RESUMO
INTRODUCTION: Quantitation of ADAMTS13 activity, functional inhibitors, and autoantibodies is crucial in diagnosis and management of thrombotic thrombocytopenic purpura. We compared and optimized commercial assay kits and validated a testing panel. METHODS: Citrated plasma specimens from healthy volunteers and residual samples submitted for clinical testing were used in the study. Commercially available ADAMTS13 activity assays including ACTIFLUOR(™) ADAMTS13 (Sekisui Diagnostics, Stamford, CT, USA), LIFECODES ATS-13 (Gen-Probe Inc., San Diego, CA, USA), and TECHNOZYM(®) ADAMTS-13 (Technoclone, Vienna, Austria) were evaluated. Functional inhibitor assays were performed using internally developed mixing protocols. Two autoantibody assays were also evaluated: IMUBIND(®) (Sekisui Diagnostics) and TECHNOZYM(®) ADAMTS-13 INH ELISA kits (Technoclone). RESULTS: A laboratory-developed assay using ACTIFLUOR(™) reagents showed best agreement with the reference method, and full validation showed a reportable range of 5% (LLOQ) to 114% with a reference interval of ≥68%. Both intra- and interassay coefficients of variation were <10%. Inhibitor assays performed with the kits showed 95% overall agreement with the reference method. A modification of the TECHNOZYM(®) autoantibody assay showed 85% overall agreement with the reference method with imprecision approximately 20%. CONCLUSION: ADAMTS13 activity and inhibitor tests using ACTIFLUOR(™) reagents and modified TECHNOZYM(®) autoantibody ELISA showed superior performance compared to the other kits for clinical use in this study.
Assuntos
Proteína ADAMTS13/antagonistas & inibidores , Autoanticorpos/sangue , Inibidores de Proteases/sangue , Púrpura Trombocitopênica Trombótica , Kit de Reagentes para Diagnóstico , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Púrpura Trombocitopênica Trombótica/sangue , Púrpura Trombocitopênica Trombótica/diagnósticoRESUMO
INTRODUCTION: The 2008 WHO criteria for the diagnosis and classification of myeloproliferative neoplasms (MPN) rely in part upon the assessment of mutations in JAK2 and MPL genes. Recently, mutations in calreticulin (CALR) have been identified in MPN lacking JAK2 and MPL mutations. We have validated a sensitive fragment analysis assay to detect CALR mutations. METHODS: Genomic DNA from peripheral blood, bone marrow, and FFPE bone marrow clot preparations from 52 MPN specimens with known JAK2 and MPL mutation status and 29 non-MPN specimens was analyzed. CALR mutation testing was performed by fragment length analysis, and the results were confirmed by sequencing. Accuracy, precision, sensitivity, specificity, and robustness of the assay were determined. RESULTS: Forty specimens (32 JAK2+, 2 JAK2-/MPL+, and 6 JAK2-/MPL-) were negative for CALR mutations. Twelve specimens had CALR mutations including 52 bp deletion (5), 5 bp insertion (6), and a novel 9 bp deletion (1). This 9 bp inframe deletion occurring at an allelic burden of 50% would delete three amino acids. One specimen with a 52 bp deletion also had JAK2 V617F mutation. All 29 non-MPN specimens were negative for CALR mutations. The assay accurately identified the mutation status of all 52 MPN specimens and had a coefficient of variation <3% for the fragment size and mutant peaks with a sensitivity of 5% for indels. CONCLUSIONS: Fragment analysis is an accurate and sensitive method for the detection of CALR indels. The novel 9 bp deletion is likely a germline variant. Consequence of coexisting JAK2 V617F and CALR mutations requires careful interpretation.
