Papers by Leonardo Vargas
Immunology, 2007
Bruton's tyrosine kinase (Btk), a member of the Tec family of tyrosine kinases, plays an importan... more Bruton's tyrosine kinase (Btk), a member of the Tec family of tyrosine kinases, plays an important role in the differentiation and activation of B cells. Mutations affecting Btk cause immunodeficiency in both humans and mice. In this study we set out to investigate the potential role of Btk in Toll-like receptor 9 (TLR9) activation and the production of pro-inflammatory cytokines such as interleukin (IL)-6, tumour necrosis factor (TNF)-α and IL-12p40. Our data show that Btk-deficient B cells respond more efficiently to CpG-DNA stimulation, producing significantly higher levels of pro-inflammatory cytokines but lower levels of the inhibitory cytokine IL-10. The quantitative reverse transcription–polymerase chain reaction (RT-PCR) analysis presented in this work shows that mRNA production of one of the important new members of the IL-12 family, IL-27, was significantly increased in Btk-deficient B cells after CpG-DNA stimulation. In this study, we demonstrate significant differences in CpG responsiveness between transitional 1 (T1) and T2 B cells for survival and maturation. Furthermore, TLR9 expression, measured both as protein and as mRNA, was increased in Btk-defective cells, especially after TLR9 stimulation. Collectively, these data provide evidence in support of the theory that Btk regulates both TLR9 activation and expression in mouse splenic B cells.
European Journal of Immunology, 2000
Bruton's tyrosine kinase (Btk) is a member of the Tec family of protein tyrosine kinases (PTK) ch... more Bruton's tyrosine kinase (Btk) is a member of the Tec family of protein tyrosine kinases (PTK) characterized by an N-terminal pleckstrin homology domain (PH) thought to directly interact with phosphoinositides. We report here that wild-type (wt) and also a gain-of-function mutant of Btk are redistributed following a wide range of receptor-mediated stimuli through phosphatidylinositol 3-kinase (PI 3-K) activation. Employing chimeric Btk with green fluorescent protein in transient transfections resulted in Btk translocation to the cytoplasmic membrane of live cells through various forms of upstream PI 3-K activation. The redistribution was blocked by pharmacological and biological inhibitors of PI 3-K. A gain-of-function mutant of Btk was found to be a potent inducer of lamellipodia and/or membrane ruffle formation. In the presence of constitutively active forms of Rac1 and Cdc42, Btk is co-localized with actin in these regions. Formation of the membrane structures was blocked by the dominant negative form of N17-Rac1. Therefore, Btk forms a link between a vast number of cell surface receptors activating PI 3-K and certain members of the Rho-family of small GTPases. In the chicken B cell line, DT40, cells lacking Btk differed from wt cells in the actin pattern and showed decreased capacity to form aggregates, further suggesting that cytoskeletal regulation mediated by Btk may be of physiological relevance.
European Journal of Immunology, 2000
Bruton's tyrosine kinase (Btk) is a member of the Tec family of protein tyrosine kinases (PTK) ch... more Bruton's tyrosine kinase (Btk) is a member of the Tec family of protein tyrosine kinases (PTK) characterized by an N-terminal pleckstrin homology domain (PH) thought to directly interact with phosphoinositides. We report here that wild-type (wt) and also a gain-of-function mutant of Btk are redistributed following a wide range of receptor-mediated stimuli through phosphatidylinositol 3-kinase (PI 3-K) activation. Employing chimeric Btk with green fluorescent protein in transient transfections resulted in Btk translocation to the cytoplasmic membrane of live cells through various forms of upstream PI 3-K activation. The redistribution was blocked by pharmacological and biological inhibitors of PI 3-K. A gain-of-function mutant of Btk was found to be a potent inducer of lamellipodia and/or membrane ruffle formation. In the presence of constitutively active forms of Rac1 and Cdc42, Btk is co-localized with actin in these regions. Formation of the membrane structures was blocked by the dominant negative form of N17-Rac1. Therefore, Btk forms a link between a vast number of cell surface receptors activating PI 3-K and certain members of the Rho-family of small GTPases. In the chicken B cell line, DT40, cells lacking Btk differed from wt cells in the actin pattern and showed decreased capacity to form aggregates, further suggesting that cytoskeletal regulation mediated by Btk may be of physiological relevance.
