References of "Rahmouni, Souad"
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See detailDUSP3 Phosphatase Deficiency or Inhibition Limits Platelet Activation and Arterial Thrombosis
Tautz, Lutz; Rahmouni, Souad ULg; Oury, Cécile ULg et al

Conference (2015, June 27)

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See detailDUSP3 Phosphatase Deficiency or Inhibition Limits Platelet Activation and Arterial Thrombosis
Rahmouni, Souad ULg; MUSUMECI, Lucia ULg; Kuipers, Marijke J et al

Conference (2015, June 24)

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See detailDUSP3 Phosphatase Deficiency or Inhibition Limits Platelet Activation and Arterial Thrombosis
Rahmouni, Souad ULg; MUSUMECI, Lucia ULg; Kuijpers, Marijke J et al

Conference (2015, June 24)

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See detailCLEC-2 is required for the activation of mouseplatelets by bacterial DNA mimetics
Delierneux, Céline ULg; Hego, Alexandre ULg; LECUT, Christelle ULg et al

Conference (2015, June 22)

Background: Short nuclease-resistant phosphorothioate synthetic CpG motif-bearing oligonucleotides (CpG ODNs) mimicking bacterial DNA display potent immunostimulatory activity and are therefore being used ... [more ▼]

Background: Short nuclease-resistant phosphorothioate synthetic CpG motif-bearing oligonucleotides (CpG ODNs) mimicking bacterial DNA display potent immunostimulatory activity and are therefore being used in clinical trials as vaccine adjuvants. Cellular uptake and activation depends on the interaction of CpG ODNs with the C-type lectin receptor DEC-205 and subsequent stimulation of the Toll-like receptor 9 (TLR9) and myeloid differentiation primary response 88 (MyD88) signaling cascade. Platelets express TLR9, MyD88, and the C-type lectin-like receptor 2 (CLEC-2). However, the impacts of CpG ODNs on platelet function have been elusive. Aims: To evaluate whether CpG ODNs affect platelet activation and thrombus formation via CLEC-2 and TLR9. Methods: We incubated washed platelets or whole blood from TLR9-, MyD88- or CLEC-2- deficient mice with CpG ODNs. We performed platelet aggregometry, flow cytometric binding and platelet activation assays as well as signal transduction analyses. Thrombus formation and fibrin generation were also analyzed by intravital microscopy in mouse microcirculation upon intravenous injection of CpG ODNs. Results: We show that CpG ODNs bind on platelet surface and are internalized. They activate platelets and induce their aggregation. TLR9- or MyD88-deficient platelets aggregated normally in response to CpG ODN. Interestingly, platelets deficient for the C-type lectin receptor CLEC-2 were unable to capture and internalize CpG ODN. CLEC-2 deficiencyabolished CpG ODN-induced platelet activation and aggregation. CpG ODN stimulated CLEC-2 dependent tyrosine kinase pathway and Syk phosphorylation. In vivo, intravenously injected CpG ODN interacted with platelets adhered to laser injured arteriolar endothelia and promoted fibrin generation and thrombus growth. Conclusion: CLEC-2 mediates CpG ODN uptake and subsequent platelet activation, independently of TLR9, which may serve an important role in the interplay between platelets and immunity. [less ▲]

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See detailCLEC-2 is required for the activation of mouse platelets by bacterial DNA mimetics
Delierneux, Céline ULg; Hego, Alexandre ULg; LECUT, Christelle ULg et al

Conference (2015, June 22)

Aims: To evaluate whether CpG ODNs affect platelet activation and thrombus formation via CLEC-2 and TLR9. Methods: We incubated washed platelets or whole blood from TLR9-, MyD88- or CLEC-2- deficient mice ... [more ▼]

