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1.  Soluble TLT-1 modulates platelet-endothelial cell interactions and actin polymerization 
TREM like transcript-1 (TLT-1) is a membrane protein receptor found in α-granules of platelets and megakaryocytes. Upon platelet activation TLT-1 is rapidly brought to the surface of platelets. Recently, we demonstrated that activated platelets release a soluble form of TLT-1 (sTLT-1) that is found in serum but not in the plasma of healthy individuals and can enhance platelet aggregation in vitro. Furthermore, evaluation of patients diagnosed with inflammatory diseases, such as sepsis, show that these patients have significantly elevated levels of sTLT-1 in their blood. Accordingly, mice deficient in TLT-1 are predisposed to bleeding in response to an inflammatory challenge; however the mechanism of TLT-1 function remains unknown. In this investigation we demonstrate an increase in the amount of platelets that adhere to endothelial cell monolayers in the presence of recombinant sTLT-1 (rsTLT-1). Additionally we present evidence that rsTLT-1 increases platelet adherence to glass slides by stimulating actin polymerization in platelets as determined by increased staining of rodamine phalloidin. These results suggest that during inflammation, sTLT-1 may mediate hemostasis by enhancing actin polymerization, resulting in increased platelet aggregation and adherence to the endothelium.
doi:10.1097/MBC.0b013e3283358116
PMCID: PMC2886849  PMID: 20093931
2.  Visualization and Identification of IL-7 Producing Cells in Reporter Mice 
PLoS ONE  2009;4(11):e7637.
Interleukin-7 (IL-7) is required for lymphocyte development and homeostasis although the actual sites of IL-7 production have never been clearly identified. We produced a bacterial artificial chromosome (BAC) transgenic mouse expressing ECFP in the Il7 locus. The construct lacked a signal peptide and ECFP (enhanced cyan fluorescent protein ) accumulated inside IL-7-producing stromal cells in thoracic thymus, cervical thymus and bone marrow. In thymus, an extensive reticular network of IL-7-containing processes extended from cortical and medullary epithelial cells, closely contacting thymocytes. Central memory CD8 T cells, which require IL-7 and home to bone marrow, physically associated with IL-7-producing cells as we demonstrate by intravital imaging.
doi:10.1371/journal.pone.0007637
PMCID: PMC2770321  PMID: 19907640
3.  Phenotypic Heterogeneity in the Gray Platelet Syndrome Extends to the Expression of TREM Family Member, TLT-1 
Thrombosis and haemostasis  2008;100(1):45-51.
The Gray platelet syndrome (GPS) is a rare inherited disorder linked to undefined molecular abnormalities that prevent the formation and maturation of α-granules. Here, we report studies on two patients from unrelated families that confirm phenotypic heterogeneity in the disease. First we used immunoelectron microscopy (I-EM) to confirm that TREM-like transcript-1 (TLT-1) is mostly localized to α-granule membranes of normal platelets. Then we performed Western blotting (WB) and flow cytometry with permeabilized platelets to show that TLT-1 is selectively reduced in the platelets of patient 1, previously noted to be deficient in glycoprotein (GP)VI (Nurden et al, Blood 2004; 104:107−114). Yet both TLT-1 and GPVI were normally expressed in platelets of patient 2. Usual levels of JAM-C and claudin-5, also members of the immunoglobulin receptor family, were detected in platelets of both patients. In contrast, P-selectin was markedly decreased for patient 1 but not patient 2. Two metalloproteases, MMP-2 and MMP-9 were normally present. As predicted, platelets of patient 1 showed little labelling for TLT-1 in I-EM, whereas residual Fg was seen in small vesicular structures and P-selectin lining vacuoles or channels of what may be elements of the surface-connected canalicular system. Our results identify TLT-1 as a glycoprotein potentially targeted in platelets of GPS patients, while decreases in at least three membrane glycoproteins suggest that an unidentified proteolytic activity may contribute to the phenotype in some patients with this rare disease.
doi:10.1160/TH08-02-0067
PMCID: PMC2694056  PMID: 18612537
4.  TREM-like transcript-1 protects against inflammation-associated hemorrhage by facilitating platelet aggregation in mice and humans 
The Journal of Clinical Investigation  2009;119(6):1489-1501.
