RESUMO
Patients who relapse after High dose therapy and autologous stem cell transplant (ASCT) for Diffuse large B cell Lymphoma (DLBCL) have a poor prognosis with a median survival of only 3-6 month.1-2 This case demonstrates the ability of thalidomide at low doses to induce durable response in a patient with DLBCL who relapsed after full intensity allogeneic transplantation.
Assuntos
Antineoplásicos/uso terapêutico , Linfoma de Células B/terapia , Linfoma Difuso de Grandes Células B/terapia , Transplante de Células-Tronco , Talidomida/uso terapêutico , Adulto , Terapia Combinada , Humanos , Linfoma de Células B/diagnóstico por imagem , Linfoma de Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/diagnóstico por imagem , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Masculino , Radiografia , Indução de Remissão , Transplante HomólogoRESUMO
Circular non-polyadenylated RNA molecules have been identified as stable transcription products of the human ETS-1 and mouse Sry genes. RNA circularization has been proposed to require two steps. The first step utilizes intramolecular base pairing to produce a transient stem-loop structure. The second step involves splicing a downstream donor splice site (DSS) to a now closely appositioned upstream acceptor splice site (ASS) within the loop. We demonstrate that the presence of long inverted repeats (IR) flanking the mouse Sry gene leads to the formation of the Sry circular transcript in cultured cells. Circularization requires the presence of both IR. As few as 400 complementary nt are necessary for this process. The presence of the IR does not significantly stimulate intermolecular annealing and trans-splicing in vivo.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas Nucleares , RNA/química , Sequências Repetitivas de Ácido Nucleico , Fatores de Transcrição , Animais , Sequência de Bases , Primers do DNA/química , Expressão Gênica , Camundongos , Dados de Sequência Molecular , Splicing de RNA , RNA Circular , RNA Mensageiro/química , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Proteína da Região Y Determinante do SexoRESUMO
A method for high-level expression of a functionally active, recombinant human red cone opsin was developed by adding the coding sequence for the C-terminal epitope of bovine rhodopsin onto the C terminus of the cone opsin and cloning the resulting construct into the vector pMEP4 beta. The recombinant pMEP4 beta vector was transfected stably into 293-EBNA cells, and expression of the cone opsin was induced by the addition of CdCl2 into the medium. The recombinant cone opsin was reconstituted with 11-cis retinal and purified by immunoaffinity chromatography. Spectral analysis prior to and following photobleaching confirmed its identity as a red cone opsin. The protein was targeted to the cell membrane and activated bovine transducin.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/biossíntese , Rodopsina/biossíntese , Opsinas de Bastonetes/biossíntese , Animais , Western Blotting , Cloreto de Cádmio/farmacologia , Bovinos , Células Cultivadas , Cromatografia de Afinidade , DNA Complementar/genética , Escherichia coli/genética , Genes Reporter , Vetores Genéticos/genética , Humanos , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Testes de Precipitina , Proteínas Recombinantes de Fusão/genética , Retinaldeído/química , Rodopsina/genética , Opsinas de Bastonetes/genética , Sensibilidade e Especificidade , Transducina/metabolismo , Transfecção , beta-Galactosidase/análise , beta-Galactosidase/genéticaRESUMO
PURPOSE: To test the effects of disruption of a conserved cysteine in the green cone opsin molecule on light-activated isomerization, transducin activation, folding, transport, and protein half-life. METHODS: Stable cell lines were established by transfecting 293-EBNA cells with a plasmid containing wild-type or mutant (C203R, C203S, C126S, C126S/C203S) green opsin cDNA molecules. The proteins were induced by culturing the cells in the presence of cadmium chloride and analyzed by spectra, transducin activation, Western blotting, pulse-labeling with immunoprecipitation, and immunocytochemistry. RESULTS: The C203R mutation disrupts the folding and half-life of the green opsin molecule and its abilities to absorb light at the appropriate wavelength and to activate transducin. Similar disruption of folding, half-life, and light activation occurs when Cys203 or its presumed partner for formation of a disulfide bond (Cys126) is replaced by serine residues. CONCLUSIONS: Like rhodopsin, the folding of the cone opsins appears to be dependent on the formation of a disulfide bond between the third transmembrane helix and the second extracellular loop. Disruption of this disulfide bond represents a cause of color vision deficiencies that is unrelated to spectral shifts of the photopigment.