Aims: To evaluate whether CpG ODNs affect platelet activation and thrombus formation via CLEC-2 and TLR9. Methods: We incubated washed platelets or whole blood from TLR9-, MyD88- or CLEC-2- deficient mice with CpG ODNs. We performed platelet aggregometry, flow cytometric binding and platelet activation assays as well as signal transduction analyses. Thrombus formation and fibrin generation were also analyzed by intravital microscopy in mouse microcirculation upon intravenous injection of CpG ODNs. Results: We show that CpG ODNs bind on platelet surface and are internalized. They activate platelets and induce their aggregation. TLR9- or MyD88-deficient platelets aggregated normally in response to CpG ODN. Interestingly, platelets deficient for the C-type lectin receptor CLEC-2 were unable to capture and internalize CpG ODN. CLEC-2 deficiency abolished CpG ODN-induced platelet activation and aggregation. CpG ODN stimulated CLEC-2 dependent tyrosine kinase pathway and Syk phosphorylation. In vivo, intravenously injected CpG ODN interacted with platelets adhered to laser injured arteriolar endothelia and promoted fibrin generation and thrombus growth. Conclusion: CLEC-2 mediates CpG ODN uptake and subsequent platelet activation, independently of TLR9, which may serve an important role in the interplay between platelets and immunity. [less ▲]

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See detailGenetic depletion of the dual specificity protein phosphatase DUSP3 promotes LLC Lung tumour metastasis
Vandereyken, Maud ULg; Amand, Mathieu; Van Overmeire, Eva et al

Poster (2015, June)

DUSP3, also called Vaccinia-H1 Related (VHR) is a small dual specificity phosphatase dephosphorylating both tyrosine and serine/threonine phosphorylated residues. DUSP3 plays an important role in cell ... [more ▼]

DUSP3, also called Vaccinia-H1 Related (VHR) is a small dual specificity phosphatase dephosphorylating both tyrosine and serine/threonine phosphorylated residues. DUSP3 plays an important role in cell cycle regulation and is up-regulated in several human cancers. The physiological role of this phosphatase is, however, poorly understood. We have recently generated a DUSP3 knockout mouse by homologous recombination. The obtained mice have no spontaneous phenotype or pathology. However, DUSP3 deficiency prevented neo-vascularization of subcutaneously transplanted Matrigel plugs and Lung Lewis Carcinoma (LLC) tumours, suggesting an involvement of DUSP3 in tumour angiogenesis. Considering the importance of angiogenesis in metastatic formation, our study aimed to investigate the role of DUSP3 in metastatic dissemination. To do so, we used the LLC experimental metastasis model that shortcuts the intravasation/extravasation processes by injecting intravenously the LLC and the B16 (metastatic melanoma cell line) cells. Surprisingly, LLC, but not B16, lung metastasis developed twice faster in DUSP3-KO than WT mice. The enhanced LLC metastatic growth in DUSP3-/- mice was transferable to WT mice via DUSP3-/- bone marrow adoptive transfer, suggesting an involvement of the hematopoietic compartment in the observed phenotype. This was confirmed by a higher infiltration of neutrophils and macrophages in the lungs of DUSP3-KO compared to WT mice after LLC injection. This infiltration was correlated with higher expression of the chemokine receptor CCR2 in LLC-bearing DUSP3-KO lungs macrophages. Interestingly, LLC, but not B16 cells, were found to secrete high level of CCL2/MCP1, the CCR ligand chemokine. In line with this observation, we found that DUSP3-/- bone marrow derived-macrophages have a higher migration potential in response to LLC, but not B16, -conditionned medium. Altogether, our results suggest that DUSP3 plays an important role in metastatic dissemination/growth by a mechanism involving the control of CCR2-CCL2 chemoattraction axis in macrophages. [less ▲]

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See detailGenetic depletion of the dual specificity protein phosphatase DUSP3 promotes LLC Lung tumour metastasis
Vandereyken, Maud ULg; Amand, Mathieu ULg; Van Overmeire, Eva et al

Conference (2015, June)

DUSP3, also called Vaccinia-H1 Related (VHR) is a small dual specificity phosphatase dephosphorylating both tyrosine and serine/threonine phosphorylated residues. DUSP3 plays an important role in cell ... [more ▼]

