RESUMO
Here we describe TZM-gfp, a novel HIV-1 reporter cell derived from the same parental clone JC.53, used previously to generate the widely-utilized indicator cell line TZM-bl. We re-engineered JC.53 cells to express GFP under regulation of HIV Tat and Rev. We characterize the new reporter cell line to show that TZM-gfp cells are equally susceptible to HIV infection, exhibit minimal background signal, and can report HIV infection in rare cells from a bulk population of experimentally-infected human monocyte-derived macrophages. We demonstrate the utility and sensitivity of the cells in detection of even a single HIV-positive macrophage by fluorescence-assisted correlative electron microscopy, using the GFP signal to guide imaging of HIV virions in primary co-culture. Finally, we used TZM-gfp cells for viral capture during co-culture with human peripheral blood mononuclear cells, showing that TZM-gfp can support outgrowth and analyses of patient-derived primary HIV-1 isolates.
Assuntos
Proteínas de Fluorescência Verde/metabolismo , Infecções por HIV/diagnóstico , HIV-1/isolamento & purificação , Leucócitos Mononucleares/virologia , Macrófagos/virologia , Replicação Viral , Células Cultivadas , Proteínas de Fluorescência Verde/genética , Infecções por HIV/virologia , HIV-1/metabolismo , HIV-1/patogenicidade , Humanos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
Lubricin is an important boundary lubricant and chondroprotective glycoprotein in synovial fluid. Both increased and decreased synovial fluid lubricin concentrations have been reported in experimental post-traumatic osteoarthritis (PTOA) animal models and in naturally occurring joint injuries in humans and animals, with no consensus about how lubricin is altered in different species or injury types. Increased synovial fluid lubricin has been observed following intra-articular fracture in humans and horses and in human late-stage osteoarthritis; however, it is unknown how synovial lubricin is affected by knee-destabilizing injuries in large animals. Spontaneous rupture of cranial cruciate ligament (RCCL), the anterior cruciate ligament equivalent in quadrupeds, is a common injury in dogs often accompanied by OA. Here, clinical records, radiographs, and synovial fluid samples from 30 dogs that sustained RCCL and 9 clinically healthy dogs were analyzed. Synovial fluid lubricin concentrations were nearly 16-fold greater in RCCL joints as compared to control joints, while IL-2, IL-6, IL-8, and TNF-α concentrations did not differ between groups. Synovial fluid lubricin concentrations were correlated with the presence of radiographic OA and were elevated in three animals sustaining RCCL injury prior to the radiographic manifestation of OA, indicating that lubricin may be a potential biomarker for early joint injury.
Assuntos
Lesões do Ligamento Cruzado Anterior/veterinária , Doenças do Cão/metabolismo , Glicoproteínas/metabolismo , Osteoartrite/veterinária , Líquido Sinovial/metabolismo , Animais , Ligamento Cruzado Anterior/diagnóstico por imagem , Ligamento Cruzado Anterior/metabolismo , Lesões do Ligamento Cruzado Anterior/diagnóstico por imagem , Lesões do Ligamento Cruzado Anterior/metabolismo , Citocinas/metabolismo , Doenças do Cão/diagnóstico por imagem , Cães , Osteoartrite/diagnóstico por imagem , Osteoartrite/metabolismo , Radiografia , Ruptura Espontânea/diagnóstico por imagem , Ruptura Espontânea/metabolismo , Ruptura Espontânea/veterinária , Líquido Sinovial/diagnóstico por imagemRESUMO
Understanding the mechanisms involved in HIV infection and latency, and development of a cure, rely on the availability of sensitive research tools such as indicator cells, which allow rigorous quantification of viral activity. Here we describe the construction and validation of a novel dual-indicator cell line, Sup-GGR, which offers two different readouts to quantify viral replication. A construct expressing both Gaussia luciferase and hrGFP in a Tat- and Rev-dependent manner was engineered into SupT1-CCR5 to create Sup-GGR cells. This cell line supports the replication of both X4 and R5-tropic HIV as efficiently as its parental cell line, SupT1-CCR5, and allows repeated sampling without the need to terminate the culture. Sup-GGR demonstrates comparable sensitivity and similar kinetics in virus outgrowth assays (VOA) to SupT1-CCR5 using clinical samples. However the Gaussia luciferase reporter is significantly less labor-intensive and allows earlier detection of reactivated latent viruses compared to the conventional HIV p24 ELISA assay. The Sup-GGR cell line constitutes a versatile new tool for HIV research and clinical trials.
