Pseudomonas aeruginosa is a leading cause of community-acquired and hospital-acquired infections.... more Pseudomonas aeruginosa is a leading cause of community-acquired and hospital-acquired infections. Successful treatment is hampered by its remarkable ability to rapidly develop resistance to antimicrobial agents, mostly through mutation. In response, the World Health Organization listed carbapenem-resistant P. aeruginosa as a Priority 1 (Critical) pathogen for research and development of new treatments. A key resource in developing effective countermeasures is access to diverse and clinically relevant strains for testing. Herein we describe a panel of 100 diverse P. aeruginosa strains to support this endeavor.Whole genome sequencing was performed on 3,785 P. aeruginosa housed in our repository. Isolates were cultured from clinical samples collected from healthcare facilities around the world between 2003 and 2017. Core-genome multi-locus sequence typing and high-resolution SNP-based phylogenetic analyses were used to select a panel of 100 strains that captured the genetic diversity o...
Differential contributions of the glycosylphosphatidylinositol (GPI)-anchor and GPI-anchored prot... more Differential contributions of the glycosylphosphatidylinositol (GPI)-anchor and GPI-anchored proteins (GPI-AP) to signalling remain poorly understood. Here we show that GPI-AP deficient murine clones produce on average 18 and 181-fold more IL-2 mRNA and protein, respectively, upon T cell receptor (TCR) stimulation, in a cell-intrinsic fashion. This phenotype is formally attributed to a mutation within the transferase complex that predicates the initial step in GPI-anchor biosynthesis. Conditional disruption of the transferase complex enabled the generation of primary GPI-AP deficient CD4 + T cells, which produce on average 10-and 23-fold more IL-2 mRNA and protein, respectively, upon TCR stimulation. Conditional disruption of the transamidase complex yields GPI-sufficient, GPI-AP deficient primary CD4 + T cells. TCR stimulation of these cells yields levels of IL-2 mRNA and protein ranging from 1-3 and 3-fold, respectively, of controls. These results provide the first evidence of a profound impact of GPI in the regulation of TCR signalling.
Pajak merupakan iuran rakyat kepada kas negara berdasarkan undang-undang (yang dapat dipaksakan) ... more Pajak merupakan iuran rakyat kepada kas negara berdasarkan undang-undang (yang dapat dipaksakan) dengan tidak mendapat jasa timbal balik (kontraprestasi) yang langsung dapat ditunjukkan, dan yang digunakan untuk membayar pengeluaran umum. Walaupun demikian tidak semua Wajib Pajak mau dengan sukarela membayar pajaknya karena menganggap bahwa pajak sebagai suatu beban yang sama dengan biaya lainnya yang terjadi pada perusahaan yang perlu diperlakukan secara efisien. Pemerintah dalam rangka menanggulangi keresahan Wajib Pajak membuat suatu kebijakan baru, yaitu Sunset Policy yang hanya berlaku untuk tahun 2008 dan dibantu oleh Direktorat Jenderal Pajak untuk mensosialisasikan kebijakan ini dan memberikan arahan bagi Wajib Pajak yang memanfaatkan kebijakan ini untuk membayar pajak. Penelitian ini dilakukan dengan judul “Peranan Sunset Policy Terhadap Peningkatan Penerimaan Pajak Dari Wajib Pajak Orang Pribadi” dengan Wajib Pajak yang diambil adalah Wajib Pajak Orang Pribadi, pada KPP Pr...
Over the past two decades, Acinetobacter baumannii has emerged as a leading cause of nosocomial i... more Over the past two decades, Acinetobacter baumannii has emerged as a leading cause of nosocomial infections worldwide. Of particular concern are panresistant strains, leading the World Health Organization (WHO) to designate carbapenem-resistant A. baumannii as a priority 1 (critical) pathogen for research and development of new antibiotics. A key component in supporting this effort is accessibility to diverse and clinically relevant strains for testing. Here, we describe a panel of 100 diverse A. baumannii strains for use in this endeavor.