DUSP3, also called Vaccinia-H1 Related (VHR) is a small dual specificity phosphatase dephosphorylating both tyrosine and serine/threonine phosphorylated residues. DUSP3 plays an important role in cell cycle regulation and is up-regulated in several human cancers. The physiological role of this phosphatase is, however, poorly understood. We have recently generated a DUSP3 knockout mouse by homologous recombination. The obtained mice have no spontaneous phenotype or pathology. However, DUSP3 deficiency prevented neo-vascularization of subcutaneously transplanted Matrigel plugs and Lung Lewis Carcinoma (LLC) tumours, suggesting an involvement of DUSP3 in tumour angiogenesis. Considering the importance of angiogenesis in metastatic formation, our study aimed to investigate the role of DUSP3 in metastatic dissemination. To do so, we used the LLC experimental metastasis model that shortcuts the intravasation/extravasation processes by injecting intravenously the LLC and the B16 (metastatic melanoma cell line) cells. Surprisingly, LLC, but not B16, lung metastasis developed twice faster in DUSP3-KO than WT mice. The enhanced LLC metastatic growth in DUSP3-/- mice was transferable to WT mice via DUSP3-/- bone marrow adoptive transfer, suggesting an involvement of the hematopoietic compartment in the observed phenotype. This was confirmed by a higher infiltration of neutrophils and macrophages in the lungs of DUSP3-KO compared to WT mice after LLC injection. This infiltration was correlated with higher expression of the chemokine receptor CCR2 in LLC-bearing DUSP3-KO lungs macrophages. Interestingly, LLC, but not B16 cells, were found to secrete high level of CCL2/MCP1, the CCR ligand chemokine. In line with this observation, we found that DUSP3-/- bone marrow derived-macrophages have a higher migration potential in response to LLC, but not B16, -conditionned medium. Altogether, our results suggest that DUSP3 plays an important role in metastatic dissemination/growth by a mechanism involving the control of CCR2-CCL2 chemoattraction axis in macrophages. [less ▲]

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See detailDUSP3 genetic deletion confers M2-like−macrophage-dependent tolerance to septic shock
Singh, Pratibha; Dejager, Lien; Amand, Mathieu ULg et al

in Journal of Immunology (2015)

DUSP3 is a small dual-specificity protein phosphatase with an unknown physiological function. We report that DUSP3 is strongly expressed in human and mouse monocytes and macrophages and that its ... [more ▼]

DUSP3 is a small dual-specificity protein phosphatase with an unknown physiological function. We report that DUSP3 is strongly expressed in human and mouse monocytes and macrophages and that its deficiency in mice promotes tolerance to lipopolysaccharide (LPS)-induced endotoxin shock and to polymicrobial septic shock following cecal ligation and puncture. By using adoptive transfer experiments, we demonstrate that resistance to endotoxin is macrophage-dependent and transferable and that this protection is associated with a striking increase of M2-like macrophages in DUSP3-/- mice in both the LPS and cecal ligation and puncture models. We show that the altered response of DUSP3-/- mice to sepsis is reflected in decreased TNF production and impaired ERK1/2 activation. Our results demonstrate that DUSP3 plays a key and non-redundant role as a regulator of innate immune responses by mechanisms involving the control of ERK1/2 activation, TNF secretion and macrophage polarization. [less ▲]

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See detailPerspective: Tyrosine Phosphatases As Novel Targets For Antiplatelet Therapy
Tautz, Lutz; Senis, Yotis; Oury, Cécile ULg et al

in Bioorganic & Medicinal Chemistry (2015), Epub ahead of print

Arterial thrombosis is the primary cause of most cases of myocardial infarction and stroke, the leading causes of death in the developed world. Platelets, highly specialized cells of the circulatory ... [more ▼]

Arterial thrombosis is the primary cause of most cases of myocardial infarction and stroke, the leading causes of death in the developed world. Platelets, highly specialized cells of the circulatory system, are key contributors to thrombotic events. Antiplatelet drugs, which prevent platelets from aggregating, have been very effective in reducing the mortality and morbidity of these conditions. However, approved antiplatelet therapies have adverse side effects, most notably the increased risk of bleeding. Moreover, there remains a considerable incidence of arterial thrombosis in a subset of patients receiving currently available drugs. Thus, there is a pressing medical need for novel antiplatelet agents with a more favorable safety profile and less patient resistance. The discovery of novel antiplatelet targets is the matter of intense ongoing research. Recent findings demonstrate the potential of targeting key signaling molecules, including kinases and phosphatases, to prevent platelet activation and aggregation. Here, we offer perspectives to targeting members of the protein tyrosine phosphatase (PTP) superfamily, a major class of enzymes in signal transduction. We give an overview of previously identified PTPs in platelet signaling, and discuss their potential as antiplatelet drug targets. We also introduce VHR (DUSP3), a PTP that we recently identified as a major player in platelet biology and thrombosis. We review our data on genetic deletion as well as pharmacological inhibition of VHR, providing proof-of-principle for a novel and potentially safer VHR-based antiplatelet therapy. [less ▲]