Biochemical and Biophysical Research Communications, 2002
Bruton&am... more Bruton's tyrosine kinase (Btk) is necessary for B-lymphocyte development. Mutation in the gene coding for Btk causes X-linked agammaglobulinemia (XLA) in humans. Similar to Btk, c-Abl is a tyrosine kinase shuttling between the cytoplasm and the nucleus where it is involved in different functions depending on the localization. In this report we describe for the first time that c-Abl and Btk physically interact and that c-Abl can phosphorylate tyrosine 223 in the SH3 domain of Btk. Interestingly, the Btk sequence matched a v-Abl substrate [correction] identified from a randomized peptide library and was also highly related to a number of previously found c-Abl substrates.
Immunological Reviews, 2005
Bruton's tyrosine kinase (Btk) is encoded by the gene that when mutated causes the primary immuno... more Bruton's tyrosine kinase (Btk) is encoded by the gene that when mutated causes the primary immunodeficiency disease X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Btk is a member of the Tec family of protein tyrosine kinases (PTKs) and plays a vital, but diverse, modulatory role in many cellular processes. Mutations affecting Btk block B-lymphocyte development. Btk is conserved among species, and in this review, we present the sequence of the full-length rat Btk and find it to be analogous to the mouse Btk sequence. We have also analyzed the wealth of information compiled in the mutation database for XLA (BTKbase), representing 554 unique molecular events in 823 families and demonstrate that only selected amino acids are sensitive to replacement (P < 0.001). Although genotype-phenotype correlations have not been established in XLA, based on these findings, we hypothesize that this relationship indeed exists. Using short interfering-RNA technology, we have previously generated active constructs downregulating Btk expression. However, application of recently established guidelines to enhance or decrease the activity was not successful, demonstrating the importance of the primary sequence. We also review the outcome of expression profiling, comparing B lymphocytes from XLA-, Xid-, and Btk-knockout (KO) donors to healthy controls. Finally, in spite of a few genes differing in expression between Xid-and Btk-KO mice, in vivo competition between cells expressing either mutation shows that there is no selective survival advantage of cells carrying one genetic defect over the other. We conclusively demonstrate that for the R28C-missense mutant (Xid), there is no biologically relevant residual activity or any dominant negative effect versus other proteins.
Allergy & Clinical Immunology International-journal of The World Allergy Organization, 2000
Mutations in the cytoplasmic tyrosine kinase, Bruton's tyrosine kinase (Btk), were identified in ... more Mutations in the cytoplasmic tyrosine kinase, Bruton's tyrosine kinase (Btk), were identified in humans diagnosed as X-linked agammaglobulinemia (XLA) and as X-linked immunodeficiency (Xid) in mice. The functional alteration of the Btk mutation results in a block in B-cell development and defects in the cellular signaling. Btk is a member of the Tec family of protein tyrosine kinases (Tec kinases) which includes Itk, Tec, Bmx, Txk, Drosophila Tec (dTec29), Raja eglanteria Tec (sTec), and zebrafish Tec (zTec). Apart from Txk and dTec29, these kinases possess an N-terminal pleckstrin homology domain (PH) which is essential for membrane targeting and also for biological activity. The PH domains are capable of binding phosphoinositides in a specific manner and function mainly as membrane localization signals. In vitro, Btk binds strongly to PIP 3 and IP 4 . Moreover, lipid specificity for other Tec kinase members is not known. We propose a model in which PH domains of Tec family kinases exhibit a switching mechanism regulating activation and deactivation states (ON-OFF states). This process is determined by exchange of lipid specificity where the membrane anchoring signal is an activation state of that mechanism.