Assuntos
Sequência de Bases , Calreticulina/genética , Éxons , Neoplasias Hematológicas/genética , Janus Quinase 2/genética , Mutação de Sentido Incorreto , Transtornos Mieloproliferativos/genética , Deleção de Sequência , Substituição de Aminoácidos , Feminino , Humanos , MasculinoRESUMO
Analysis and dissemination of transplant patient safety data are essential to understanding key issues facing the transplant community and fostering a "culture of safety." The Organ Procurement and Transplantation Network's (OPTN) Operations and Safety Committee de-identified safety situations reported through several mechanisms, including the OPTN's online patient safety portal, through which the number of reported cases has risen sharply. From 2012 to 2013, 438 events were received through either the online portal or other reporting pathways, and about half were self-reports. Communication breakdowns (22.8%) and testing issues (16.0%) were the most common types. Events included preventable errors that led to organ discard as well as near misses. Among events reported by Organ Procurement Organization (OPOs), half came from just 10 of the 58 institutions, while half of events reported by transplant centers came from just 21 of 250 institutions. Thirteen (23%) OPOs and 155 (62%) transplant centers reported no events, suggesting substantial underreporting of safety-related errors to the national database. This is the first comprehensive, published report of the OPTN's safety efforts. Our goals are to raise awareness of safety data recently reported to the OPTN, encourage additional reporting, and spur systems improvements to mitigate future risk.
Assuntos
Bases de Dados Factuais , Transplante de Órgãos , Segurança do Paciente/normas , Obtenção de Tecidos e Órgãos/tendências , Comunicação , Coleta de Dados , Humanos , Sistema de Registros , Estados UnidosRESUMO
Assessing the pharmacodynamics (PD) of a potential therapeutic through the use of a downstream biomarker is essential. This is traditionally performed in the target tissue but limited volume and invasiveness of sampling pose challenges with solid tumours. Currently, there are several small molecule receptor kinase inhibitors and large molecule therapeutic antibodies in clinical trials that interfere with TGFbeta signalling to treat various forms of cancer. With the advent of these new therapies, there is a need for a surrogate tissue that is easily accessible and indicative of tumour response. We propose the use of an ex vivo TGFbeta1 stimulation of peripheral blood mononuclear cells (PBMCs) coupled with the measurement of phosphorylated SMAD2 (Sma/Mothers Against dpp, a downstream transcriptional activator) using a sandwich ELISA. TGFbeta is involved in many different cellular responses, such as proliferation, angiogenesis, migration, invasion and immunomodulation. SMAD2 and SMAD3 are phosphorylated as a result of the canonical cascade through ligand binding and receptor kinase activation. These phosphorylated SMADs (pSMAD) associate with SMAD4, a co-SMAD, and transcriptionally activate TGFbeta-mediated genes. This paper describes the novel method for measuring the downstream effects of inhibiting canonical TGFbeta signalling using ex vivo stimulation of surrogate tissue to predict tumour response. In addition, we present the assay validation rationale and data. This novel, validated assay can be used to gain insight into clinical trials regarding TGFbeta signal modulation by multiple inhibitor platforms for both large and small molecules.
Assuntos
Receptores de Ativinas Tipo I/antagonistas & inibidores , Leucócitos Mononucleares/metabolismo , Neoplasias/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Western Blotting , Linhagem Celular Tumoral , Interpretação Estatística de Dados , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Camundongos , Camundongos Nus , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fosforilação/efeitos dos fármacos , Proteínas Serina-Treonina Quinases , Ratos , Ratos Endogâmicos F344 , Receptor do Fator de Crescimento Transformador beta Tipo I , Reprodutibilidade dos Testes , Proteínas Smad/análise , Proteína Smad2/análise , Proteína Smad2/metabolismo , Proteína Smad3/análise , Proteína Smad3/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Several model systems of plasmodia have demonstrated the potential of the merozoite surface protein, MSP-1, to induce protective immunity. However, little is known about the function of this protein or its interaction with other surface molecules that may also serve as immunological targets. To identify potentially significant inter- and intra-molecular interactions involving MSP-1, we have utilized the yeast two-hybrid system. A cDNA activation domain library was constructed from the erythrocytic stages of the murine malarial parasite Plasmodium yoelii yoelii 17XL. A 795 bp region of Py17XL MSP-1 (bait), homologous to the Plasmodium falciparum MSP1(33) fragment, was inserted into a Gal4p DNA binding domain vector and used to screen the activation domain library (target). Several randomly selected clones that demonstrated bait-target interaction were found to express overlapping regions of Py17XL MSP-1. Deletion constructs further localized the peptide fragments retaining interaction indicating that a region within the MSP-1(38) fragment interacts with the MSP-1 bait domain. Subsequent studies confirmed this interaction, as both peptides were co-precipitated from cell lysate by a peptide tag-specific antibody. It was observed that the interaction of these two fragments significantly increased the half-life of the MSP-1(38) within yeast cells. The specific interaction described here demonstrates the potential of this approach to elucidate additional inter- or intra-molecular interactions of Py17XL MSP1 and other malarial proteins.