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See detailDUSP3 Phosphatase Deficiency or Inhibition Limit Platelet Activation and Arterial Thrombosis
Musumeci, Lucia ULg; Kuijpers, Marijke; Gilio, Karen et al

in Circulation (2015), 131(7), 656-68

Background A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. Better understanding of the molecular mechanisms leading to platelet ... [more ▼]

Background A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. Better understanding of the molecular mechanisms leading to platelet activation is of importance for the development of improved therapies. Recently, protein tyrosine phosphatases (PTPs) have emerged as critical regulators of platelet function. Methods and Results This is the first report implicating the dual-specificity phosphatase 3 (DUSP3) in platelet signaling and thrombosis. This phosphatase is highly expressed in human and mouse platelets. Platelets from DUSP3-deficient mice displayed a selective impairment of aggregation and granule secretion mediated through the collagen receptor glycoprotein VI (GPVI) and the C-type lectin-like receptor 2 (CLEC-2). DUSP3-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism, compared to wild-type mice, and showed severely impaired thrombus formation upon ferric chloride-induced carotid artery injury. Intriguingly, bleeding times were not altered in DUSP3-deficient mice. At the molecular level, DUSP3 deficiency impaired Syk tyrosine phosphorylation, subsequently reducing phosphorylation of PLCγ2 and calcium fluxes. To investigate DUSP3 function in human platelets, a novel small-molecule inhibitor of DUSP3 was developed. This compound specifically inhibited collagen and CLEC-2-induced human platelet aggregation, thereby phenocopying the effect of DUSP3 deficiency in murine cells. Conclusions DUSP3 plays a selective and essential role in collagen- and CLEC-2-mediated platelet activation and thrombus formation in vivo. Inhibition of DUSP3 may prove therapeutic for arterial thrombosis. This is the first time a PTP, implicated in platelet signaling, has been targeted with a small-molecule drug. [less ▲]

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See detailThe RIAD peptidomimetic inhibits HIV-1 replication in humanized NSG mice
Singh, Maneesh; Singh, Pratibha; Vaira, Dolores et al

in European Journal of Clinical Investigation (2014), 44(2), 146-152

Background Increased intracellular concentration of cyclic AMP (cAMP) in T cells is associated with various immunodeficiency conditions including human immunodeficiency virus (HIV) infection. Several ... [more ▼]

Background Increased intracellular concentration of cyclic AMP (cAMP) in T cells is associated with various immunodeficiency conditions including human immunodeficiency virus (HIV) infection. Several reports indicate a critical role of activated protein kinase A (PKA) in the susceptibility of cells to HIV infection. We have used a cell permeable, stable peptidomimetic version (P3) of the RI-anchoring disruptor (RIAD), which prevents PKA interaction with A-kinase-anchoring proteins (AKAPs). It is known that RIAD peptide abrogates effects of localized cAMP signalling through anchored type I PKA in lymphocytes and prevents murine AIDS (MAIDS) infection when expressed as a transgene in mice. Methods and Results In vitro HIV-infected human peripheral blood mononuclear cells (PBMCs) show reduced levels of p24 and intracellular cAMP in T cells when treated with RIAD peptidomimetic (RIAD-P3). Humanized NOD/SCID/IL2cnull (NSG) mice infected with HIV-1 JRCSF and treated with RIAD-P3 (3􏰀5 mg) once every 2 weeks showed significantly reduced levels of viral load at +28, +42 and +56 days and increased CD4 numbers at +56 days after the start of treatment. RIAD-P3-treated humanized mice had lower levels of intracellular cAMP in T cells sorted from splenocytes. Conclusions Treatment with RIAD-P3 limits HIV-1 viral replication and stabilizes CD4 levels by mechanisms involving cAMP/PKA-I pathway in human PBMCs and humanized NSG mice. [less ▲]