Healthcare management forum / Canadian College of Health Service Executives = Forum gestion des soins de santé / Collège canadien des directeurs de services de santé, 2016
Precision medicine aims to fix what is wrong with today's healthcare: a lack of targeted inte... more Precision medicine aims to fix what is wrong with today's healthcare: a lack of targeted interventions tailored to the person. It encompasses many aspects of health; chief among these is one's genetic profile. Researchers are making progress as gene-sequencing technologies get better and cheaper. Although there is cause for optimism, as several initiatives at Sunnybrook Research Institute show, scientific, systemic, and logistical challenges must be surmounted before advances can be integrated into the clinic. Despite these barriers, precision medicine is the only way forward.
The most efficient approach to monitoring and improving cleaning outcomes remains unresolved. We ... more The most efficient approach to monitoring and improving cleaning outcomes remains unresolved. We sought to extend the findings of a previous study by determining whether cleaning thoroughness (dye removal) correlates with cleaning efficacy (absence of molecular or cultivable biomaterial) and whether one brief educational intervention improves cleaning outcomes. Before-after trial. Newly built community hospital. 90 minute training refresher with surface-specific performance results. Dye removal, measured by fluorescence, and biomaterial removal and acquisition, measured with culture and culture-independent PCR-based assays, were clandestinely assessed for eight consecutive months. At this midpoint, results were presented to the cleaning staff (intervention) and assessments continued for another eight consecutive months. 1273 surfaces were sampled before and after terminal room cleaning. In the short-term, dye removal increased from 40.3% to 50.0% (not significant). For the entire st...
In this paper I examine the reasons for, and the impact of, presidential campaigning on the votin... more In this paper I examine the reasons for, and the impact of, presidential campaigning on the voting behavior of the U.S. Congress. Building off prior research in this area, I look at a particular aspect of campaigning — the endorsement of congressional candidates — as manifested in the public statements of presidents, and show how varied levels of campaigning — both in frequency and intensity — produce different levels of presidential support in Congress. In doing so I seek to push this line of inquiry so as not only develop a theory of presidential campaign behavior, but also to further understand how the actions of the ‘modern presidency’ have interacted with and transformed deep-seated notions of the separation of powers.
Journal of immunology (Baltimore, Md. : 1950), 1998
Binding of the HIV envelope glycoprotein gp120 to CD4 inhibits T cell activation. We have used a ... more Binding of the HIV envelope glycoprotein gp120 to CD4 inhibits T cell activation. We have used a murine T cell clone transfected with either wild-type human CD4 or mutated forms of CD4 to characterize the pathways involved in this inhibitory effect of gp120. Ag-induced proliferation of T cell clones transfected with human CD4 was completely inhibited in the presence of gp120, even though stimulation of this clone is independent of a CD4/MHC class II interaction. In addition, our results demonstrate that the inhibition by gp120 is not due to the sequestration of lck from TCR and does not require activation of lck by gp120. This suggests that CD4 can regulate the initiation of T cell activation independently of its interaction with lck. Moreover, we demonstrate that the nonresponsiveness induced by gp120 can be reversed by soluble CD4 when added early after onset of stimulation and that gp120 exerts its inhibitory effect when cells are in the G0 > or = 1 phase of the cell cycle.
The influence of insulin and glucagon on the release of glycolytic enzyme activities and actin fr... more The influence of insulin and glucagon on the release of glycolytic enzyme activities and actin from cultured pig kidney cells treated with digitonin has been studied. Both insulin and glucagon reduced the release of all glycolytic enzymes except for phosphofructokinase, and concurrently reduced the release of actin. These data have been discussed in relation to their contribution to knowledge of the interactions between glycolytic enzymes and actin filaments of the cytoskeleton, and to the influence of hormones on these interactions.