Assuntos
Reservatórios de Doenças/virologia , HIV-1/isolamento & purificação , HIV-1/fisiologia , Latência Viral/fisiologia , Replicação Viral/fisiologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Luciferases/metabolismoRESUMO
Long noncoding RNAs (lncRNAs) impart significant regulatory functions in a diverse array of biological pathways and manipulation of these RNAs provides an important avenue to modulate such pathways, particularly in disease. Our knowledge about lncRNAs' role in determination of cellular fate during HIV-1 infection remains sparse. Here, we have identified the impact of the lncRNA SAF in regulating apoptotic effector caspases in macrophages, a long-lived cellular reservoir of HIV-1, that are largely immune to virus-induced cell death. Expression of SAF is significantly up-regulated in HIV-1-infected human monocyte-derived macrophages (MDM) compared with bystander and virus-nonexposed cells. A similar enhancement in SAF RNA expression is also detected in the HIV-1-infected airway macrophages obtained by bronchoalveolar lavage of HIV-1-infected individuals. Down-regulation of SAF with siRNA treatment increases caspase-3/7 activity levels in virus-infected MDMs. This induction of apoptotic caspases occurs exclusively in HIV-1-infected macrophages and not in bystander cells, leading to a significant reduction in HIV-1 replication and overall viral burden in the macrophage culture. This study identifies targeting of the lncRNA SAF as a potential means to specifically induce cell death in HIV-1-infected macrophages.
Assuntos
Apoptose , Caspase 3/metabolismo , Caspase 7/metabolismo , Infecções por HIV/metabolismo , HIV-1/fisiologia , Macrófagos/metabolismo , RNA Longo não Codificante/metabolismo , Replicação Viral/fisiologia , Caspase 3/genética , Caspase 7/genética , Infecções por HIV/genética , Infecções por HIV/patologia , Humanos , Macrófagos/patologia , Macrófagos/virologia , RNA Longo não Codificante/genéticaRESUMO
8E5/LAV cells harbor a single HIV provirus, and are used frequently to generate standards for HIV genome quantification. Using flow cytometry-based in situ mRNA hybridization validated by qPCR, we find that different batches of 8E5 cells contain varying numbers of cells lacking viral mRNA and/or viral genomes. These findings raise concerns for studies employing 8E5 cells for quantitation, and highlight the value of mRNA FISH and flow cytometry in the detection and enumeration of HIV-positive cells.
Assuntos
DNA Viral/genética , HIV-1/genética , Provírus/genética , RNA Viral/análise , Fatores de Transcrição/metabolismo , Transcrição Gênica , Linhagem Celular Tumoral , DNA Viral/análise , Citometria de Fluxo , Genoma Viral , Humanos , Hibridização in Situ Fluorescente , Leucemia-Linfoma Linfoblástico de Células Precursoras , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The dorsal mesentery (DM) is the major conduit for blood and lymphatic vessels in the gut. The mechanisms underlying their morphogenesis are challenging to study and remain unknown. Here we show that arteriogenesis in the DM begins during gut rotation and proceeds strictly on the left side, dependent on the Pitx2 target gene Cxcl12. Although competent Cxcr4-positive angioblasts are present on the right, they fail to form vessels and progressively emigrate. Surprisingly, gut lymphatics also initiate in the left DM and arise only after-and dependent on-arteriogenesis, implicating arteries as drivers of gut lymphangiogenesis. Our data begin to unravel the origin of two distinct vascular systems and demonstrate how early left-right molecular asymmetries are translated into organ-specific vascular patterns. We propose a dual origin of gut lymphangiogenesis in which prior arterial growth is required to initiate local lymphatics that only subsequently connect to the vascular system.