Assuntos
Sequência Conservada , Cisteína/genética , Mutação , Células Fotorreceptoras Retinianas Cones/metabolismo , Opsinas de Bastonetes/genética , Sequência de Aminoácidos , Linhagem Celular , Percepção de Cores , Estabilidade de Medicamentos , Retículo Endoplasmático/metabolismo , Ligação Genética , Humanos , Isomerismo , Luz , Dados de Sequência Molecular , Dobramento de Proteína , Opsinas de Bastonetes/metabolismo , Transducina/fisiologia , Transducina/efeitos da radiação , Transtornos da Visão/genética , Cromossomo XRESUMO
PURPOSE: To test the effects of disruption of a conserved proline in the green cone opsin molecule on light-activated isomerization, transducin activation, protein accumulation, glycosylation, and transport. METHODS: Stable cell lines were established by transfecting EBNA-293 cells with a plasmid containing wild-type or mutant (P307L) green opsin cDNA molecules. The proteins were induced by culturing the cells in the presence of CdCl2 and analyzed by spectra, transducin activation, Western blotting, and immunocytochemistry. RESULTS: The P307L mutation diminished ability of the visual pigment to absorb light at the appropriate wavelength and to activate transducin. Protein glycosylation and transport to the cell membrane were unaffected. Although there was some diminution in the accumulation of the opsin, this was insufficient to account for the observed effect. CONCLUSIONS: Like rhodopsin, the formation of the cone opsins visual pigments is dependent on the binding of retinal into a hydrophobic pocket that is formed by the second and fourth transmembranous loops. Disruption of a conserved proline near the retinal binding site represents a cause of color vision deficiency that is unrelated to spectral shifts of the photopigment.
Assuntos
Prolina/genética , Células Fotorreceptoras Retinianas Cones/fisiologia , Opsinas de Bastonetes/fisiologia , Transporte Biológico , Western Blotting , Células Cultivadas , Ligação Genética , Glicosilação , Humanos , Imuno-Histoquímica , Mutagênese , Células Fotorreceptoras Retinianas Cones/metabolismo , Opsinas de Bastonetes/genética , Opsinas de Bastonetes/metabolismo , Análise Espectral , Transducina/metabolismo , Transfecção , Cromossomo XRESUMO
PURPOSE: This study was designed to test whether palmitoylation and glycosylation are required for the formation of the green opsin visual pigment. METHODS: Stable cell lines were established by transfecting EBNA-293 cells with a pMEP4ss recombinant plasmid containing wild-type bovine rhodopsin or wild-type or mutant (N32S) green opsin cDNA molecules that included a tag for the eight amino acid residues located at the C-terminus of rhodopsin. The opsins were induced by addition of CdCl2 into the medium and then reconstituted with 11-cis-retinal. The reconstituted opsins were purified by immunoaffinity chromatography, then analyzed by difference spectra, and by binding 35S-GTP in the presence of bovine transducin. Non-reconstituted opsins were analyzed by Western blotting and by pulse-labeling with 3H-palmitic acid followed by immunoprecipitation. RESULTS: Elimination of glycosylation by mutagenesis of the N-linked glycosylation site did not impair the ability of the resulting cone opsin to absorb light at the appropriate wavelength nor to activate transducin. Furthermore, as judged by pulse-labeling with 3H-palmitic acid and immunoprecipitation and by gas chromatography-mass spectroscopy, the wild type green opsin differs from rhodopsin by not being palmitoylated. CONCLUSIONS: Glycosylation and palmitoylation are not required for the formation of cone opsin visual pigments. For the previously described green opsin C203R mutation, disruption of folding and transport, rather than altered glycosylation is sufficient to explain the associated color vision deficiency.