Proceedings of the National Academy of Sciences of the United States of America, Jan 30, 2007
The Wiskott-Aldrich syndrome protein (WASp) plays a major role in coupling T cell antigen recepto... more The Wiskott-Aldrich syndrome protein (WASp) plays a major role in coupling T cell antigen receptor (TCR) stimulation to induction of actin cytoskeletal changes required for T cell activation. Here, we report that WASp inducibly binds the sorting nexin 9 (SNX9) in T cells and that WASp, SNX9, p85, and CD28 colocalize within clathrin-containing endocytic vesicles after TCR/CD28 costimulation. SNX9, implicated in clathrin-mediated endocytosis, binds WASp via its SH3 domain and uses its PX domain to interact with the phosphoinositol 3-kinase regulatory subunit p85 and product, phosphoinositol (3,4,5)P3. The data reveal ligation-induced CD28 endocytosis to be clathrin- and phosphoinositol 3-kinase-dependent and TCR/CD28-evoked CD28 internalization and NFAT activation to be markedly enhanced by SNX9 overexpression, but severely impaired by expression of an SNX9 mutant (SNX9DeltaPX) lacking p85-binding capacity. CD28 endocytosis and CD28-evoked actin polymerization also are impaired in WAS...
The thymus-independent T lymphopoietic capacity of the murine intestinal mucosa has been establis... more The thymus-independent T lymphopoietic capacity of the murine intestinal mucosa has been established. Cryptopatches have now been identified as the location of the elusive precursors for gut-derived T cells. These cryptopatch cells have been shown to give rise to intestinal T cells expressing either TCRgammadelta or TCRalphabeta. Here we discuss the role of MHC in the development and selection of gut-derived T cells. Through the analysis of iIEL selection in animals expressing a transgenic TCRalphabeta, in the presence or absence of p56(lck), we discuss lineage relationships among CD4(-)8(+) iIEL subsets, and their possible function(s).
Proceedings of the National Academy of Sciences, 2001
Induction of resting B cell growth and differentiation requires a complex series of temporally co... more Induction of resting B cell growth and differentiation requires a complex series of temporally coordinated signals that are initiated on contact with activated helper T cells. These signals complement one another, each rendering the B cell susceptible to factors supporting progressive activation. Here, we demonstrate that soluble CD14 (sCD14) bypasses the physiological sequelae of events that limit B cell activation. B cell growth and differentiation in vitro is induced by both native and recombinant forms of sCD14 at nanomolar concentrations. sCD14-mediated cellular activation does not require membrane CD14 expression, depends on a region of CD14 that is not involved in lipopolysaccharide binding, and requires functional Toll-like receptor 4. Consistent with biological activity of sCD14 in vitro , its administration to neonatal mice enhances Ig secretion. The results presented establish sCD14 as a naturally occurring soluble B cell mitogen of mammalian origin.
While both lck and fyn are essential to generating the full sequelae of proximal signals emanatin... more While both lck and fyn are essential to generating the full sequelae of proximal signals emanating from the TcR/CD3 complex, thus far, modeling the temporal and spatial involvement of fyn has been problematic. The absence of a binding partner, analogous to the role of CD4 in targeting lck, commonalties of structure, common modes of activation and overlapping substrate specificities support the conclusion that many aspects of their roles may be redundant. Whether or how their functions are coordinated remains obscure. Recent experiments incorporating membrane partitioning towards resolving the distinct roles of lck and fyn in TcR/CD3 mediated cellular activation demonstrate that the membrane microdomains, termed lipid rafts, function to segregate the two kinases in unstimulated primary cells, and offers resolution in modeling their non-redundant contributions to the proximal TcR/CD3 signaling. Their activation, while interdependent, is temporally and spatially uncoupled. Lck activation is upstream of fyn activation. Moreover lck-dependent fyn activation is unidirectional, predicting the existence of distinct kinase specific regulatory mechanisms operating in lipid rafts. Thus, more than merely vehicles supporting protein concentration, these microdomains provide an environment in which the regulation of enzymatic activities is coupled to and regulated by a temporal coordination of protein translocations subsequent to TcR/CD3/CD4 engagement.