Assuntos
Proteína 1 de Superfície de Merozoito/química , Proteína 1 de Superfície de Merozoito/metabolismo , Plasmodium yoelii/metabolismo , Sequência de Aminoácidos , Animais , Eritrócitos/parasitologia , Biblioteca Gênica , Malária/parasitologia , Masculino , Proteína 1 de Superfície de Merozoito/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Plasmodium yoelii/química , Estrutura Terciária de Proteína , Análise de Sequência de DNA , Técnicas do Sistema de Duplo-HíbridoRESUMO
For many inborn errors of metabolism, early treatment is critical to prevent long-term developmental sequelae. We have previously shown that systemic treatment of neonatal mucopolysaccharidosis type VII (MPS VII) mice with recombinant adeno-associated virus (AAV) vectors results in relatively long-term expression of beta-glucuronidase (GUSB) in multiple tissues, and a reduction in lysosomal storage. Here, we demonstrate that therapeutic levels of enzyme persist for at least 1 year following a single intravenous injection of virus in neonatal MPS VII mice. The level and distribution of GUSB expression achieved is sufficient to prevent the development of many aspects of clinical disease over the life of the animal. Following treatment, bone lengths, weights and retinal function were maintained at nearly normal levels throughout the life of the animal. In addition, significant improvements in survival and auditory function were seen in AAV-treated MPS VII mice when compared with untreated mutant siblings. These data suggest that AAV-mediated gene transfer in the neonatal period can lead to prevention of many of the clinical symptoms associated with MPS VII in the murine model of this disease.
Assuntos
Terapia Genética/métodos , Glucuronidase/genética , Mucopolissacaridose VII/terapia , Transdução Genética , Animais , Animais Recém-Nascidos , Surdez/fisiopatologia , Eletrorretinografia , Potenciais Evocados Auditivos do Tronco Encefálico , Expressão Gênica , Camundongos , Camundongos Mutantes , Modelos Animais , Mucopolissacaridose VII/enzimologia , Mucopolissacaridose VII/fisiopatologia , Retina/fisiopatologia , Taxa de SobrevidaRESUMO
Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by the lack of beta-glucuronidase (GUSB) activity. GUSB deficiency leads to the progressive accumulation of undegraded glycosaminoglycans (GAGs) in cells of most tissues, including the brain, and is associated with mental retardation. Reduction of lysosomal storage in the central nervous system and prevention of cognitive dysfunction may require intracranial delivery of a therapeutic agent during the newborn period that provides a continuous source of GUSB. Therefore, we injected recombinant adeno-associated virus encoding human GUSB into both the anterior cortex and the hippocampus of newborn MPS VII mice. Total GUSB activity in the brain approached normal levels by 18 weeks. Although GUSB activity was concentrated near the injection sites, lysosomal distension was reduced in most areas of the brain. In addition to histopathologic evidence of GAG reduction, the previously undescribed accumulation of GM2 and GM3 gangliosides in the brain was also prevented. Furthermore, GUSB expression and reduced lysosomal distension correlated with improvements in cognitive function as measured in the Morris Water Maze test. These findings indicate that localized overexpression of GUSB has positive effects on the pathology and cognitive function and does not have overt toxicity.
