RESUMO
Retroviral vectors including lentiviral vectors are commonly used tools to stably express transgenes or RNA molecules in mammalian cells. Their utilities are roughly divided into two categories, stable overexpression of transgenes and RNA molecules, which requires maximal transduction efficiency, or functional selection with retrovirus (RV)-based libraries, which takes advantage of retroviral superinfection resistance. However, the dynamic features of RV-mediated transduction are not well characterized. Here, we engineered two murine stem cell virus-based retroviral vectors expressing dual fluorescence proteins and antibiotic markers, and analyzed virion production efficiency and virion stability, dynamic infectivity and superinfection resistance in different cell types, and strategies to improve transduction efficiency. We found that the highest virion production occurred between 60 and 72 h after transfection. The stability of the collected virion supernatant decreased by >60% after 3 days in storage. We found that RV infectivity varied drastically in the tested human cancer lines, while low transduction efficiency was partially overcome with increased virus titer, prolonged infection duration and/or repeated infections. Furthermore, we demonstrated that RV receptors PIT1 and PIT2 were lowly expressed in the analyzed cells, and that PIT1 and/or PIT2 overexpression significantly improved transduction efficiency in certain cell lines. Thus, our findings provide resourceful information for the optimal conditions of retroviral-mediated gene delivery.
Assuntos
Retroviridae/genética , Transfecção/métodos , Animais , Linhagem Celular Tumoral , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Camundongos , Receptores Virais/genética , Receptores Virais/metabolismo , Retroviridae/metabolismo , Retroviridae/patogenicidade , Transfecção/normas , Vírion/genética , Vírion/metabolismoRESUMO
Three cases of congenital arteriovenous malformations localized to the flexor digitorum superficialis presented to a single surgeon. In all three cases, the tumours were resectable with negligible morbidity to the upper extremity.
Assuntos
Malformações Arteriovenosas/cirurgia , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/cirurgia , Artéria Ulnar/anormalidades , Artéria Ulnar/cirurgia , Adulto , Malformações Arteriovenosas/diagnóstico , Feminino , Humanos , Masculino , Radiografia , Artéria Ulnar/diagnóstico por imagemAssuntos
Subpopulações de Linfócitos B/citologia , Deleção Clonal/fisiologia , Ativação do Complemento/imunologia , Receptores de Complemento 3b/imunologia , Receptores de Complemento 3d/imunologia , Animais , Antígenos CD19/imunologia , Autoanticorpos/imunologia , Autoantígenos/imunologia , Subpopulações de Linfócitos B/imunologia , Complemento C3/deficiência , Complemento C3/genética , Complemento C3b/imunologia , Via Clássica do Complemento , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Células Dendríticas Foliculares/imunologia , Endotélio Vascular/imunologia , Membro Posterior/irrigação sanguínea , Humanos , Imunidade Inata , Imunoglobulina M/imunologia , Intestinos/irrigação sanguínea , Isquemia/imunologia , Isquemia/patologia , Camundongos , Camundongos Knockout , Modelos Imunológicos , Traumatismo por Reperfusão Miocárdica/imunologia , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/imunologia , Proteínas Nucleares , Ratos , Receptores de Complemento 3d/deficiência , Receptores de Complemento 3d/genética , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/patologiaRESUMO
While the significance of natural Ab is not entirely clear, one proposed role is clearance of bacterial Ags. To determine whether natural Ab was involved in clearance of endotoxin, we have examined novel strains of mice with either a total or selective deficiency in Ig. Recombinase-activating gene-2 (RAG-2(-/-))-deficient mice, which have no serum Ig due to arrested development of B cells at the pro-B stage, demonstrate increased sensitivity to endotoxin that correlates with an impaired clearance. When RAG-2(-/-) mice are reconstituted with pooled sera from normal mice, both survival and clearance of circulating endotoxin are enhanced. To further define the nature of the protective Ab, Bruton's tyrosine kinase (Btk)-deficient mice were characterized in the high dose LPS model. Like RAG-2(-/-) mice, they are highly sensitive to endotoxin and have an impaired clearance of LPS. Reconstitution of Btk(-/-) mice, which have reduced levels of IgG3 and IgM, with purified normal mouse IgM dramatically enhances their ability to clear endotoxin compared with mock (saline)-reconstituted littermates. The cellular source of natural anti-LPS IgM was identified as the peritoneal-residing B-1 cell by enzyme-linked immunospot (ELISPOT) assay. Taken together, these studies demonstrate the important role of natural Ab and complement in the clearance of pathogenic substances from the circulation.