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See detailDUSP3/VHR is a pro-angiogenic atypical dual-specificity phosphatase
Amand, Mathieu ULg; Erpicum, Charlotte ULg; BAJOU, Khalid ULg et al

Poster (2014, January 27)

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See detailDUSP3/VHR is a pro-angiogenic atypical dual-specificity phosphatase
Amand, Mathieu ULg; Erpicum, Charlotte ULg; BAJOU, Khalid ULg et al

in Molecular Cancer (2014)

Background DUSP3 phosphatase, also known as Vaccinia-H1 Related (VHR) phosphatase, encoded by DUSP3/Dusp3 gene, is a relatively small member of the dual-specificity protein phosphatases. In vitro studies ... [more ▼]

Background DUSP3 phosphatase, also known as Vaccinia-H1 Related (VHR) phosphatase, encoded by DUSP3/Dusp3 gene, is a relatively small member of the dual-specificity protein phosphatases. In vitro studies showed that DUSP3 is a negative regulator of ERK and JNK pathways in several cell lines. On the other hand, DUSP3 is implicated in human cancer. It has been alternatively described as having tumor suppressive and oncogenic properties. Thus, the available data suggest that DUSP3 plays complex and contradictory roles in tumorigenesis that could be cell type-dependent. Since most of these studies were performed using recombinant proteins or in cell-transfection based assays, the physiological function of DUSP3 has remained elusive. Results Using immunohistochemistry on human cervical sections, we observed a strong expression of DUSP3 in endothelial cells (EC) suggesting a contribution for this phosphatase to EC functions. DUSP3 downregulation, using RNA interference, in human EC reduced significantly in vitro tube formation on Matrigel and spheroid angiogenic sprouting. However, this defect was not associated with an altered phosphorylation of the documented in vitro DUSP3 substrates, ERK1/2, JNK1/2 and EGFR but was associated with an increased PKC phosphorylation. To investigate the physiological function of DUSP3, we generated Dusp3-deficient mice by homologous recombination. The obtained DUSP3-/- mice were healthy, fertile, with no spontaneous phenotype and no vascular defect. However, DUSP3 deficiency prevented neo-vascularization of transplanted b-FGF containing Matrigel and LLC xenograft tumors as evidenced by hemoglobin (Hb) and FITC-dextran quantifications. Furthermore, we found that DUSP3 is required for b-FGF-induced microvessel outgrowth in the aortic ring assay. Conclusions All together, our data identify DUSP3 as a new important player in angiogenesis. [less ▲]

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See detailMinocycline attenuates HIV-1 infection and suppresses chronic immune activation in humanized NOD/LtsZ-scidIL-2Rgamma(null) mice.
Singh, Maneesh; Singh, Pratibha; Vaira, Dolores et al

in Immunology (2014), 142(4), 562-72

More than a quarter of a century of research has established chronic immune activation and dysfunctional T cells as central features of chronic HIV infection and subsequent immunodeficiency. Consequently ... [more ▼]

More than a quarter of a century of research has established chronic immune activation and dysfunctional T cells as central features of chronic HIV infection and subsequent immunodeficiency. Consequently, the search for a new immunomodulatory therapy that could reduce immune activation and improve T-cell function has been increased. However, the lack of small animal models for in vivo HIV study has hampered progress. In the current study, we have investigated a model of cord blood haematopoietic progenitor cells (CB-HPCs) -transplanted humanized NOD/LtsZ-scidIL-2Rgamma(null) mice in which progression of HIV infection is associated with widespread chronic immune activation and inflammation. Indeed, HIV infection in humanized NSG mice caused up-regulation of several T-cell immune activation markers such as CD38, HLA-DR, CD69 and co-receptor CCR5. T-cell exhaustion markers PD-1 and CTLA-4 were found to be significantly up-regulated on T cells. Moreover, increased plasmatic levels of lipopolysaccharide, sCD14 and interleukin-10 were also observed in infected mice. Treatment with minocycline resulted in a significant decrease of expression of cellular and plasma immune activation markers, inhibition of HIV replication and improved T-cell counts in HIV-infected humanized NSG mice. The study demonstrates that minocycline could be an effective, low-cost adjunctive treatment to regulate chronic immune activation and replication of HIV. [less ▲]