Assuntos
Dependovirus/genética , Terapia Genética , Vetores Genéticos , Glucuronidase/genética , Mucopolissacaridose VII/terapia , Animais , Animais Recém-Nascidos , Córtex Cerebral , Expressão Gênica , Técnicas de Transferência de Genes , Glucuronidase/deficiência , Glucuronidase/uso terapêutico , Hipocampo , Injeções , Camundongos , Modelos Animais , Mucopolissacaridose VII/fisiopatologia , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Lysosomal storage diseases are a class of inborn errors of metabolism that lead to widespread disease in multiple tissues. The murine model of mucopolysaccharidosis type VII (MPS VII) closely parallels the human syndrome and has been extensively used to investigate the natural history and therapeutic strategies for lysosomal storage diseases in general. Here we demonstrate a previously undescribed immune defect in the MPS VII mouse. Although the normal populations of cells are present in lymph nodes of these mice, MPS VII mice show a blunted T cell proliferative response and decreased antibody production after immunization with antigens. One mechanism of this defect is ineffective processing of protein antigens, as responses to peptide antigens are normal. This phenotype is presumably caused by the lysosomal disorder, as the defect can be corrected in vivo by direct enzyme replacement therapy. These findings have implications for the use of this animal model, and may have clinical significance for other, more-common lysosomal storage diseases.
Assuntos
Mucopolissacaridose VII/imunologia , Animais , Divisão Celular , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Glucuronidase/uso terapêutico , Humanos , Subpopulações de Linfócitos , Camundongos , Proteínas Recombinantes/uso terapêuticoRESUMO
The fragile X syndrome is the most commonly inherited cause of mental retardation. Genetic diagnosis of this disease relies on the detection of triplet repeat expansion in the FMR1 gene on the X chromosome. Although the majority of disease in fragile X patients is due to mutations involving triplet repeat expansion, deletion of various portions of FMR1 has also been described in association with the fragile X syndrome. Here we describe a rare polymorphism in the noncoding region of FMR1 that mimics detection of a deletion in a commonly used assay for fragile X syndrome, which can result in misdiagnosis of the disease.
Assuntos
Erros de Diagnóstico , Síndrome do Cromossomo X Frágil/diagnóstico , Síndrome do Cromossomo X Frágil/genética , Proteínas do Tecido Nervoso/genética , Polimorfismo Genético , Proteínas de Ligação a RNA , Deleção de Sequência/genética , Artefatos , Sequência de Bases , Southern Blotting , Desoxirribonuclease EcoRI/metabolismo , Proteína do X Frágil da Deficiência Intelectual , Frequência do Gene , Humanos , Masculino , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNARESUMO
For many inborn errors of metabolism, early treatment is critical to prevent long-term developmental sequelae. We have used a gene-therapy approach to demonstrate this concept in a murine model of mucopolysaccharidosis type VII (MPS VII). Newborn MPS VII mice received a single intravenous injection with 5.4 x 10(6) infectious units of recombinant adeno-associated virus encoding the human beta-glucuronidase (GUSB) cDNA. Therapeutic levels of GUSB expression were achieved by 1 week of age in liver, heart, lung, spleen, kidney, brain, and retina. GUSB expression persisted in most organs for the 16-week duration of the study at levels sufficient to either reduce or prevent completely lysosomal storage. Of particular significance, neurons, microglia, and meninges of the central nervous system were virtually cleared of disease. In addition, neonatal treatment of MPS VII mice provided access to the central nervous system via an intravenous route, avoiding a more invasive procedure later in life. These data suggest that gene transfer mediated by adeno-associated virus can achieve therapeutically relevant levels of enzyme very early in life and that the rapid growth and differentiation of tissues does not limit long-term expression.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética , Glucuronidase/genética , Doenças por Armazenamento dos Lisossomos/terapia , Mucopolissacaridose VII/terapia , Animais , Animais Recém-Nascidos , DNA Complementar , Dependovirus , Vetores Genéticos , Glucuronidase/biossíntese , Glicosaminoglicanos/metabolismo , Humanos , Fígado/metabolismo , Fígado/patologia , Doenças por Armazenamento dos Lisossomos/patologia , Lisossomos/enzimologia , Camundongos , Camundongos Mutantes , Mucopolissacaridose VII/metabolismo , Mucopolissacaridose VII/patologia , Reação em Cadeia da Polimerase , Proteínas Recombinantes/biossínteseRESUMO
