially augmenting B cell cognate Ag density on FDCs does not alter the outcome of the maturation of the B cell response. The significance of this finding in terms of the currently accepted model for the generation of B cell memory is discussed.


Assuntos
Complexo Antígeno-Anticorpo/imunologia , Linfócitos B/citologia , Deleção Clonal/fisiologia , Células Dendríticas/imunologia , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Memória Imunológica/fisiologia , Receptores de IgG/deficiência , Animais , Linfócitos B/imunologia , Sequência de Bases , Diferenciação Celular , Feminino , Genes de Imunoglobulinas , Genótipo , Centro Germinativo/citologia , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Mutagênese Insercional , Receptores de Antígenos de Linfócitos B/imunologia , Receptores de IgG/genética , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Transdução de Sinais , Organismos Livres de Patógenos Específicos
17.
Mol Immunol ; 34(5): 367-78, 1997 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9293770

RESUMO

Previous work on the cis-acting elements that control heavy chain variable region (VH) gene somatic hypermutation has indicated the presence of an as yet unidentified element(s) 3' of the intron enhancer that is necessary for high rate mutation. Examination of cis-acting elements involved in kappa light chain V gene hypermutation has demonstrated a requirement for both the intronic and 3' kappa enhancers in this process. To examine whether the 3'alpha heavy chain enhancer [3'alpha E(hs1,2)] is required for somatic hypermutation of VH genes, we generated two types of transgenic mice. One type was generated using a construct containing a VH promoter, a rearranged VDJ, the heavy chain intronic enhancer, and the murine heavy chain 3'alpha E(hs1,2). The transgenes in the second lines were similar to the transgenes in the first with the addition of a second complete matrix attachment region (MAR) 3' of the heavy chain intronic enhancer, and splice acceptor and polyadenylation sites between the two enhancers. Analysis of both transgenes revealed levels of mutation at least 10-fold lower than endogenous VH genes. These data suggest that the 3'alpha E(hs1,2) does not play a role analogous to the 3' kappa enhancer in the regulation of the hypermutation process. Moreover, in one of the transgenes, the presence of the 3'alpha E(hs1,2) resulted in a lack of transcription in vivo, suggesting a negative regulatory role for this enhancer in certain contexts.


Assuntos
Elementos Facilitadores Genéticos/imunologia , Genes de Imunoglobulinas , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Mutação/imunologia , Animais , Linfócitos B/metabolismo , Haptenos/administração & dosagem , Hibridomas/metabolismo , Cadeias Pesadas de Imunoglobulinas/biossíntese , Região Variável de Imunoglobulina/biossíntese , Camundongos , Camundongos Endogâmicos A , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nitrofenóis/administração & dosagem , Nitrofenóis/imunologia , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/imunologia , Fenilacetatos , Transcrição Gênica/imunologia , Transgenes/imunologia , p-Azobenzenoarsonato/administração & dosagem , p-Azobenzenoarsonato/imunologia
18.
Semin Immunol ; 8(3): 141-50, 1996 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8738913

RESUMO

We have used conventional transgenic technology and a novel transgene homologous recombination pathway to investigate the cis-acting elements necessary for murine antibody V-gene somatic hypermutation. These studies show that an undefined element 3' of the IgH intronic enhancer is required for VH hypermutation. This element appears not to be the 3' alpha IgH enhancer, at least in its "minimal' form. Elements 5' of the natural VH promoter are not necessary for hypermutation, and this promoter can be replaced by a non-Ig promoter, resulting in a reduction, but not ablation of the rate of hypermutation in the adjacent VH gene. Our analyses provide no support for "gene conversion' models of hypermutation, and support models that propose a role for transcription in hypermutation over purely DNA-based models. Analysis of a CD72/kappa chimeric transgene suggests that all of the factors that influence hypermutation of kappa transgenes have yet to be defined.