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See detailDual-specificity phosphatase 3 knockout female mice are resistant to LPS and to polymicrobial induced septic shock in TNF dependent manner.
Rahmouni, Souad ULg; Singh; Dejager, Lien et al

in Journal of Immunology (2013, May)

We report the generation of dual-specificity phosphatase 3 (DUSP3) deficient mice. These mice develop normally and do not exhibit any spontaneous phenotype. However, VHR-/- females, but not males, are ... [more ▼]

We report the generation of dual-specificity phosphatase 3 (DUSP3) deficient mice. These mice develop normally and do not exhibit any spontaneous phenotype. However, VHR-/- females, but not males, are resistant to LPS- and to polymicrobial infection-induced septic shock. After LPS injection, while VHR-/- males and VHR+/+ mice of both genders, displayed an increased serum levels of TNF-α and IFN, the levels of these cytokines remained significantly low in the VHR-/- females. In vitro experiments using peritoneal macrophages showed the same results suggesting that the systemic cytokines profiles observed are macrophages-dependent. Adoptive transfer of VHR-/- females bone marrow to irradiated VHR+/+ female mice, but not to VHR-/- or VHR+/+ males, protected them from death after administration of LPS. Interestingly, VHR-/- females were sensitive to TNF-α- induced lethality. We also report that the decrease of TNF-α production observed in VHR-/- female’s macrophages after LPS activation was associated with a decreased ERK1/2, but not MEK1/2, activation. Interestingly, pervanadate (PTP pan inhibitor) treatment prior to LPS activation restored ERK1/2 activation in the VHR-deficient macrophages, suggesting that VHR is targeting one of the ERK1/2 PTPs or DUSPs. These results, together with our observation that DUSP3 is the most highly expressed phosphatase in macrophages, suggest a key non-redundant role of VHR as positive regulator of TNF-α in innate immune response in females. [less ▲]

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See detailImpact of bone marrow-derived mesenchymal stromal cells on experimental xenogeneic graft-versus-host disease
Bruck, France; Belle, Ludovic ULg; LECHANTEUR, Chantal ULg et al

in Cytotherapy (2013), 15(3), 267-279

Background aims. Graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation caused by donor T cells reacting against host tissues. Previous ... [more ▼]

Background aims. Graft-versus-host disease (GVHD) is a life-threatening complication of allogeneic hematopoietic cell transplantation caused by donor T cells reacting against host tissues. Previous studies have suggested that mesenchymal stromal cells (MSCs) could exert potent immunosuppressive effects. Methods. The ability of human bone marrow derived MSCs to prevent xenogeneic GVHD in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice and in NOD/SCID/interleukin-2Rg(null) (NSG) mice transplanted with human peripheral blood mononuclear cells (PBMCs) was assessed. Results. Injection of 200 106 human PBMCs intraperitoneally (IP) into sub-lethally (3.0 Gy) irradiated NOD/SCID mice also given anti-asialo GM1 antibodies IP 1 day prior and 8 days after transplantation induced lethal xenogeneic GVHD in all tested mice. Co-injection of 2 106 MSCs IP on day 0 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs. Similarly, injection of 30 106 human PBMCs IP into sub-lethally (2.5 Gy) irradiated NSG mice induced a lethal xenogeneic GVHD in all tested mice. Injection of 3 106 MSCs IP on days 0, 7, 14 and 21 did not prevent lethal xenogeneic GVHD induced by injection of human PBMCs. Conclusions. Injection of MSCs did not prevent xenogeneic GVHD in these two humanized mice models. [less ▲]

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See detailEvaluating Effects of Tyrosine Phosphatase Inhibitors on T Cell Receptor Signaling
Rahmouni, Souad ULg; Delacroix, Laurence ULg; Liu, Wallace et al

in Phosphatase Modulators, Methods in Molecular Biology (2013)

The importance of tyrosine phosphorylation in normal cell physiology is well established, highlighted by the many human diseases that stem from abnormalities in protein tyrosine kinase (PTK) and protein ... [more ▼]