For many metabolic diseases, early correction of the inherited deficiency is required to prevent long-term sequelae. We examined the ability of adeno-associated virus (AAV) to mediate efficient gene transfer during the neonatal period in mice with the lysosomal storage disease mucopolysaccharidosis type VII (MPS VII). Quadriceps of newborn MPS VII mice were injected with an AAV vector containing human beta-glucuronidase (GUSB) cDNA. High-level intramuscular GUSB expression was seen as early as 2 weeks of age, and persisted for at least 16 weeks with no reduction in activity. In addition, GUSB activity was detected in both liver and spleen at later time points. The level of GUSB activity resulted in a significant reduction in lysosomal storage in the liver and a minimal reduction in the spleen at 16 weeks. However, the temporal and spatial pattern of hepatic GUSB activity, coupled with the presence of GUSB cDNA in liver sections, suggests that hematogenous dissemination of virus at the time of injection led to gene transfer to hepatic cells. These results demonstrate that AAV vectors can successfully infect neonatal muscle and persist through the rapid growth phase following birth. However, GUSB secretion from an intramuscular source is inefficient, limiting the therapeutic efficacy of this approach.
Assuntos
Animais Recém-Nascidos , Terapia Genética , Glucuronidase/genética , Glucuronidase/metabolismo , Mucopolissacaridose VII/terapia , Fatores Etários , Animais , Southern Blotting , Dependovirus/genética , Regulação Enzimológica da Expressão Gênica , Vetores Genéticos/administração & dosagem , Glicosaminoglicanos/química , Humanos , Injeções Intramusculares , Fígado/citologia , Fígado/enzimologia , Fígado/patologia , Lisossomos/química , Camundongos , Camundongos Mutantes , Mucopolissacaridose VII/patologia , Baço/citologia , Baço/enzimologia , beta-N-Acetil-Hexosaminidases/químicaRESUMO
A leading candidate for a vaccine targeted at the erythrocytic stages of plasmodial parasite development is the merozoite surface protein-1 (MSP-1). We have previously shown that the carboxyl-terminal region of MSP-1 derived from Plasmodium yoelii yoelii 17XL, expressed as a fusion protein with glutathione S-transferase (GST-PYC2), can immunize mice against an otherwise lethal homologous challenge infection. This protection has been shown to be predominantly mediated by antibodies. We report here on the efficacy of immunization with MSP-1 carboxyl regions when the challenge is a heterologous rodent parasite species. The course of parasitemia was not altered in mice immunized with GST-PYC2 and challenged with 10(4) heterologous Plasmodium chabaudi adami parasites, as both control and immunized mice developed infections that peaked at day 7 and then rapidly declined. Similarly, mice immunized with GST-PYC2 and challenged with 10(5) Plasmodium berghei ANKA parasites displayed virulence similar to that seen in infection control mice. The homologous region of the P. chabaudi adami MSP-1 gene was similarly expressed as a fusion protein with GST. Mice immunized with GST-PCC2 and challenged with 10(4) parasites showed significant protection against homologous P. chabaudi adami infection but no protection whatsoever against heterologous P. yoelii yoelii 17XL infection. These in vivo results correlate with the observation that sera generated by immunization with the carboxyl region of MSP-1 recognizes this protein from homologous, but not heterologous, radiolabeled parasite protein preparations.