Assuntos
Ácido Palmítico/metabolismo , Opsinas de Bastonetes/biossíntese , Western Blotting , Linhagem Celular , Glicosilação , Humanos , Mutagênese , Ácido Palmítico/análise , Proteínas Recombinantes de Fusão , Rodopsina/análise , Rodopsina/metabolismo , Opsinas de Bastonetes/genética , Opsinas de Bastonetes/metabolismo , Transducina/metabolismo , TransfecçãoRESUMO
The distinction between a specific factor inactivator and a non-specific inhibitor is important when confronted by a patient with a history of bleeding and abnormal in-vitro coagulation tests. We report on two patients who presented with bleeding and a prolonged activated partial thromboplastin time. Initial factor assays suggested combined deficiency of factors VIII and IX as a result of the presence of inactivators. The use of dilution studies, chromogenic assays, a novel in-house enzyme-linked-immunosorbent-assay-based technique and phospholipid neutralization, demonstrated that Case 1 had a genuine factor VIII inactivator resulting in factor VIII levels of less than 1 IU/dl but no factor IX deficiency. Case 2 had normal levels of factor VIII on further testing and no specific inactivator to either factor VIII or IX but a potent antiphospholipid antibody which had interfered with the phospholipid-dependent in-vitro assays. Care must be taken in the interpretation of laboratory assays in the presence of antiphospholipid antibodies to ensure that the correct diagnosis is made and inappropriate treatment avoided.
Assuntos
Anticorpos Antifosfolipídeos/imunologia , Fator VIII/imunologia , Hemofilia A/diagnóstico , Hemofilia A/imunologia , Lúpus Eritematoso Sistêmico/diagnóstico , Lúpus Eritematoso Sistêmico/imunologia , Adulto , Idoso , Anticorpos/imunologia , Diagnóstico Diferencial , Fator IX/imunologia , Feminino , Hemofilia A/sangue , Humanos , Imunoensaio/métodos , Lúpus Eritematoso Sistêmico/sangue , Masculino , Sensibilidade e EspecificidadeRESUMO
Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is a clinical syndrome of a chronic progressive or relapsing and remitting, symmetrical, sensory and motor radiculoneuropathy. The immune reaction in CIDP is characterised by selective inflammation of peripheral nerves and is probably due to the interaction of cellular and humoral responses. Only three treatments for CIDP have demonstrated benefit in randomised studies, corticosteroids, plasma exchange and intravenous immunoglobulin. 25% of patients fail to respond or do not respond adequately to these treatments. Experimental data in animal models have shown that several autoimmune disorders, either congenital or acquired, can be transferred and/or treated by the transplantation of bone marrow stem cells. Haematopoietic stem cell transplantation (HSCT) has been performed with varying success in over 700 patients with autoimmune disorders throughout Europe. The experience in CIDP is very limited. This article will review current understanding of CIDP and experience of the use of HSCT in refractory CIDP.
Assuntos
Transplante de Células-Tronco Hematopoéticas/métodos , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/imunologia , Polirradiculoneuropatia Desmielinizante Inflamatória Crônica/terapia , Corticosteroides/uso terapêutico , Humanos , Imunoglobulinas Intravenosas/uso terapêutico , Troca Plasmática/métodosRESUMO
Disease relapse following an allogeneic transplant remains a major cause of treatment failure, often with a poor outcome. Second allogeneic transplant procedures have been associated with high TRM, especially with myeloablative conditioning. We hypothesized that the use of reduced-intensity conditioning (RIC) would decrease the TRM. We performed a retrospective national multicentre analysis of 71 patients receiving a second allogeneic transplant using RIC after disease relapse following an initial allogeneic transplant. The majority of patients had leukaemia/myelodysplasia (MDS) (N=57), nine had lymphoproliferative disorders, two had myeloma and three had myeloproliferative diseases. A total of 25% of patients had unrelated donors. The median follow-up was 906 days from the second allograft. The predicted overall survival (OS) and TRM at 2 years were 28 and 27%, respectively. TRM was significantly lower in those who relapsed late (>11 months) following the first transplant (2 years: 17 vs 38% in early relapses; P=0.03). Two factors were significantly associated with a better survival: late relapse (P=0.014) and chronic GVHD following the second transplant (P=0.014). These data support our hypothesis that the second RIC allograft results in a lower TRM than using MA. A proportion of patients achieved a sustained remission even when relapsing after a previous MA transplant.