Immunological Reviews, 2009
Summary: Bruton’s agammaglobulinemia tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase impo... more Summary: Bruton’s agammaglobulinemia tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase important in B-lymphocyte development, differentiation, and signaling. Btk is a member of the Tec family of kinases. Mutations in the Btk gene lead to X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Activation of Btk triggers a cascade of signaling events that culminates in the generation of calcium mobilization and fluxes, cytoskeletal rearrangements, and transcriptional regulation involving nuclear factor-κB (NF-κB) and nuclear factor of activated T cells (NFAT). In B cells, NF-κB was shown to bind to the Btk promoter and induce transcription, whereas the B-cell receptor-dependent NF-κB signaling pathway requires functional Btk. Moreover, Btk activation is tightly regulated by a plethora of other signaling proteins including protein kinase C (PKC), Sab/SH3BP5, and caveolin-1. For example, the prolyl isomerase Pin1 negatively regulates Btk by decreasing tyrosine phosphorylation and steady state levels of Btk. It is intriguing that PKC and Pin1, both of which are negative regulators, bind to the pleckstrin homology domain of Btk. To this end, we describe here novel mutations in the pleckstrin homology domain investigated for their transforming capacity. In particular, we show that the mutant D43R behaves similar to E41K, already known to possess such activity.
Current Chemical Genomics, 2010
Myotonic dystrophy type 1 (DM1) is a genetic disorder characterized by muscle wasting, myotonia, ... more Myotonic dystrophy type 1 (DM1) is a genetic disorder characterized by muscle wasting, myotonia, cataracts, cardiac arrhythmia, hyperinsulinism and intellectual deficits, and is caused by expansion of a CTG repeat in the 3'UTR of the Dystrophia Myotonica-Protein Kinase (DMPK) gene. The DMPK transcripts containing expanded CUG repeats accumulate in nuclear foci and ultimately cause mis-splicing of secondary genes through the dysregulation of RNA-binding proteins including Muscleblind 1 (MBNL1) and CUG binding protein 1 (CUGBP1). Correction of mis-splicing of genes such as the Skeletal muscle-specific chloride channel 1 (CLCN1), Cardiac troponin T (TNNT2), Insulin receptor (INSR) and Sarcoplasmic/endoplasmic reticulum Ca 2+ ATPase 1 (SERCA1) may alleviate some of the symptoms of DM1; hence identification of small molecule modulators is an important step towards a therapy for DM1 patients. Here we describe the generation of immortalized myoblast cell lines derived from healthy (DMPK CTG 5 ) and DM1 patient (DMPK CTG 1000 ) fibroblasts by constitutive overexpression of human telomerase reverse transcriptase (hTERT) and inducible overexpression of the Myoblast determination factor (MYOD). MBNL1-containing nuclear foci, mis-splicing events and defective myotube differentiation defects characteristic of DM1 were observed in these cells. A CLCN1 luciferase minigene construct (CLCN1-luc) was stably introduced to monitor intron 2 retention in the DM1 cellular context (a reported splicing defect in DM1). The assay was validated by performing a high-throughput screen (HTS) of ~13,000 low molecular weight compounds against the CLCN1-luc DM1 myoblast cell line, providing an ideal system for conducting HTS to better understand and treat DM1.
Immunobiology, 2006
Dendritic cells (DCs) are the only professional antigen-presenting cells endowed with the ability... more Dendritic cells (DCs) are the only professional antigen-presenting cells endowed with the ability to stimulate naı¨ve T cells and initiate a primary immune response. For this reason, DC-based immunization has been shown to be highly effective in eliciting CTL responses to viruses and tumor-associated antigens. Here we report on the use of DC immunization to enhance the B cell-mediated humoral immune response to highly conserved proteins and the application of this approach to the generation of monoclonal antibodies (mAbs) against these proteins. To illustrate the technique we describe the production of mAbs to class II transactivator (CIITA), the major histocompatibility complex (MHC) CIITA, a difficult immunogen owing to its high degree of identity among species. We show that mice immunized with a combination of an intravenous injection of DCs pulsed with recombinant fragments of CIITA followed by intraperitoneal injection of the antigen in incomplete Freund's adjuvant induced a detectable antibody response against CIITA, while sera from mice immunized using the traditional method (i.e. intraperitoneal immunization with 50 mg of protein in complete Freund's adjuvant) gave an almost undetectable response. Furthermore, a total of four fusion experiments demonstrate that immunization with Ag-pulsed DCs is necessary for the efficient generation of hybridomas and a good yield of mAbs specific for the recombinant and the native endogenous CIITA protein. Conversely, four independent fusions carried out with splenocytes from mice immunized using the traditional method failed to produce anti-CIITA hybridomas. We propose that immunization with antigenloaded DCs should be the method of preference when attempting to raise mAbs against weak self-immunogens.
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Papers by Leonardo Vargas