Assuntos
Anticorpos Antiprotozoários/imunologia , Imunização , Malária/prevenção & controle , Proteína 1 de Superfície de Merozoito/imunologia , Plasmodium/imunologia , Sequência de Aminoácidos , Animais , Especificidade de Anticorpos , Malária/imunologia , Malária/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Parasitemia , Plasmodium berghei/imunologia , Plasmodium chabaudi/imunologia , Plasmodium yoelii/imunologia , Proteínas Recombinantes de Fusão/imunologia , Especificidade da EspécieRESUMO
Immunization with DNA vaccines encoding relevant Ags can induce not only cell-mediated immune response but also humoral immune responses against pathogenic microorganisms in several animal models. Our previous results demonstrated that, when the C terminus (PyC2) of Plasmodium yoelii merozoite surface protein-1 (MSP-1), a leading vaccine candidate against erythrocytic stages of malaria, was expressed as a fusion protein (GST-PyC2) with glutathione S-transferase (GST), it elicited Ab-mediated protective immune responses in BALB/c mice. In our present study, we wished to examine the humoral responses to a DNA vaccine (V3) encoding GST-PyC2. The GST-PyC2 expressed in V3-transfected Cos 7 cells was recognized by a protective monoclonal Ab to PyC2 (mAb302), although the secreted product had undergone N-linked glycosylation. When BALB/c mice were immunized with V3 plasmid, anti-PyC2 Abs were successfully induced. These Abs immunoprecipitated native PyMSP-1 protein and competed with mAb302 for binding to its epitope at a level similar to those elicited by GST-PyC2 protein immunization. However, these Abs had significantly lower titers and avidities, and different isotype profiles and protective capacities against a lethal erythrocytic stage challenge, than those resulting from immunization with GST-PyC2 protein. Most surprising was the finding that, in contrast to protein immunization, there was no significant increase in the avidity of either GST-specific or PyC2-specific IgG Abs during the course of DNA immunization. This suggests that there may be little or no affinity maturation of specific Abs during DNA immunization in this system.
Assuntos
Formação de Anticorpos , DNA de Protozoário/imunologia , Vacinas Antimaláricas/imunologia , Malária/imunologia , Malária/prevenção & controle , Proteína 1 de Superfície de Merozoito/imunologia , Plasmodium yoelii/imunologia , Animais , Proteína 1 de Superfície de Merozoito/genética , Camundongos , Vacinas de DNA/imunologiaRESUMO
The mechanisms by which Abs mediate protection during blood-stage malaria infections is controversial, with some evidence pointing to the direct effect of Abs on parasite invasion and growth, while other studies suggest that Abs act in cooperation with monocytes to achieve parasite inhibition. To determine whether the effector phase of protection in vivo to the rodent parasite Plasmodium yoelii yoelii requires Fc receptor bearing cells, we passively transferred immune sera into FcR gamma-chain knockout mice. Inflammatory macrophages from these knockout mice were unable to mediate phagocytosis or Ab-dependent cell-mediated cytotoxicity (ADCC) through Fc gamma RI, Fc gamma RII, or Fc gamma RIII. Passive transfer of either P. y. yoelii hyperimmune sera or anti-GST-PYC2 sera directed to the major merozoite surface protein (MSP-1) of this parasite enabled both BALB/cByJ mice and FcR gamma-chain-deficient mice to resist lethal P. y. yoelii 17XL (Py17XL) challenge. mAb302, a protective IgG3 Ab, also passively protected both strains of mice. Most of these samples contain Ab isotypes that would not be able to protect mice if their protective effects required Ab-dependent cell-mediated cytotoxicity. These results establish that, in this infection, protection is directly mediated by Abs and does not require the participation of Fc receptors.
