Disruption of the murine procollagen C-proteinase enhancer 2 gene causes accumulation of pro-apoA-I and increased HDL levels.

J Lipid Res. 2011 Nov;52(11):1974-83

Authors: Francone OL, Ishida BY, de la Llera-Moya M, Royer L, Happe C, Zhu J, Chalkey RJ, Schaefer P, Cox C, Burlingame A, Kane JP, Rothblat GH

Abstract
Given the increased prevalence of cardiovascular disease in the world, the search for genetic variations that impact risk factors associated with the development of this disease continues. Multiple genetic association studies demonstrate that procollagen C-proteinase enhancer 2 (PCPE2) modulates HDL levels. Recent studies revealed an unexpected role for this protein in the proteolytic processing of pro-apolipoprotein (apo) A-I by enhancing the cleavage of the hexapeptide extension present at the N-terminus of apoA-I. To investigate the role of the PCPE2 protein in an in vivo model, PCPE2-deficient (PCPE2 KO) mice were examined, and a detailed characterization of plasma lipid profiles, apoA-I, HDL speciation, and function was done. Results of isoelectric focusing (IEF) electrophoresis together with the identification of the amino terminal peptides DEPQSQWDK and WHVWQQDEPQSQWDVK, representing mature apoA-I and pro-apoA-I, respectively, in serum from PCPE2 KO mice confirmed that PCPE2 has a role in apoA-I maturation. Lipid profiles showed a marked increase in plasma apoA-I and HDL-cholesterol (HDL-C) levels in PCPE2 KO mice compared with wild-type littermates, regardless of gender or diet. Changes in HDL particle size and electrophoretic mobility observed in PCPE2 KO mice suggest that the presence of pro-apoA-I impairs the maturation of HDL. ABCA1-dependent cholesterol efflux is defective in PCPE2 KO mice, suggesting that the functionality of HDL is altered.

PMID: 21771977 [PubMed - indexed for MEDLINE]

HIF-1-mediated up-regulation of cardiotrophin-1 is involved in the survival response of cardiomyocytes to hypoxia.

Cardiovasc Res. 2011 Nov 1;92(2):247-55

Authors: Robador PA, San José G, Rodríguez C, Guadall A, Moreno MU, Beaumont J, Fortuño A, Díez J, Martínez-González J, Zalba G

Abstract
AIMS: Cardiotrophin-1 (CT-1) is a cytokine of the interleukin-6 superfamily which is up-regulated in cardiac diseases, in part via hypoxia-dependent mechanisms. However, no evidence for a direct regulation of CT-1 gene (CTF1) promoter by hypoxia inducible factor-1 (HIF-1) has been provided.
METHODS AND RESULTS: Hypoxia increased CT-1 mRNA levels in the murine adult cardiomyocyte cell line HL-1 in a time-dependent manner. Interestingly, in a murine model (C57BL/6), we show that systemic hypoxia also significantly up-regulated CT-1 in myocardial tissue. The effect of hypoxia on CT-1 expression was mediated through a transcriptional mechanism, since hypoxia increased luciferase activity of constructs containing CTF1 promoter sequences. The increase in CT-1 levels was significantly reduced by drugs that prevent calcium mobilization, such as lercanidipine, or that inhibit the activation of the PI3K/Akt pathway (wortmannin) or mammalian target of rapamycin (rapamycin). The CT-1 elevation was similarly induced by HIF-1α over-expression in co-transfection experiments and prevented by HIF-1α silencing. The direct interaction of HIF-1α with the CTF1 promoter was confirmed through site-directed mutagenesis of hypoxia response elements, electrophoreric mobility shift, and ChIP assays. Hypoxia induced HL-1 apoptosis (measured as annexin-V binding or caspase 3/7 activity) which was increased when CT-1 was silenced in knocked-down cells by lentiviral vectors.
CONCLUSION: Hypoxia increased CT-1 levels in cardiac cells (in vitro and in vivo) through a direct regulation of CTF1 promoter by HIF-1α. This CT-1 activation by hypoxia may protect cells from apoptosis, thus supporting a protective role for CT-1 as a survival factor for cardiomyocytes.