Assuntos
Proteínas do Sistema Complemento/imunologia , Imunidade Inata , Imunoglobulinas/deficiência , Lipopolissacarídeos/imunologia , Choque Séptico/imunologia , Animais , Proteínas de Ligação a DNA/genética , Imunidade Inata/genética , Imunoglobulinas/genética , Imunoglobulinas/imunologia , Lipopolissacarídeos/administração & dosagem , Camundongos , Camundongos Mutantes , Choque Séptico/genéticaRESUMO
Endotoxin shock is a life-threatening syndrome associated with a Gram-negative infection and mediated by a systemic inflammatory response. As a major effector of inflammation, the complement system has been implicated in both the pathogenesis and the protection from endotoxin shock. To clarify the role of complement in endotoxin shock, we have used mice totally deficient in either complement component C3 or C4. We found that both the C3- and C4-deficient mice were significantly more sensitive to endotoxin than wild-type controls. The endotoxin-challenged complement-deficient mice failed to clear endotoxin efficiently from the circulation and this led to excess consumption of C1 inhibitor protein (C1 INH), a major regulator of both complement and the contact system of blood coagulation. Replacement of C1 INH rescued the endotoxin-challenged complement-deficient mice from shock and death. These findings suggest a novel therapy for treatment of endotoxemia with C1 INH protein.
Assuntos
Proteínas Inativadoras do Complemento 1/imunologia , Complemento C3/imunologia , Complemento C4/imunologia , Choque Séptico/imunologia , Animais , Proteínas Inativadoras do Complemento 1/genética , Proteína Inibidora do Complemento C1 , Complemento C3/deficiência , Complemento C3/genética , Complemento C4/deficiência , Complemento C4/genética , Suscetibilidade a Doenças , Camundongos , Camundongos Mutantes , Choque Séptico/genéticaRESUMO
Within the new field of performing arts medicine is an active group of researchers and clinicians who are dedicated to advancing our understanding of musculoskeletal problems of performers. Among these problems are those of the upper extremity in instrumental musicians such as overuse syndrome, entrapment neuropathies, focal dystonia, osteoarthritis, and problems related to joint hypermobility. The epidemiology of these problems is presented as well as results of recent studies investigating their etiology as they relate to musicians. A brief discussion of the treatment of injured musicians also is included.
Assuntos
Doenças Musculoesqueléticas/etiologia , Música , Doenças Profissionais/etiologia , Distonia/epidemiologia , Distonia/etiologia , Humanos , Incidência , Instabilidade Articular/epidemiologia , Instabilidade Articular/etiologia , Doenças Musculoesqueléticas/epidemiologia , Doenças Musculoesqueléticas/terapia , Síndromes de Compressão Nervosa/epidemiologia , Síndromes de Compressão Nervosa/etiologia , Doenças Profissionais/epidemiologia , Doenças Profissionais/terapia , Síndrome do Desfiladeiro Torácico/epidemiologia , Síndrome do Desfiladeiro Torácico/etiologiaRESUMO
This brief review of musculoskeletal injuries in musicians serves as an introduction to performing arts medicine and to the role rheumatologists can play in managing injured musicians. This niche is an area of practice that the rheumatologist may find very gratifying that, at the same time, provides a valuable service to the community. Common problems include overuse syndromes, nerve entrapment, thoracic outlet syndrome, osteoarthritis, motor dysfunction, fibromyalgia, and complications of hypermobility. Important points in the history and physical examination of the injured musician must include details of practice and performance patterns. Treatment involves many techniques used in nonmusicians but also often important is direct contact with the musicians' teachers.
RESUMO
We administered pulsatile low doses of gonadotropin-releasing hormone (GnRH) (1 to 5 micrograms) to patients whose anovulation was caused by relative and absolute deficiency of endogenous GnRH. Eight such patients, including one with previous pituitary stalk transection, were treated during a total of 23 cycles; pulses of GnRH were administered via a portable pump every 96 or 120 minutes. Activation of pituitary-ovarian function with orderly development of a single dominant follicle, a luteinizing hormone surge, and ovulation occurred in 20 of the 23 cycles. The other three cycles were anovulatory. All patients responded, and five (62%) of the eight conceived, for a total of seven pregnancies and four full-term deliveries of normal infants. This study demonstrates that small pulsatile doses of GnRH can activate cyclic pituitary-ovarian function in hypogonadotropin-acyclic women and induce ovulation resulting in pregnancy and live birth.