Assuntos
Anticorpos Antiprotozoários/fisiologia , Malária/imunologia , Malária/prevenção & controle , Plasmodium yoelii/imunologia , Receptores de IgG/fisiologia , Animais , Anticorpos Antiprotozoários/sangue , Especificidade de Anticorpos , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Modelos Animais de Doenças , Glutationa Transferase/genética , Glutationa Transferase/imunologia , Soros Imunes/biossíntese , Imunoglobulina G/sangue , Isotipos de Imunoglobulinas/sangue , Malária/mortalidade , Vacinas Antimaláricas/genética , Vacinas Antimaláricas/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Plasmodium yoelii/patogenicidade , Receptores de IgG/sangue , Receptores de IgG/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Dopamine and dobutamine have recently been shown to produce a negative interference in several biochemical tests that use peroxide and peroxidase to generate a chromophore. To define the chemical mechanism of this interference, we examined the effects of dopamine and dobutamine in various peroxidase-based biochemical tests. Dopamine interfered stoichiometrically with peroxidase-based tests that use 4-aminophenazone to form chromophore but interfered little in those that use other compounds to generate chromophore. Dopamine reacts with 4-aminophenazone in the presence of peroxide and peroxidase to form a novel quinone-imine dye, with a smaller absorptivity than the chromophore formed in the absence of dopamine. The smaller absorptivity of this novel chromophore results in negative interference by reducing the total absorbance at the wavelength used to measure analyte. In contrast, dobutamine interfered stoichiometrically with all peroxidase-based tests studied, regardless of whether 4-aminophenazone was used to form the chromophore. Dobutamine was rapidly oxidized by peroxide in the presence of peroxidase, thus depleting the peroxide necessary to generate chromophore. Dopamine and dobutamine demonstrate two distinct general mechanisms of interference in peroxidase-based biochemical tests.
Assuntos
Compostos Cromogênicos/química , Dobutamina/química , Dopamina/química , Peroxidase do Rábano Silvestre , Peróxidos , Ampirona/química , Colesterol/sangue , Creatinina/sangue , Dobutamina/sangue , Dopamina/sangue , Humanos , Oxirredução , Espectrofotometria Ultravioleta , Triglicerídeos/sangue , Ácido Úrico/sangueRESUMO
The lysosomal storage diseases are a family of inherited disorders usually caused by a deficiency in a single lysosomal enzyme, and are characterised by progressive intralysosomal storage in multiple cell types. Although individual syndromes can be uncommon, as a whole this family of diseases affects approximately 1 in 3,000 live births. The severity of disease can be variable, ranging from minimal evidence of lysosomal storage to widespread multi-system involvement and early mortality. Although the enzymatic defects responsible for most of these diseases are known, treatment options for the majority of these disorders are limited to supportive care and genetic counselling. Knowledge of the genetic defects underlying these diseases, coupled with advances in the fields of gene transfer and expression, provide an opportunity to utilise gene therapy strategies in order to treat these disorders. Here we provide a description of the biochemical and molecular basis of gene therapy for lysosomal storage diseases, as well as an overview of some of the in vitro and in vivo studies that have been performed.
