RESUMO
The zebrafish enjoys several advantages over other model organisms. It is small, easy to maintain, prolific, and numerous genetic tools are available for it. For example, forward genetic screens have allowed investigators to identify important genes potentially involved in a variety of functions from embryogenesis to cancer. However, despite its sophisticated behavioral repertoire, behavioral methods have rarely been utilized in forward genetic screens. Here, we employ a two-tiered strategy, a proof of concept study, to explore the feasibility of behavioral screens. We generated mutant lines using transposon-based insertional mutagenesis, allowing us to bias mutant selection with target genes expressed within the brain. Furthermore, we employed an efficient and fast behavioral pre-selection in which we investigated the locomotory response of 5-day post-fertilization old larval fish to hyperosmotic shock. Based on this assay, we selected five lines for our lower throughput secondary adult behavioral screen. The latter screen utilized tests in which computer animated image presentation and video-tracking-based automated quantification of behavior allowed us to compare heterozygous zebrafish with their wild-type siblings on their responses to a variety of stimuli. We found significant mutation induced adult behavioral alterations in 4 out of the 5 lines analyzed, including changes in response to social or fear inducing stimuli, to handling and novelty, or in habituation to novelty. We discuss the pros and cons of behavioral phenotyping and of the use of different forward genetic methods in biomedical research with zebrafish.
Assuntos
Comportamento Animal , Testes Genéticos , Mutação/genética , Peixe-Zebra/genética , Animais , Regulação da Expressão Gênica , Habituação Psicofisiológica , Larva/genética , Atividade Motora , Osmose , Fatores de TempoRESUMO
Light chain (AL) amyloidosis is a devastating disease characterized by amyloid deposits formed by immunoglobulin light chains. Current available treatments involve conventional chemotherapy and autologous stem cell transplant. We have recently concluded a phase III trial comparing these two treatments. AL amyloidosis patients who achieve hematological complete response (CR) do not necessarily achieve organ response regardless of the treatment they received. In order to investigate the possible correlation between amyloid formation kinetics and organ response, we selected AL amyloidosis patients from the trial with kidney involvement and CR after treatment. Six patients were selected and their monoclonal immunoglobulin light chains were characterized. The proteins showed differences in their stability and their kinetics of amyloid formation. A correlation was detected at pH 7.4, showing that less stable proteins are more likely to form amyloid fibrils. AL-T03 is too unstable to form amyloid fibrils at pH 7.4. This protein was found in the only patient in the study that had organ response, suggesting that partially folded species are required for amyloid formation to occur in AL amyloidosis.
Assuntos
Amiloide/química , Amiloidose/metabolismo , Cadeias Leves de Imunoglobulina/química , Amiloide/genética , Amiloide/metabolismo , Amiloidose/genética , Amiloidose/patologia , Ensaios Clínicos Fase III como Assunto , Humanos , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/metabolismo , Cinética , Dobramento de Proteína , Estabilidade Proteica , Análise de Sequência de ProteínaRESUMO
Monoclonal tumor plasma cells as well as non-terminally differentiated B cells having a clonal relationship to the tumor cells have been detected in the peripheral blood (PB) of some multiple myeloma (MM) patients but rarely in light chain (primary systemic) amyloidosis (AL) patients. Previously, our group found these peripheral clonotypic B cells in three AL patients. Here, we report detailed analysis of a larger cohort of AL patients to validate the prior findings and to investigate the effect of this cell population on clinical outcome. Fourteen AL patients were selected from a clinical prospective trial, and the relationship between immunoglobulin light chain variable gene (V(L)) representation in PB B cells and the clonal population in the bone marrow (BM) was investigated. A clonal relationship was not detected, and the present study provides important insights into the disparity with the earlier data, including clinical history of the patients and methodological analysis.
Assuntos
Amiloidose/imunologia , Subpopulações de Linfócitos B/metabolismo , Cadeias Leves de Imunoglobulina/genética , Região Variável de Imunoglobulina/genética , Mieloma Múltiplo/imunologia , Plasmócitos/metabolismo , Amiloidose/genética , Amiloidose/patologia , Antígenos CD19/metabolismo , Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/patologia , Circulação Sanguínea/imunologia , Medula Óssea/imunologia , Medula Óssea/patologia , Ensaios Clínicos Fase III como Assunto , Células Clonais , DNA/análise , Primers do DNA , Humanos , Cadeias Leves de Imunoglobulina/imunologia , Linfopoese , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Plasmócitos/imunologia , Plasmócitos/patologia , Estudos ProspectivosRESUMO
Renal dysfunction commonly occurs in multiple myeloma (MM) and is caused by deposition of abnormal light chain within various compartments of the kidney. Renal pathologic findings are diverse and include cast nephropathy (CN), amyloidosis and light-chain deposition disease (LCDD). We report a case of renal failure in a patient with MM caused by concurrent CN, amyloidosis and LCDD which has not been previously described.
Assuntos
Amiloidose/complicações , Hipergamaglobulinemia/complicações , Cadeias Leves de Imunoglobulina , Nefropatias/complicações , Mieloma Múltiplo/complicações , Insuficiência Renal/etiologia , Adulto , Feminino , Humanos , Insuficiência Renal/terapiaRESUMO
BACKGROUND: The amyloidoses are protein misfolding diseases characterized by the deposition of amyloid that leads to cell death and tissue degeneration. In immunoglobulin light chain amyloidosis (AL), each patient has a unique monoclonal immunoglobulin light chain (LC) that forms amyloid deposits. Somatic mutations in AL LCs make these proteins less thermodynamically stable than their non-amyloidogenic counterparts, leading to misfolding and ultimately the formation of amyloid fibrils. We hypothesize that location rather than number of non-conservative mutations determines the amyloidogenicity of light chains. METHODOLOGY/PRINCIPAL FINDINGS: We performed sequence alignments on the variable domain of 50 kappa and 91 lambda AL light chains and calculated the number of non-conservative mutations over total number of patients for each secondary structure element in order to identify regions that accumulate non-conservative mutations. Among patients with AL, the levels of circulating immunoglobulin free light chain varies greatly, but even patients with very low levels can have very advanced amyloid deposition. CONCLUSIONS: Our results show that in specific secondary structure elements, there are significant differences in the number of non-conservative mutations between normal and AL sequences. AL sequences from patients with different levels of secreted light chain have distinct differences in the location of non-conservative mutations, suggesting that for patients with very low levels of light chains and advanced amyloid deposition, the location of non-conservative mutations rather than the amount of free light chain in circulation may determine the amyloidogenic propensity of light chains.