To assess the respective contribution of the extracellular and intracellular domains of CD4 in re... more To assess the respective contribution of the extracellular and intracellular domains of CD4 in regulating early T cell activation events, we have used a CD4-independent murine T cell clone transfected with human CD4. Stimulation of CD4 positive clones could only be observed if CD4 molecules associated to lck were co-aggregated with the TCR complex, con®rming that the simultaneous interaction of MHC class II molecules with the CD4/lck complex and the TCR is required to initiate T cell activation. To assess the involvement of the extracellular portion of CD4 in this process, we transfected a chimeric molecule (EGFRCD4) consisting of the extracellular portion of the epidermal growth factor receptor (EGFR), and of the transmembrane and cytoplasmic domains of human CD4. Although this chimeric molecule associates with lck, transfected clones were induced to proliferate by mAb speci®c for TCR in the absence of co-aggregation. A new regulatory role for the extracellular domain of CD4 which is independent of its interaction with MHC class II molecules is thus revealed in these experiments. Taken together, our results demonstrate that, in a CD4-independent cell line, two domains of CD4 regulate early T cell activation events: (1) its association with lck and (2) its extracellular domain, independently of its interaction with MHC class II molecules.
Recent results provide insight into the temporal and spatial relationship governing lck-dependent... more Recent results provide insight into the temporal and spatial relationship governing lck-dependent fyn activation and demonstrate TCR/CD4-induced activation and translocation of lck into lipid rafts and the ensuing activation of colocalized fyn. The prediction follows that directly targeting lck to lipid rafts will bypass the requirement for juxtaposing TCR and CD4-lck, and rescue cellular activation mediated by Ab specific for the constant region of TCR chain. The present study uses a family of murine IL-2dependent CD4 ؉ T cell clonal variants in which anti-TCRC signaling is impaired in an lck-dependent fashion. Importantly, these variants respond to Ag-and mAb-mediated TCR-CD4 coaggregation, both of which enable the coordinated interaction of CD4associated lck with the TCR/CD3 complex. We have previously demonstrated that anti-TCRC responsiveness in this system correlates with the presence of kinase-active, membrane-associated lck and preformed hypophosphorylated TCR:-associated protein of 70 kDa complexes, a phenotype recapitulated in primary resting CD4 ؉ T cells. We show in this study that forced expression of wild-type lck achieved the same basal composition of the TCR/CD3 complex and yet did not rescue anti-TCRC signaling. In contrast, forced expression of C20S/C23S-mutated lck (double-cysteine lck), unable to bind CD4, rescues anti-TCRC proximal signaling and cellular growth. Double-cysteine lck targets lipid rafts, colocalizes with >98% of cellular fyn, and results in a 7-fold increase in basal fyn kinase activity. Coaggregation of CD4 and TCR achieves the same outcome. These results underscore the critical role of lipid rafts in spatially coordinating the interaction between lck and fyn that predicates proximal TCR/CD3 signaling.
Whether or how the activation of Lck and Fyn during T cell receptor (TCR) signaling is coordinate... more Whether or how the activation of Lck and Fyn during T cell receptor (TCR) signaling is coordinated, and their delivery of function integrated, is unknown. Here we show that lipid rafts function to segregate Lck and Fyn in T cells before activation. Coaggregation of TCR and CD4 leads to Lck activation within seconds outside lipid rafts, followed by its translocation into lipid rafts and the activation of colocalized Fyn. Genetic evidence demonstrates that Fyn activation is strictly dependent on receptor-induced translocation of Lck. These results characterize the interdependence of Lck and Fyn function and establish the spatial and temporal distinctions of their roles in the cellular activation process.