The importance of tyrosine phosphorylation in normal cell physiology is well established, highlighted by the many human diseases that stem from abnormalities in protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) function. Contrary to earlier assumptions, it is now clear that both PTKs and PTPs are highly specific, non-redundant, and tightly regulated enzymes. Hematopoietic cells express particularly high numbers of PTKs and PTPs, and aberrant function of these proteins have been linked to many hematopoietic disorders. While PTK inhibitors are among FDA approved drugs for the treatment of leukemia and other cancers, efforts to develop therapeutics that target specific PTPs are still in its infancy. Here, we describe methods on how to evaluate effects of PTP inhibitors on T cell receptor signaling. Moreover, we provide a comprehensive strategy for compound prioritization, applicable to any drug discovery project involving T cells. We present a testing funnel that starts with relatively high-throughput luciferase reporter assays, followed by immunoblot, calcium flux, flow cytometry, and proliferation assays, continues with cytokine bead arrays, and finishes with specificity assays that involve RNA interference. We provide protocols for experiments in the Jurkat T cell line, but more importantly give detailed instructions, paired with numerous tips, on how to prepare and work with primary human T cells. [less ▲]

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See detailDynamic interaction between lymphoid tyrosine phosphatase and C-terminal Src kinase controls T cell activation
Tautz, Lutz; Vang, Torkel; Liu, Wallace et al

in FASEB Journal (2012, April), 26

Lymphoid tyrosine phosphatase (LYP) and C-terminal Src kinase (CSK) are negative regulators of signaling mediated through the T cell antigen receptor (TCR) and are thought to act in a cooperative manner ... [more ▼]

Lymphoid tyrosine phosphatase (LYP) and C-terminal Src kinase (CSK) are negative regulators of signaling mediated through the T cell antigen receptor (TCR) and are thought to act in a cooperative manner when forming a complex. Here, we show that dissociation of the LYP/CSK complex is necessary for recruitment of LYP to lipid rafts, where it down-modulates TCR-mediated signaling. Our findings may also explain the reduced TCR signaling associated with a single nucleotide polymorphism, which confers increased risk for autoimmunity and results in the expression of a LYP allele that can no longer bind CSK. Development of a potent and selective chemical probe of LYP allowed us to confirm that the observed down-modulation of TCR-induced signaling was due to the LYP catalytic activity. Our compound also represents a starting point for the development of a LYP-based treatment of autoimmunity. [less ▲]

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See detailAn improved protocol for efficient engraftment in NOD/LTSZ-SCIDIL-2Rgammanull mice allows HIV replication and development of anti-HIV immune responses.
Singh, Maneesh; Singh, Pratibha; Gaudray, Gilles et al

in PLoS ONE (2012), 7(6), 38491

Cord blood hematopoietic progenitor cells (CB-HPCs) transplanted immunodeficient NOD/LtsZ-scidIL2Rgamma(null) (NSG) and NOD/SCID/IL2Rgamma(null) (NOG) mice need efficient human cell engraftment for long ... [more ▼]

Cord blood hematopoietic progenitor cells (CB-HPCs) transplanted immunodeficient NOD/LtsZ-scidIL2Rgamma(null) (NSG) and NOD/SCID/IL2Rgamma(null) (NOG) mice need efficient human cell engraftment for long-term HIV-1 replication studies. Total body irradiation (TBI) is a classical myeloablation regimen used to improve engraftment levels of human cells in these humanized mice. Some recent reports suggest the use of busulfan as a myeloablation regimen to transplant HPCs in neonatal and adult NSG mice. In the present study, we further ameliorated the busulfan myeloablation regimen with fresh CB-CD34+cell transplantation in 3-4 week old NSG mice. In this CB-CD34+transplanted NSG mice engraftment efficiency of human CD45+cell is over 90% in peripheral blood. Optimal engraftment promoted early and increased CD3+T cell levels, with better lymphoid tissue development and prolonged human cell chimerism over 300 days. These humanized NSG mice have shown long-lasting viremia after HIV-1JRCSF and HIV-1Bal inoculation through intravenous and rectal routes. We also saw a gradual decline of the CD4+T cell count, widespread immune activation, up-regulation of inflammation marker and microbial translocation after HIV-1 infection. Humanized NSG mice reconstituted according to our new protocol produced, moderate cellular and humoral immune responses to HIV-1 postinfection. We believe that NSG mice reconstituted according to our easy to use protocol will provide a better in vivo model for HIV-1 replication and anti-HIV-1 therapy trials. [less ▲]

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