RESUMO
Most malariologists believe that optimal malaria vaccines will induce protective immune responses against different stages of the parasite's life cycle. A multiple antigen peptide (MAP) vaccine based on the Plasmodium yoelii circumsporozoite protein (PyCSP) protects mice against sporozoite challenge by inducing antibodies that prevent sporozoites from invading hepatocytes. A purified recombinant protein vaccine based on the P. yoelii merozoite surface protein-1 (PyMSP-1) protects mice against challenge with infected erythrocytes, presumably by inducing antibodies against the erythrocytic stage of the parasite. We now report studies designed to determine if the PyMSP-1 vaccine protects against challenge with sporozoites, the stage encountered in the field, and if immunization with a combination of the PyCSP and PyMSP-1 vaccines provides additive or synergistic protection against sporozoite challenge. In two experiments, using TiterMax or Ribi R-700 as adjuvant, 3 of 19 mice immunized with the PyMSP-1 vaccine were completely protected against sporozoite challenge. The remaining mice had significantly delayed onset and lower levels of peak parasitemia than did control mice (11.1 +/- 2.8% vs. 36.7 +/- 1.6% in experiment #2, P < 0.01). Immunization with the combination vaccine reduced by approximately 50% the level of antibodies induced to PyCSP and PyMSP-1, as compared to that induced by the individual components. However, in two experiments, there was evidence of additive protection. Six of 19 (31.6%) immunized with the PyCSP vaccine, 3 of 19 (15.8%) immunized with the PyMSP-1 vaccine, and 10 of 19 (52.6%) immunized with the combination were completely protected against sporozoit challenge. This modest increase in protection in the combination group may be a reflection of additive anti-PyCSP and anti-PyMSP-1 immunity, since mice in the combination group had diminished levels of antibodies to each components. These studies indicate that considerable work may be required to optimize the construction, delivery, and assessment of multi-stage malaria vaccines.
Assuntos
Malária/imunologia , Malária/prevenção & controle , Plasmodium yoelii/imunologia , Vacinas Combinadas/imunologia , Vacinas Sintéticas/imunologia , Animais , Anticorpos Antiprotozoários/análise , Esqueleto da Parede Celular/farmacologia , Fatores Corda/farmacologia , Ensaio de Imunoadsorção Enzimática , Feminino , Immunoblotting , Isotipos de Imunoglobulinas/análise , Lipídeo A/análogos & derivados , Lipídeo A/farmacologia , Proteína 1 de Superfície de Merozoito , Camundongos , Camundongos Endogâmicos BALB C , Parasitemia/imunologia , Poloxaleno/farmacologia , Precursores de Proteínas/imunologia , Proteínas de Protozoários/imunologia , VacinaçãoAssuntos
Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/imunologia , Malária/imunologia , Precursores de Proteínas/imunologia , Proteínas de Protozoários/imunologia , Animais , Antígenos de Protozoários/química , Fator de Crescimento Epidérmico/química , Imunização , Proteína 1 de Superfície de Merozoito , Camundongos , Plasmodium yoelii , Precursores de Proteínas/química , Proteínas de Protozoários/química , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Previously, we described a protective immune response induced by the carboxyl-terminal region of the merozoite surface protein-1 (MSP-1) from the rodent malarial parasite Plasmodium yoelii yoelii 17XL, expressed as a fusion protein and designated glutathione S-transferase (GST)-PYC2. We also demonstrated that the humoral response induced by GST-PYC2 was the primary mechanism by which immunized animals controlled their blood-stage infections. We have now examined the influence of several adjuvants on the immune response to the GST-PYC2 fusion protein. While alum, Freund's adjuvant, Ribi adjuvant system, and TiterMax were efficacious in eliciting a protective response with GST-PYC2 in BALB/c mice, saponin failed to induce protection, although significant levels of PYC2-specific antibodies were produced in all immunized animals. This protection depended on the mouse strain since immunization of Swiss Webster mice with GST-PYC2 in alum did not produce levels of PYC2-specific antibodies comparable to those in BALB/c mice nor did it induce any demonstrable level of protection against parasite challenge. Swiss Webster mice were protected, however, when immunized with GST-PYC2 in other adjuvants. Immunization with PYC2, isolated free of GST induced lower levels of antigen-specific antibody; only those animals given PYC2 in Freund's adjuvant demonstrated a significant degree of protection, suggesting the possibility (of additional cellular effector mechanisms. These findings demonstrate that adjuvant, host genotype, and the fine specificity of the response significantly influence the protection induced by the carboxyl terminus of MSP-1 in vivo and illustrate the need to consider these factors in evaluating MSP-1 as a vaccine component.