Pseudomonas aeruginosa is a leading cause of community-acquired and hospital-acquired infections.... more Pseudomonas aeruginosa is a leading cause of community-acquired and hospital-acquired infections. Successful treatment is hampered by its remarkable ability to rapidly develop resistance to antimicrobial agents, mostly through mutation. In response, the World Health Organization listed carbapenem-resistant P. aeruginosa as a Priority 1 (Critical) pathogen for research and development of new treatments. A key resource in developing effective countermeasures is access to diverse and clinically relevant strains for testing. Herein we describe a panel of 100 diverse P. aeruginosa strains to support this endeavor.Whole genome sequencing was performed on 3,785 P. aeruginosa housed in our repository. Isolates were cultured from clinical samples collected from healthcare facilities around the world between 2003 and 2017. Core-genome multi-locus sequence typing and high-resolution SNP-based phylogenetic analyses were used to select a panel of 100 strains that captured the genetic diversity o...
Differential contributions of the glycosylphosphatidylinositol (GPI)-anchor and GPI-anchored prot... more Differential contributions of the glycosylphosphatidylinositol (GPI)-anchor and GPI-anchored proteins (GPI-AP) to signalling remain poorly understood. Here we show that GPI-AP deficient murine clones produce on average 18 and 181-fold more IL-2 mRNA and protein, respectively, upon T cell receptor (TCR) stimulation, in a cell-intrinsic fashion. This phenotype is formally attributed to a mutation within the transferase complex that predicates the initial step in GPI-anchor biosynthesis. Conditional disruption of the transferase complex enabled the generation of primary GPI-AP deficient CD4 + T cells, which produce on average 10-and 23-fold more IL-2 mRNA and protein, respectively, upon TCR stimulation. Conditional disruption of the transamidase complex yields GPI-sufficient, GPI-AP deficient primary CD4 + T cells. TCR stimulation of these cells yields levels of IL-2 mRNA and protein ranging from 1-3 and 3-fold, respectively, of controls. These results provide the first evidence of a profound impact of GPI in the regulation of TCR signalling.
Pajak merupakan iuran rakyat kepada kas negara berdasarkan undang-undang (yang dapat dipaksakan) ... more Pajak merupakan iuran rakyat kepada kas negara berdasarkan undang-undang (yang dapat dipaksakan) dengan tidak mendapat jasa timbal balik (kontraprestasi) yang langsung dapat ditunjukkan, dan yang digunakan untuk membayar pengeluaran umum. Walaupun demikian tidak semua Wajib Pajak mau dengan sukarela membayar pajaknya karena menganggap bahwa pajak sebagai suatu beban yang sama dengan biaya lainnya yang terjadi pada perusahaan yang perlu diperlakukan secara efisien. Pemerintah dalam rangka menanggulangi keresahan Wajib Pajak membuat suatu kebijakan baru, yaitu Sunset Policy yang hanya berlaku untuk tahun 2008 dan dibantu oleh Direktorat Jenderal Pajak untuk mensosialisasikan kebijakan ini dan memberikan arahan bagi Wajib Pajak yang memanfaatkan kebijakan ini untuk membayar pajak. Penelitian ini dilakukan dengan judul “Peranan Sunset Policy Terhadap Peningkatan Penerimaan Pajak Dari Wajib Pajak Orang Pribadi” dengan Wajib Pajak yang diambil adalah Wajib Pajak Orang Pribadi, pada KPP Pr...
Over the past two decades, Acinetobacter baumannii has emerged as a leading cause of nosocomial i... more Over the past two decades, Acinetobacter baumannii has emerged as a leading cause of nosocomial infections worldwide. Of particular concern are panresistant strains, leading the World Health Organization (WHO) to designate carbapenem-resistant A. baumannii as a priority 1 (critical) pathogen for research and development of new antibiotics. A key component in supporting this effort is accessibility to diverse and clinically relevant strains for testing. Here, we describe a panel of 100 diverse A. baumannii strains for use in this endeavor.