Assuntos
Padronização Corporal , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Intestinos/embriologia , Sistema Linfático/embriologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Animais , Artérias/embriologia , Quimiocina CXCL12/metabolismo , Galinhas , Proteínas de Fluorescência Verde/metabolismo , Linfangiogênese , Vasos Linfáticos/embriologia , Mesentério , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Análise de Sequência com Séries de Oligonucleotídeos , Codorniz , Receptores CXCR4/metabolismo , Proteína Homeobox PITX2RESUMO
A critical aspect of gut morphogenesis is initiation of a leftward tilt, and failure to do so leads to gut malrotation and volvulus. The direction of tilt is specified by asymmetric cell behaviors within the dorsal mesentery (DM), which suspends the gut tube, and is downstream of Pitx2, the key transcription factor responsible for the transfer of left-right (L-R) information from early gastrulation to morphogenesis. Although Pitx2 is a master regulator of L-R organ development, its cellular targets that drive asymmetric morphogenesis are not known. Using laser microdissection and targeted gene misexpression in the chicken DM, we show that Pitx2-specific effectors mediate Wnt signaling to activate the formin Daam2, a key Wnt effector and itself a Pitx2 target, linking actin dynamics to cadherin-based junctions to ultimately generate asymmetric cell behaviors. Our work highlights how integration of two conserved cascades may be the ultimate force through which Pitx2 sculpts L-R organs.
Assuntos
Gastrulação , Proteínas de Homeodomínio/metabolismo , Intestinos/embriologia , Proteínas dos Microfilamentos/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Via de Sinalização Wnt , Proteínas rho de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Caderinas/metabolismo , Embrião de Galinha , Proteínas de Homeodomínio/genética , Mucosa Intestinal/metabolismo , Mesentério/embriologia , Mesentério/metabolismo , Mesoderma/metabolismo , Camundongos , Proteínas dos Microfilamentos/genética , Fatores de Transcrição/genética , Proteínas rho de Ligação ao GTP/genética , Proteína Homeobox PITX2RESUMO
The tumor-initiating cell (TIC) frequency of bulk tumor cell populations is one of the criteria used to distinguish malignancies that follow the cancer stem cell model from those that do not. However, tumor-initiating cell frequencies may be influenced by experimental conditions and the extent to which tumors have progressed, parameters that are not always addressed in studies of these cells. We employed limiting dilution cell transplantation of minimally manipulated tumor cells from mammary tumors of several transgenic mouse models to determine their tumor-initiating cell frequency. We determined whether the tumors that formed following tumor cell transplantation phenocopied the primary tumors from which they were isolated and whether they could be serially transplanted. Finally we investigated whether propagating primary tumor cells in different tissue culture conditions affected their resident tumor-initiating cell frequency. We found that tumor-initiating cells comprised between 15% and 50% of the bulk tumor cell population in multiple independent mammary tumors from three different transgenic mouse models of breast cancer. Culture of primary mammary tumor cells in chemically-defined, serum-free medium as non-adherent tumorspheres preserved TIC frequency to levels similar to that of the primary tumors from which they were established. By contrast, propagating the primary tumor cells in serum-containing medium as adherent populations resulted in a several thousand-fold reduction in their tumor-initiating cell fraction. Our findings suggest that experimental conditions, including the sensitivity of the transplantation assay, can dramatically affect estimates of tumor initiating cell frequency. Moreover, conditional on cell culture conditions, the tumor-initiating cell fraction of bulk mouse mammary tumor cell preparations can either be maintained at high or low frequency in vitro thus permitting comparative studies of tumorigenic and non-tumorigenic cancer cells.
Assuntos
Neoplasias Mamárias Experimentais , Animais , Feminino , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Neoplasias Mamárias Experimentais/fisiopatologia , Camundongos , Camundongos Transgênicos , Transplante de Neoplasias , Transplante Homólogo , Células Tumorais CultivadasRESUMO
Cleavage activation of the hemagglutinin (HA) precursor is an essential step in the influenza virus replication cycle that is driven by host cell proteases. HA cleavage activation is required for virus-endosome membrane fusion and the subsequent release of the influenza virus genome into the cytoplasm. Previous studies have determined that HA cleavage is most likely driven by either membrane-bound or extracellular trypsin-like proteases that reside in the respiratory tract. However, there is still uncertainty regarding which proteases are critical for HA cleavage in vivo. Therefore, further investigation of HA cleavage activation is needed in order to gain insight into the critical proteases involved. Matriptase is a member of the type II transmembrane serine protease family that is highly expressed in a membrane-bound form throughout the respiratory tract. One feature of matriptase is that, once activated, the catalytic domain is secreted into the extracellular space and so serves as a functional extracellular protease. In this study, we have determined that the secreted, catalytic domain of matriptase has the ability to cleave and activate HA from the influenza virus H1 subtype but not the H2 and H3 subtypes. Furthermore, matriptase selectively cleaved the HA of particular strains within the H1 subtype, revealing both subtype and H1 strain specificity. Matriptase was also found to activate thrombolytic zymogens that have been shown to cleave and activate the influenza virus HA. Our data demonstrate that matriptase has the ability to cleave HA directly or indirectly by activating HA-cleaving zymogens.