Assuntos
Neoplasias Hematológicas/terapia , Transplante de Células-Tronco Hematopoéticas , Recidiva Local de Neoplasia/terapia , Sistema de Registros , Condicionamento Pré-Transplante/métodos , Adolescente , Adulto , Idoso , Criança , Doença Enxerto-Hospedeiro , Humanos , Pessoa de Meia-Idade , Estudos Retrospectivos , Análise de Sobrevida , Transplante Homólogo , Adulto JovemRESUMO
PIP: The proposal for Pakistan's family planning program in the 7th 5-year plan (1988-1993) is based on the experience gained during the previous plans. The current program emphasizes better and wider delivery of birth control services and a more intensive and varied motivational program. During the 7th plan, the emphasis will be on lowering the population growth rate. Overall policy will attempt to bring about a behavioral change in favor of safe delivery. Fertility management will be the key development objective. A multi-sectoral approach will be followed by involving all ministries and departments in dealing with population-related issues and by incorporating population components in their activities. These ministries and departments are expected to play a vital role in contributing to the communication strategy, population education, and service delivery. The objectives of the plan are 1) to raise contraceptive prevalence from 12.9% to 23.4% by 1992-1993, 2) to provide reproductive care to mothers and child health care to children under 5, 3) to reduce the crude birth rate from 42 to 38/1000, and 4) to prevent 3.1 million births. Strategies aim at strengthening field implementation and service delivery systems, introducing mobile service units, and emphasizing effective contraceptive methods. The monitoring and evaluation section lacks training resources, does not provide sufficient information to policy makers, and lacks good monitoring and evaluation at the operational level.^ieng
Assuntos
Administração Financeira , Objetivos , Diretrizes para o Planejamento em Saúde , Planejamento em Saúde , Centros de Saúde Materno-Infantil , Organização e Administração , Avaliação de Programas e Projetos de Saúde , Planejamento Social , Ásia , Atenção à Saúde , Países em Desenvolvimento , Economia , Serviços de Planejamento Familiar , Saúde , Serviços de Saúde , Paquistão , Atenção Primária à SaúdeRESUMO
We have addressed the problem of distinguishing angiogenesis induced in the chick chorioallantoic membrane by injury and inflammation from angiogenesis induced by primary stimulation. Focal, slow-release application of trypsin stimulated a localized spoke-wheel pattern of vascularity. In comparison, a range of doses up to a sublethal amount of trypsin applied generally, in liquid form, resulted in no change in DNA synthesis or vessel content, despite a transient influx of inflammatory cells. This contrasts with previous work with fibrin degradation products, histamine and heparin which each produce characteristic patterns of increased DNA synthesis leading to angiogenesis in the entire 'dropped' area of the chorioallantoic membrane. Such general application, therefore, avoids the danger of misinterpretation of focal, toxic effects.
Assuntos
Alantoide/efeitos dos fármacos , Córion/efeitos dos fármacos , Membranas Extraembrionárias/efeitos dos fármacos , Neovascularização Patológica/patologia , Tripsina/farmacologia , Alantoide/irrigação sanguínea , Alantoide/patologia , Animais , Embrião de Galinha , Córion/irrigação sanguínea , Córion/patologia , Inflamação/induzido quimicamente , Tripsina/toxicidadeRESUMO
High dose chemoradiotherapy with autologous peripheral blood progenitor cell transplantation (PBPCT) may improve outcome in myeloma. Melphalan is an effective drug in the treatment of myeloma, but is potentially toxic to progenitor cells. We studied 8 patients receiving intermittent intravenous melphalan (25 mg/m2) as induction therapy before PBPCT to assess engraftment characteristics post-transplantation. Comparison was made with an age-matched control group of patients with non-Hodgkins lymphoma who had not received melphalan during induction therapy. There was correlation (P=0.037) between the dose of melphalan per kg body weight given, premobilization, and days to neutrophil engraftment, but no significant difference between the two groups in neutrophil recovery. The study group had delayed platelet recovery (P=0.01) and required more platelet support post-transplantation (P=0.05). 3-4 weekly melphalan (25 mg/m2) up to 6 courses was delivered to patients who went on to PBPCT without significantly influencing neutrophil recovery but with a negative impact on platelet recovery.