Healthcare management forum / Canadian College of Health Service Executives = Forum gestion des soins de santé / Collège canadien des directeurs de services de santé, 2016
Precision medicine aims to fix what is wrong with today's healthcare: a lack of targeted inte... more Precision medicine aims to fix what is wrong with today's healthcare: a lack of targeted interventions tailored to the person. It encompasses many aspects of health; chief among these is one's genetic profile. Researchers are making progress as gene-sequencing technologies get better and cheaper. Although there is cause for optimism, as several initiatives at Sunnybrook Research Institute show, scientific, systemic, and logistical challenges must be surmounted before advances can be integrated into the clinic. Despite these barriers, precision medicine is the only way forward.
The most efficient approach to monitoring and improving cleaning outcomes remains unresolved. We ... more The most efficient approach to monitoring and improving cleaning outcomes remains unresolved. We sought to extend the findings of a previous study by determining whether cleaning thoroughness (dye removal) correlates with cleaning efficacy (absence of molecular or cultivable biomaterial) and whether one brief educational intervention improves cleaning outcomes. Before-after trial. Newly built community hospital. 90 minute training refresher with surface-specific performance results. Dye removal, measured by fluorescence, and biomaterial removal and acquisition, measured with culture and culture-independent PCR-based assays, were clandestinely assessed for eight consecutive months. At this midpoint, results were presented to the cleaning staff (intervention) and assessments continued for another eight consecutive months. 1273 surfaces were sampled before and after terminal room cleaning. In the short-term, dye removal increased from 40.3% to 50.0% (not significant). For the entire st...
In this paper I examine the reasons for, and the impact of, presidential campaigning on the votin... more In this paper I examine the reasons for, and the impact of, presidential campaigning on the voting behavior of the U.S. Congress. Building off prior research in this area, I look at a particular aspect of campaigning — the endorsement of congressional candidates — as manifested in the public statements of presidents, and show how varied levels of campaigning — both in frequency and intensity — produce different levels of presidential support in Congress. In doing so I seek to push this line of inquiry so as not only develop a theory of presidential campaign behavior, but also to further understand how the actions of the ‘modern presidency’ have interacted with and transformed deep-seated notions of the separation of powers.
Journal of immunology (Baltimore, Md. : 1950), 1998
Binding of the HIV envelope glycoprotein gp120 to CD4 inhibits T cell activation. We have used a ... more Binding of the HIV envelope glycoprotein gp120 to CD4 inhibits T cell activation. We have used a murine T cell clone transfected with either wild-type human CD4 or mutated forms of CD4 to characterize the pathways involved in this inhibitory effect of gp120. Ag-induced proliferation of T cell clones transfected with human CD4 was completely inhibited in the presence of gp120, even though stimulation of this clone is independent of a CD4/MHC class II interaction. In addition, our results demonstrate that the inhibition by gp120 is not due to the sequestration of lck from TCR and does not require activation of lck by gp120. This suggests that CD4 can regulate the initiation of T cell activation independently of its interaction with lck. Moreover, we demonstrate that the nonresponsiveness induced by gp120 can be reversed by soluble CD4 when added early after onset of stimulation and that gp120 exerts its inhibitory effect when cells are in the G0 > or = 1 phase of the cell cycle.
The influence of insulin and glucagon on the release of glycolytic enzyme activities and actin fr... more The influence of insulin and glucagon on the release of glycolytic enzyme activities and actin from cultured pig kidney cells treated with digitonin has been studied. Both insulin and glucagon reduced the release of all glycolytic enzymes except for phosphofructokinase, and concurrently reduced the release of actin. These data have been discussed in relation to their contribution to knowledge of the interactions between glycolytic enzymes and actin filaments of the cytoskeleton, and to the influence of hormones on these interactions.