Assuntos
Genes de Imunoglobulinas , Região Variável de Imunoglobulina/genética , Mutação , Regiões Promotoras Genéticas , Transgenes , Animais , Camundongos , Recombinação Genética
19.
J Inflamm ; 47(4): 190-205, 1995.
Artigo em Inglês | MEDLINE | ID: mdl-9144076

RESUMO

It has been clearly demonstrated in rodents that nitric oxide (NO) plays an important role in host defense and immunity. However, evidence that human leukocytes express inducible nitric oxide synthase (iNOS) or its products has been inconclusive and a source of controversy. We report that iNOS could not be detected in human monocytes, HL-60 cells, neutrophils, and T cells by Western blotting analysis (< or = 10 pg) or by radiolabeled L-arginine-to-L-citrulline conversion (< or = 20 pmol L-citrulline) under conditions sufficient to induce iNOS in the rodent system and in human hepatocytes, which include activation with cytokines, endotoxins, and/or chemoattractants. However, sensitive methods such as RT-PCR and Northern blot analysis show "constitutively expressed" iNOS mRNA from human monocytes, neutrophils, Jurkat cells, and HL-60 cells. This iNOS mRNA is 4.4 kb and is similar to that seen in human hepatocytes and rodent macrophages. In spite of the constitutive expression of mRNA in neutrophils and the lack of detectable NOS activity (based on Western blotting and L-arginine-to-L-citrulline conversion assay), stimulation of human neutrophils unit FMLP in vitro induced the ADP-ribosylation of an intracellular NO target, glyceraldehyde-3-PO4 dehydrogenase (GAPDH), in a NO-dependent manner. These studies indicate that low levels of NOS protein are expressed in neutrophils (and perhaps T cells and monocytes) and produce NO following stimulation. The data indicate that, in addition to its phagocytic and tumoricidal activity. NO may also function as an autacoid signaling molecule within the cells.


Assuntos
Leucócitos Mononucleares/enzimologia , Neutrófilos/enzimologia , Óxido Nítrico Sintase/sangue , Adenosina Difosfato Ribose/sangue , Animais , Sequência de Bases , Linhagem Celular , Separação Celular/métodos , Primers do DNA/genética , DNA Complementar/sangue , DNA Complementar/genética , Regulação Enzimológica da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenases/sangue , Humanos , Técnicas In Vitro , Inflamação/enzimologia , Leucócitos Mononucleares/metabolismo , Camundongos , Dados de Sequência Molecular , Neutrófilos/metabolismo , Óxido Nítrico/sangue , Óxido Nítrico Sintase/genética , Reação em Cadeia da Polimerase , RNA Mensageiro/sangue , RNA Mensageiro/genética , Homologia de Sequência do Ácido Nucleico
20.
J Exp Med ; 181(1): 271-81, 1995 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-7807007

RESUMO

Antibody VH transgenes containing small amounts of natural 5' and 3' flanking DNA undergo nonreciprocal homologous recombination with the endogenous Igh locus in B cells. The resulting "hybrid" heavy chain loci are generated at a low frequency but are fully functional, undergoing somatic hypermutation and isotype class switching. We have used this recombination pathway to introduce a somatically mutated variable (V) region with an unusually high affinity for the hapten p-azophenylarsonate (Ars) into the preimmune antibody repertoire. The affinity of this V region for Ars is 100-fold higher than any unmutated anti-Ars antibody previously characterized. Expression of the transgene-encoded V region did not affect many aspects of antigen-driven B cell differentiation, including somatic hypermutation, in either Ars-specific transgene- or endogenous V gene-expressing clones. Thus, the regulation of these processes appears to operate in a "global" fashion, in that the mechanisms involved are imperceptive of the relative affinities for antigen of the antibodies expressed by B cell clones participating in the immune response. In contrast, the selection of V region mutants leading to affinity maturation and memory cell formation was found to be strongly influenced by the transgenic V region, but only in clones expressing this V region. Hybridomas derived from transgene- and endogenous V region-expressing memory cells were isolated at similar frequencies from individual transgenic mice. The V regions expressed by hybridomas in both of these groups had 2- to 30-fold greater affinity for Ars than their unmutated precursors, despite the fact that the transgene-encoded precursors had 100-fold higher affinity than their endogenous counterparts. These results show that the criterion for entry into the memory compartment is established not by the affinity of a B cell's V region relative to all other V regions expressed during the response, but by the affinity of this V region relative to its unmutated precursor. Thus, the development of B cell memory is regulated in a "clone-autonomous" fashion.


Assuntos
Antígenos , Linfócitos B/imunologia , Genes de Imunoglobulinas , Animais , Afinidade de Anticorpos , Diversidade de Anticorpos , Linfócitos B/citologia , Sequência de Bases , Diferenciação Celular , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Mutação , Recombinação Genética , p-Azobenzenoarsonato/imunologia
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