PMID: 21771897 [PubMed - indexed for MEDLINE]

On the use of micron-sized iron oxide particles (MPIOS) to label resting monocytes in bone marrow.

Mol Imaging Biol. 2011 Oct;13(5):819-24

Authors: Tang KS, Hann B, Shapiro EM

Abstract
PURPOSE: The use of MRI to monitor immune cell infiltration into various pathologies is well established. In an effort to boost the magnetic material within immune cells, this work attempted to label resting monocytes within bone marrow, in mice, by intravenous administration of micron-sized iron oxide particles (MPIOs), similar in fashion to the administration of (U)SPIO.
PROCEDURES: MPIOs were incubated with various immune cells both in culture, and in whole blood. Flow cytometry and histology were used to analyze magnetic cell labeling. Also, MPIOs were injected intravenously into mice. In vivo, high-resolution 3-D MRI was performed on mouse legs, and signal changes were quantified. Flow cytometry and histology were used to analyze magnetic cell labeling of bone marrow resident cells.
RESULTS: It is demonstrated here that monocytes and neutrophils can indeed endocytose MPIOs both in cell culture and ex vivo in whole blood. However, despite rapid accumulation of MPIOs within the bone marrow following injection, MPIOs did not label monocytes or any other hematopoietic cell type in the marrow. Hypotheses are drawn to explain these results in light of recent usage of MPIOs for immune cell tracking.
CONCLUSIONS: Systemic administration of various MPIO formulations showed that MPIOs arrive in bone marrow rapidly following injection and remain there for at least 7 days. Data also shows slow clearance of some particles from the tissue over this period. While MPIOs can efficiently label monocytes in culture and in whole blood ex vivo, they were not found to label bone marrow resident monocytes.

PMID: 20936363 [PubMed - indexed for MEDLINE]

The Epidermal Growth Factor Receptor Is Involved in Angiotensin II But Not Aldosterone/Salt-Induced Cardiac Remodelling.

PLoS One. 2012;7(1):e30156

Authors: Messaoudi S, Zhang AD, Griol-Charhbili V, Escoubet B, Sadoshima J, Farman N, Jaisser F

Abstract
Experimental and clinical studies have shown that aldosterone/mineralocorticoid receptor (MR) activation has deleterious effects in the cardiovascular system; however, the signalling pathways involved in the pathophysiological effects of aldosterone/MR in vivo are not fully understood. Several in vitro studies suggest that Epidermal Growth Factor Receptor (EGFR) plays a role in the cardiovascular effects of aldosterone. This hypothesis remains to be demonstrated in vivo. To investigate this question, we analyzed the molecular and functional consequences of aldosterone exposure in a transgenic mouse model with constitutive cardiomyocyte-specific overexpression of a mutant EGFR acting as a dominant negative protein (DN-EGFR). As previously reported, Angiotensin II-mediated cardiac remodelling was prevented in DN-EGFR mice. However, when chronic MR activation was induced by aldosterone-salt-uninephrectomy, cardiac hypertrophy was similar between control littermates and DN-EGFR. In the same way, mRNA expression of markers of cardiac remodelling such as ANF, BNF or β-Myosin Heavy Chain as well as Collagen 1a and 3a was similarly induced in DN-EGFR mice and their CT littermates. Our findings confirm the role of EGFR in AngII mediated cardiac hypertrophy, and highlight that EGFR is not involved in vivo in the damaging effects of aldosterone on cardiac function and remodelling.

PMID: 22291909 [PubMed - in process]

Epicutaneous immunization with DNP-BSA induces CD4(+) CD25(+) Treg cells that inhibit Tc1-mediated CS.