Proceedings of the National Academy of Sciences of the United States of America, Jan 30, 2007
The Wiskott-Aldrich syndrome protein (WASp) plays a major role in coupling T cell antigen recepto... more The Wiskott-Aldrich syndrome protein (WASp) plays a major role in coupling T cell antigen receptor (TCR) stimulation to induction of actin cytoskeletal changes required for T cell activation. Here, we report that WASp inducibly binds the sorting nexin 9 (SNX9) in T cells and that WASp, SNX9, p85, and CD28 colocalize within clathrin-containing endocytic vesicles after TCR/CD28 costimulation. SNX9, implicated in clathrin-mediated endocytosis, binds WASp via its SH3 domain and uses its PX domain to interact with the phosphoinositol 3-kinase regulatory subunit p85 and product, phosphoinositol (3,4,5)P3. The data reveal ligation-induced CD28 endocytosis to be clathrin- and phosphoinositol 3-kinase-dependent and TCR/CD28-evoked CD28 internalization and NFAT activation to be markedly enhanced by SNX9 overexpression, but severely impaired by expression of an SNX9 mutant (SNX9DeltaPX) lacking p85-binding capacity. CD28 endocytosis and CD28-evoked actin polymerization also are impaired in WAS...
The thymus-independent T lymphopoietic capacity of the murine intestinal mucosa has been establis... more The thymus-independent T lymphopoietic capacity of the murine intestinal mucosa has been established. Cryptopatches have now been identified as the location of the elusive precursors for gut-derived T cells. These cryptopatch cells have been shown to give rise to intestinal T cells expressing either TCRgammadelta or TCRalphabeta. Here we discuss the role of MHC in the development and selection of gut-derived T cells. Through the analysis of iIEL selection in animals expressing a transgenic TCRalphabeta, in the presence or absence of p56(lck), we discuss lineage relationships among CD4(-)8(+) iIEL subsets, and their possible function(s).
Proceedings of the National Academy of Sciences, 2001
Induction of resting B cell growth and differentiation requires a complex series of temporally co... more Induction of resting B cell growth and differentiation requires a complex series of temporally coordinated signals that are initiated on contact with activated helper T cells. These signals complement one another, each rendering the B cell susceptible to factors supporting progressive activation. Here, we demonstrate that soluble CD14 (sCD14) bypasses the physiological sequelae of events that limit B cell activation. B cell growth and differentiation in vitro is induced by both native and recombinant forms of sCD14 at nanomolar concentrations. sCD14-mediated cellular activation does not require membrane CD14 expression, depends on a region of CD14 that is not involved in lipopolysaccharide binding, and requires functional Toll-like receptor 4. Consistent with biological activity of sCD14 in vitro , its administration to neonatal mice enhances Ig secretion. The results presented establish sCD14 as a naturally occurring soluble B cell mitogen of mammalian origin.
While both lck and fyn are essential to generating the full sequelae of proximal signals emanatin... more While both lck and fyn are essential to generating the full sequelae of proximal signals emanating from the TcR/CD3 complex, thus far, modeling the temporal and spatial involvement of fyn has been problematic. The absence of a binding partner, analogous to the role of CD4 in targeting lck, commonalties of structure, common modes of activation and overlapping substrate specificities support the conclusion that many aspects of their roles may be redundant. Whether or how their functions are coordinated remains obscure. Recent experiments incorporating membrane partitioning towards resolving the distinct roles of lck and fyn in TcR/CD3 mediated cellular activation demonstrate that the membrane microdomains, termed lipid rafts, function to segregate the two kinases in unstimulated primary cells, and offers resolution in modeling their non-redundant contributions to the proximal TcR/CD3 signaling. Their activation, while interdependent, is temporally and spatially uncoupled. Lck activation is upstream of fyn activation. Moreover lck-dependent fyn activation is unidirectional, predicting the existence of distinct kinase specific regulatory mechanisms operating in lipid rafts. Thus, more than merely vehicles supporting protein concentration, these microdomains provide an environment in which the regulation of enzymatic activities is coupled to and regulated by a temporal coordination of protein translocations subsequent to TcR/CD3/CD4 engagement.