Assuntos
Antineoplásicos Alquilantes/uso terapêutico , Transplante de Células-Tronco Hematopoéticas , Melfalan/uso terapêutico , Mieloma Múltiplo/terapia , Antineoplásicos Alquilantes/farmacologia , Terapia Combinada , Feminino , Mobilização de Células-Tronco Hematopoéticas , Humanos , Masculino , Melfalan/farmacologia , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Transplante AutólogoRESUMO
The healing wound offers a clear example of the sequence of events in chronic inflammation leading to repair. Although angiogenesis has an obvious and essential role in this process, it has been little studied. For an angiogenic factor to seem relevant, it would have to be shown to precede the peak of increased vascularity. To define this peak, the vessel content of simple, incised mouse wounds was estimated using morphometry of histological sections, and found to rise to a maximum at days 5 and 6. Total angiogenic activity of aqueous extracts was found to reach a peak at day 3. The detection of such activity on the chick chorioallantoic membrane is very dependent on the preparation technique and the choice of proteinase inhibitors. Previous in vitro work by us using purified material has shown fibrin degradation products to be effective in stimulating angiogenesis. Fibrin degradation products are prominent on immunoblotting from day 3, when macrophages are plentiful, with a similar band pattern to human granulation tissue.
Assuntos
Fibrinólise/fisiologia , Neovascularização Patológica/fisiopatologia , Cicatrização/fisiologia , Animais , Endopeptidases/metabolismo , Fibrina/análise , Produtos de Degradação da Fibrina e do Fibrinogênio/análise , Masculino , Camundongos , Camundongos Endogâmicos , Fluxo Sanguíneo Regional , Pele/irrigação sanguínea , Pele/lesões , Manejo de Espécimes/métodosRESUMO
G protein-coupled receptors (GPCRs) are seven-transmembrane (TM) helical proteins that bind extracellular molecules and transduce signals by coupling to heterotrimeric G proteins in the cytoplasm. The human D4 dopamine receptor is a particularly interesting GPCR because the polypeptide loop linking TM helices 5 and 6 (loop i3) may contain from 2 to 10 similar direct hexadecapeptide repeats. The precise role of loop i3 in D4 receptor function is not known. To clarify the role of loop i3 in G protein coupling, we constructed synthetic genes for the three main D4 receptor variants. D4-2, D4-4, and D4-7 receptors contain 2, 4, and 7 imperfect hexadecapeptide repeats in loop i3, respectively. We expressed and characterized the synthetic genes and found no significant effect of the D4 receptor polymorphisms on antagonist or agonist binding. We developed a cell-based assay where activated D4 receptors coupled to a Pertussis toxin-sensitive pathway to increase intracellular calcium concentration. Studies using receptor mutants showed that the regions of loop i3 near TM helices 5 and 6 were required for G protein coupling. The hexadecapeptide repeats were not required for G protein-mediated calcium flux. Cell membranes containing expressed D4 receptors and receptor mutants were reconstituted with purified recombinant G protein alpha subunits. The results show that each D4 receptor variant is capable of coupling to several G(i)alpha subtypes. Furthermore, there is no evidence of any quantitative difference in G protein coupling related to the number of hexadecapeptide repeats in loop i3. Thus, loop i3 is required for D4 receptors to activate G proteins. However, the polymorphic region of the loop does not appear to affect the specificity or efficiency of G(i)alpha coupling.