Immunol Cell Biol. 2012 Jan 31;

Authors: Majewska-Szczepanik M, Zemelka-Wiącek M, Ptak W, Wen L, Szczepanik M

Abstract
As we have shown previously that protein antigen applied epicutaneously (EC) in mice inhibits TNP-specific Th1-mediated contact sensitivity (CS), we postulated that the maneuver of EC immunization might also suppress Tc1-dependent CS response. Here we showed that EC immunization of normal mice with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA) applied on the skin in the form of a patch induces a state of subsequent unresponsiveness due to regulatory T cells (Treg) that inhibited sensitization and elicitation of effector T-cell responses. Suppression is transferable in vivo by TCRαβ(+) CD4(+) CD25(+) lymphocytes harvested from lymph nodes (LNs) of skin-patched animals. Flow cytometry revealed that EC immunization with DNP-BSA increased TCRαβ(+) CD4(+) CD25(+) FoxP3(+) lymphocytes in subcutaneous LNs, suggesting that observed suppression was mediated by Treg cells. Further, in vitro experiments showed that EC immunization with DNP-BSA prior to 1-fluoro-2,4-dinitrobenzen sensitization suppressed LN cell proliferation and inhibited production of TNF-α, IL-12 and IFN-γ. Using a transwell system or anti-CTLA-4 mAb, we found that EC induced suppression required direct Treg-effector cell contact and is CTLA-4-dependent.Immunology and Cell Biology advance online publication, 31 January 2012; doi:10.1038/icb.2012.1.

PMID: 22290507 [PubMed - as supplied by publisher]

A Potencial Theranostic Agent for EGF-R Expression Tumors: 177Lu-DOTA-Nimotuzumab.

Curr Radiopharm. 2012 Jan 11;

Authors: Victoria C, Zhang X, Fernández M, Díaz-Miqueli A, Iznaga-Escobar N, Deutscher SL, Balter H, Quinn TP, Cabral P

Abstract
In this work Nimotuzumab (monoclonal antibody, recognizes the EGF-R)was radiolabeled with 177Lu as a potential cancer therapy radiopharmaceutical. In-vitro cell binding studies and in-vivo biodistribution and imaging studies were performed to determine the radiochemical stability, targeting specificity and pharmacokinetics of the 177Lu-labeled antibody. Nimotuzumab was derivatized with DOTA-NHS at room temperature for 2 hours. DOTA-Nimotuzumab was radiolabeled with 177LuCl3 (15 MBq/mg) at 37ºC for 1 h. The radiochemical purity was assessed by ITLC, silica gel and by RP-HPLC. Binding specificity studies were performed with EGF-R positive A431 human epithelial carcinoma and EGF-R negative MDA-MB-435 breast carcinoma cells. Biodistribution studies were performed in healthy female CD-1 mice at 1 h, 4 h, 24 h, and A431 xenografted nude mice at 10 min, 1 h, 4 h, 24 h, 48 h, and 96 h. SPECT-CT imaging studies were performed in A431 xenografted mice at 24 h post injection. DOTA-Nimotuzumab was efficiently labeled with 177LuCl3 at 37ºC. The in vitro stability of labeled product was optimal over 24 h in buffered saline and mouse serum. Specific recognition of EGF-R by 177Lu-DOTA-Nimotuzumab was observed in A431 cell binding studies. Biodistribution studies demonstrated increasing tumor uptake of 177Lu-DOTA-Nimotuzumab over time, with tumor to muscle ratios of 6.26, 10.68, and 18.82 at 4 h, 24 h, and 96 h post injection. Imaging of A431 xenografted mice showed high uptake in th e tumor. 177Lu-DOTA-Nimotuzumab has the potential to be a promising therapy agent, which may be useful in the treatment of patients with EGF-R positive cancer.

PMID: 22280117 [PubMed - as supplied by publisher]

Integrin-targeted imaging of inflammation in vascular remodeling.