To assess the respective contribution of the extracellular and intracellular domains of CD4 in re... more To assess the respective contribution of the extracellular and intracellular domains of CD4 in regulating early T cell activation events, we have used a CD4-independent murine T cell clone transfected with human CD4. Stimulation of CD4 positive clones could only be observed if CD4 molecules associated to lck were co-aggregated with the TCR complex, con®rming that the simultaneous interaction of MHC class II molecules with the CD4/lck complex and the TCR is required to initiate T cell activation. To assess the involvement of the extracellular portion of CD4 in this process, we transfected a chimeric molecule (EGFRCD4) consisting of the extracellular portion of the epidermal growth factor receptor (EGFR), and of the transmembrane and cytoplasmic domains of human CD4. Although this chimeric molecule associates with lck, transfected clones were induced to proliferate by mAb speci®c for TCR in the absence of co-aggregation. A new regulatory role for the extracellular domain of CD4 which is independent of its interaction with MHC class II molecules is thus revealed in these experiments. Taken together, our results demonstrate that, in a CD4-independent cell line, two domains of CD4 regulate early T cell activation events: (1) its association with lck and (2) its extracellular domain, independently of its interaction with MHC class II molecules.
Recent results provide insight into the temporal and spatial relationship governing lck-dependent... more Recent results provide insight into the temporal and spatial relationship governing lck-dependent fyn activation and demonstrate TCR/CD4-induced activation and translocation of lck into lipid rafts and the ensuing activation of colocalized fyn. The prediction follows that directly targeting lck to lipid rafts will bypass the requirement for juxtaposing TCR and CD4-lck, and rescue cellular activation mediated by Ab specific for the constant region of TCR chain. The present study uses a family of murine IL-2dependent CD4 ؉ T cell clonal variants in which anti-TCRC signaling is impaired in an lck-dependent fashion. Importantly, these variants respond to Ag-and mAb-mediated TCR-CD4 coaggregation, both of which enable the coordinated interaction of CD4associated lck with the TCR/CD3 complex. We have previously demonstrated that anti-TCRC responsiveness in this system correlates with the presence of kinase-active, membrane-associated lck and preformed hypophosphorylated TCR:-associated protein of 70 kDa complexes, a phenotype recapitulated in primary resting CD4 ؉ T cells. We show in this study that forced expression of wild-type lck achieved the same basal composition of the TCR/CD3 complex and yet did not rescue anti-TCRC signaling. In contrast, forced expression of C20S/C23S-mutated lck (double-cysteine lck), unable to bind CD4, rescues anti-TCRC proximal signaling and cellular growth. Double-cysteine lck targets lipid rafts, colocalizes with >98% of cellular fyn, and results in a 7-fold increase in basal fyn kinase activity. Coaggregation of CD4 and TCR achieves the same outcome. These results underscore the critical role of lipid rafts in spatially coordinating the interaction between lck and fyn that predicates proximal TCR/CD3 signaling.
Whether or how the activation of Lck and Fyn during T cell receptor (TCR) signaling is coordinate... more Whether or how the activation of Lck and Fyn during T cell receptor (TCR) signaling is coordinated, and their delivery of function integrated, is unknown. Here we show that lipid rafts function to segregate Lck and Fyn in T cells before activation. Coaggregation of TCR and CD4 leads to Lck activation within seconds outside lipid rafts, followed by its translocation into lipid rafts and the activation of colocalized Fyn. Genetic evidence demonstrates that Fyn activation is strictly dependent on receptor-induced translocation of Lck. These results characterize the interdependence of Lck and Fyn function and establish the spatial and temporal distinctions of their roles in the cellular activation process.
Uploads
Papers by Michael Julius