Triggering receptor expressed on myeloid cells–like (TREM-like) transcript-1 (TLT-1), a type 1 single Ig domain orphan receptor specific to platelet and megakaryocyte α-granules, relocates to the platelet surface upon platelet stimulation. We found here that patients diagnosed with sepsis, in contrast to healthy individuals, had substantial levels of soluble TLT-1 (sTLT-1) in their plasma that correlated with the presence of disseminated intravascular coagulation. sTLT-1 bound to fibrinogen and augmented platelet aggregation in vitro. Furthermore, the cytoplasmic domain of TLT-1 could also bind ezrin/radixin/moesin family proteins, suggesting its ability to link fibrinogen to the platelet cytoskeleton. Accordingly, platelets of Treml1–/– mice failed to aggregate efficiently, extending tail-bleeding times. Lipopolysaccharide-treated Treml1–/– mice developed higher plasma levels of TNF and D-dimers than wild-type mice and were more likely to succumb during challenge. Finally, Treml1–/– mice were predisposed to hemorrhage associated with localized inflammatory lesions. Taken together, our findings suggest that TLT-1 plays a protective role during inflammation by dampening the inflammatory response and facilitating platelet aggregation at sites of vascular injury. Therefore, therapeutic modulation of TLT-1–mediated effects may provide clinical benefit to patients with hypercoagulatory conditions, including those associated with inflammation.
doi:10.1172/JCI36175
PMCID: PMC2689104  PMID: 19436112
5.  Advantages of q-PCR as a method of screening for gene targeting in mammalian cells using conventional and whole BAC-based constructs 
Nucleic Acids Research  2008;36(18):e117.
We evaluate here the use of real-time quantitative PCR (q-PCR) as a method for screening for homologous recombinants generated in mammalian cells from either conventional gene-targeting constructs or whole BAC-based constructs. Using gene-targeted events at different loci, we show that q-PCR is a highly sensitive and accurate method for screening for conventional gene targeting that can reduce the number of clones requiring follow-up screening by Southern blotting. We further compared q-PCR to fluorescent in situ hybridization (FISH) for the detection of gene-targeting events using full-length BAC-based constructs designed to introduce mutations either into one gene or simultaneously into two adjacent genes. We find that although BAC-based constructs appeared to have high rates of homologous recombination when evaluated by FISH, screening by FISH was prone to false positives that were detected by q-PCR. Our results demonstrate the utility of q-PCR as a screening tool for gene targeting and further highlight potential problems with the use of whole BAC-based constructs for homologous recombination.
doi:10.1093/nar/gkn523
PMCID: PMC2566865  PMID: 18710883
6.  Combined Resveratrol, Quercetin, and Catechin Treatment Reduces Breast Tumor Growth in a Nude Mouse Model1 
Translational Oncology  2008;1(1):19-27.
Grape polyphenols can act as antioxidants, antiangiogenics, and selective estrogen receptor (ER) modifiers and are therefore especially relevant for gynecological cancers such as breast cancer. The major polyphenols of red wine (resveratrol, quercetin, and catechin) have been individually shown to have anticancer properties. However, their combinatorial effect on metastatic breast cancers has not been investigated in vivo. We tested the effect of low dietary concentrations of resveratrol, quercetin, and catechin on breast cancer progression in vitro by analyzing cell proliferation and cell cycle progression. The effects of these compounds on fluorescently tagged breast tumor growth in nude mice were assessed using in situ fluorescence image analysis. Individual polyphenols at 0.5 µM neither decreased breast cancer cell proliferation nor affected cell cycle progression in vitro. However, a combination of resveratrol, quercetin, and catechin at 0.5, 5, or 20 µM each significantly reduced cell proliferation and blocked cell cycle progression in vitro. Furthermore, using in situ image analysis, we determined that combined dietary polyphenols at 0.5, 5, or 25 mg/kg reduced primary tumor growth of breast cancer xenografts in a nude mouse model. Peak inhibition was observed at 5 mg/kg. These results indicate that grape polyphenols may inhibit breast cancer progression.
PMCID: PMC2510765  PMID: 18607509

Results 1-6 (6)