Assuntos
Orthomyxoviridae/genética , Serina Endopeptidases/metabolismo , Animais , Domínio Catalítico , Bovinos , Chlorocebus aethiops , Precursores Enzimáticos/química , Células HEK293 , Humanos , Influenza Humana/virologia , Orthomyxoviridae/metabolismo , Inibidores de Proteases/química , Sistema Respiratório/virologia , Serina Endopeptidases/química , Especificidade da Espécie , Terapia Trombolítica , Fatores de Tempo , Células VeroRESUMO
BMI1 is required for the self-renewal of stem cells in many tissues including the lung epithelial stem cells, Bronchioalveolar Stem Cells (BASCs). Imprinted genes, which exhibit expression from only the maternally or paternally inherited allele, are known to regulate developmental processes, but what their role is in adult cells remains a fundamental question. Many imprinted genes were derepressed in Bmi1 knockout mice, and knockdown of Cdkn1c (p57) and other imprinted genes partially rescued the self-renewal defect of Bmi1 mutant lung cells. Expression of p57 and other imprinted genes was required for lung cell self-renewal in culture and correlated with repair of lung epithelial cell injury in vivo. Our data suggest that BMI1-dependent regulation of expressed alleles at imprinted loci, distinct from imprinting per se, is required for control of lung stem cells. We anticipate that the regulation and function of imprinted genes is crucial for self-renewal in diverse adult tissue-specific stem cells.
Assuntos
Células-Tronco Adultas/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Quinases Associadas a Fase S/metabolismo , Células-Tronco Adultas/patologia , Animais , Sobrevivência Celular/genética , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/genética , Perfilação da Expressão Gênica , Genes p16/fisiologia , Loci Gênicos , Impressão Genômica/genética , Pulmão/patologia , Camundongos , Camundongos Mutantes , Proteínas Nucleares/genética , Complexo Repressor Polycomb 1 , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/genética , Regeneração/genética , Proteínas Repressoras/genética , Proteínas Quinases Associadas a Fase S/genéticaRESUMO
The Pea3 Ets transcription factor is overexpressed in breast tumors suggesting that it plays a role in mammary oncogenesis. However, the normal biological function of Pea3 in the mammary gland is not known. Here we report that Pea3 was expressed in the epithelium of the mouse mammary anlagen commensurate with their genesis, and at later times in the nipple and mammary ducts of female embryos. In adult mice Pea3 transcripts peaked at the onset of puberty and early pregnancy, times of active epithelial cell proliferation and differentiation. Pea3 was expressed in all progenitor cap cells and rare body cells of terminal end buds, and in the myoepithelial cells of ducts and alveoli. Analyses of the mammary glands of Pea3-null mice during puberty revealed an increased number of terminal end buds and an increased fraction of proliferating progenitor cells within these structures compared to their wild type littermates. Tissue transplant experiments demonstrated that these phenotypes were intrinsic to the Pea3-null mammary epithelium. During pregnancy, mammary glands isolated from Pea3-null females had impaired alveolar development as revealed by a decreased fraction of alveolar structures. We performed in vitro colony forming assays of mammary epithelial cells and discovered that loss of Pea3 altered the distribution of specific multipotent progenitor cells. Double-immunofluorescence confirmed that multipotential progenitors co-expressing markers of the myoepithelial and luminal epithelial lineage were amplified in the mammary glands of Pea3-null mice by comparison to their wild type counterparts. We propose that Pea3 functions in multipotential progenitors to regulate their lineage-specific differentiation potential.