RESUMO
Chromothripsis is a catastrophic cellular event recently described in cancer in which chromosomes undergo massive deletion and rearrangement. Here, we report a case in which chromothripsis spontaneously cured a patient with WHIM syndrome, an autosomal dominant combined immunodeficiency disease caused by gain-of-function mutation of the chemokine receptor CXCR4. In this patient, deletion of the disease allele, CXCR4(R334X), as well as 163 other genes from one copy of chromosome 2 occurred in a hematopoietic stem cell (HSC) that repopulated the myeloid but not the lymphoid lineage. In competitive mouse bone marrow (BM) transplantation experiments, Cxcr4 haploinsufficiency was sufficient to confer a strong long-term engraftment advantage of donor BM over BM from either wild-type or WHIM syndrome model mice, suggesting a potential mechanism for the patient's cure. Our findings suggest that partial inactivation of CXCR4 may have general utility as a strategy to promote HSC engraftment in transplantation.
Assuntos
Instabilidade Cromossômica , Síndromes de Imunodeficiência/genética , Verrugas/genética , Animais , Cromossomos Humanos , Modelos Animais de Doenças , Haploinsuficiência , Células-Tronco Hematopoéticas/metabolismo , Humanos , Linfócitos/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Mosaicismo , Mutação , Células Mieloides/metabolismo , Doenças da Imunodeficiência Primária , Receptores CXCR4/genética , Remissão EspontâneaRESUMO
Human respiratory syncytial virus (HRSV), human metapneumovirus (HMPV), and human parainfluenza virus type 3 (HPIV3) are common, important respiratory pathogens, but HRSV has a substantially greater impact with regard to acute disease, long-term effects on airway function, and frequency of re-infection. It has been reported to strongly interfere with the functioning of dendritic cells (DC). We compared HRSV to HMPV and HPIV3 with regard to their effects on human monocyte-derived immature DC (IDC). Side-by-side analysis distinguished between common effects versus those specific to individual viruses. The use of GFP-expressing viruses yielded clear identification of robustly infected cells and provided the means to distinguish between direct effects of robust viral gene expression versus bystander effects. All three viruses infected inefficiently based on GFP expression, with considerable donor-to donor-variability. The GFP-negative cells exhibited low, abortive levels of viral RNA synthesis. The three viruses induced low-to-moderate levels of DC maturation and cytokine/chemokine responses, increasing slightly in the order HRSV, HMPV, and HPIV3. Infection at the individual cell level was relatively benign, such that in general GFP-positive cells were neither more nor less able to mature compared to GFP-negative bystanders, and cells were responsive to a secondary treatment with lipopolysaccharide, indicating that the ability to mature was not impaired. However, there was a single exception, namely that HPIV3 down-regulated CD38 expression at the RNA level. Maturation by these viruses was anti-apoptotic. Inefficient infection of IDC and sub-optimal maturation might result in reduced immune responses, but these effects would be common to all three viruses rather than specific to HRSV.
Assuntos
Células Dendríticas/virologia , Metapneumovirus/fisiologia , Vírus da Parainfluenza 3 Humana/fisiologia , Infecções por Paramyxoviridae/virologia , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/fisiologia , Infecções por Respirovirus/virologia , ADP-Ribosil Ciclase 1/metabolismo , Adulto , Animais , Apoptose , Linhagem Celular , Células Cultivadas , Chlorocebus aethiops , Citocinas/metabolismo , Células Dendríticas/citologia , Regulação da Expressão Gênica , Humanos , Monócitos/imunologia , Monócitos/virologia , RNA Viral/metabolismo , Células Vero , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia , Proteínas Virais de Fusão/metabolismoRESUMO
The study of cellular processes has been facilitated by the use of methods to detect molecular associations both in vivo and in vitro. An invaluable tool to study molecular associations associated with dynamic processes in living cells utilizes the phenomenon of fluorescence resonance energy transfer (FRET), together with selected fluorophores that are attached to molecules of interest. Many reports have utilized fluorophores conjugated to antibodies for FRET pairs. However, these methods are restricted to extracellular molecules and dependent upon the availability of appropriate antibodies. The recent development of green fluorescent protein (GFP) variants suitable for FRET has expanded the utility of this methodology by permitting the study of intracellular as well as extracellular processes. Combining FRET with flow cytometric analysis results in a powerful high-throughput assay for molecular associations. This article details the use of green fluorescent protein (GFP) mutants cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) to measure the association of the signaling component TRAF2 with the TNFR-2 receptor to illustrate the versatility of this methodology.
Assuntos
Citometria de Fluxo/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas Luminescentes/química , Antígenos CD/metabolismo , Proteínas de Bactérias , Variação Genética , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mutação , Ligação Proteica , Proteínas/metabolismo , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral , Fator 2 Associado a Receptor de TNFRESUMO
In previous studies amphotropic MFGS-gp91phox (murine onco-retrovirus vector) was used in a clinical trial of X-linked chronic granulomatous disease (X-CGD) gene therapy to achieve transient correction of oxidase activity in 0.1% of neutrophils. We later showed that transduced CD34+ peripheral blood stem cells (CD34+ PBSCs) from this trial transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in correction of only 2.5% of human neutrophils. However, higher rates of transduction into stem cells are required. In the current study we demonstrate that the same vector (MFGS-gp91phox) pseudo-typed with RD114 envelope in a 4-day culture/transduction regimen results in a 7-fold increase in correction of NOD/SCID mouse repopulating X-CGD CD34+ PBSCs (14%-22% corrected human neutrophils; human cell engraftment 13%-67%). This increase may result from high expression of receptor for RD114 that we demonstrate on CD34+CD38- stem cells. Using RD114-MFGS encoding cyan fluorescent protein to allow similar studies of normal CD34+ PBSCs, we show that progressively higher levels of gene marking of human neutrophils (67%-77%) can be achieved by prolongation of culture/transduction to 6 days, but with lower rates of human cell engraftment. Our data demonstrate the highest reported level of functional correction of any inherited metabolic disorder in human cells in vivo with the NOD/SCID mouse system using onco-retrovirus vector.