Arterioscler Thromb Vasc Biol. 2011 Dec;31(12):2820-6

Authors: Razavian M, Marfatia R, Mongue-Din H, Tavakoli S, Sinusas AJ, Zhang J, Nie L, Sadeghi MM

Abstract
OBJECTIVE: Inflammation plays a key role in the development of vascular diseases. Monocytes and macrophages express α(v)β(3) integrin. We used an α(v) integrin-specific tracer, (99m)Tc-NC100692, to investigate integrin-targeted imaging for detection vessel wall inflammation.
METHODS AND RESULTS: The binding of a fluorescent homologue of NC100692 to α(v)β(3) on human monocytes and macrophages was shown by flow cytometry. Vessel wall inflammation and remodeling was induced in murine carotid arteries through adventitial exposure to CaCl(2). NC100692 micro single photon computed tomography/CT imaging was performed after 2 and 4 weeks and showed significantly higher uptake of the tracer in CaCl(2)-exposed left carotids compared with sham-operated contralateral arteries. Histological analysis at 4 weeks demonstrated significant remodeling of left carotid arteries and considerable macrophage infiltration, which was confirmed by real-time polymerase chain reaction. There was no significant difference in normalized α(v), β(3), or β(5) mRNA expression between right and left carotid arteries. Finally, NC100692 uptake strongly correlated with macrophage marker expression in carotid arteries.
CONCLUSIONS: NC100692 imaging can detect vessel wall inflammation in vivo. If further validated, α(v)-targeted imaging may provide a noninvasive approach for identifying patients who are at high risk for vascular events and tracking the effect of antiinflammatory treatments.

PMID: 21940943 [PubMed - indexed for MEDLINE]

Memory T cells from minor histocompatibility antigen-vaccinated and virus-immune donors improve GVL and immune reconstitution.

Blood. 2011 Nov 24;118(22):5965-76

Authors: Li N, Matte-Martone C, Zheng H, Cui W, Venkatesan S, Tan HS, McNiff J, Demetris AJ, Roopenian D, Kaech S, Shlomchik WD

Abstract
Donor T cells contribute to the success of allogeneic hematopoietic stem cell transplantation (alloSCT). Alloreactive donor T cells attack leukemia cells, mediating the GVL effect. Donor T cells, including the memory T cells (T(M)) that are generated after infection, also promote immune reconstitution. Nonetheless, leukemia relapse and infection are major sources of treatment failure. Efforts to augment GVL and immune reconstitution have been limited by GVHD, the attack by donor T cells on host tissues. One approach to augmenting GVL has been to infuse ex vivo-generated T cells with defined specificities; however, this requires expertise that is not widely available. In the present study, we tested an alternative approach, adoptive immunotherapy with CD8+ T(M) from donors vaccinated against a single minor histocompatibility antigen (miHA) expressed by leukemia cells. Vaccination against the miHA H60 greatly augmented T(M)-mediated GVL against mouse chronic-phase (CP-CML) and blast crisis chronic myeloid leukemia (BC-CML). T(M)-mediated GVL was antigen specific and was optimal when H60 expression was hematopoietically restricted. Even when H60 was ubiquitous, donor H60 vaccination had a minimal impact on GVHD. T(M) from lymphocytic choriomeningitis virus (LCMV)-immune and H60-vaccinated donors augmented GVL and protected recipients from LCMV. These data establish a strategy for augmenting GVL and immune reconstitution without elaborate T-cell manipulation.

PMID: 21917752 [PubMed - indexed for MEDLINE]

Detection of β cell death in diabetes using differentially methylated circulating DNA.

Proc Natl Acad Sci U S A. 2011 Nov 22;108(47):19018-23

Authors: Akirav EM, Lebastchi J, Galvan EM, Henegariu O, Akirav M, Ablamunits V, Lizardi PM, Herold KC

Abstract
In diabetes mellitus, β cell destruction is largely silent and can be detected only after significant loss of insulin secretion capacity. We have developed a method for detecting β cell death in vivo by amplifying and measuring the proportion of insulin 1 DNA from β cells in the serum. By using primers that are specific for DNA methylation patterns in β cells, we have detected circulating copies of β cell-derived demethylated DNA in serum of mice by quantitative PCR. Accordingly, we have identified a relative increase of β cell-derived DNA after induction of diabetes with streptozotocin and during development of diabetes in nonobese diabetic mice. We have extended the use of this assay to measure β cell-derived insulin DNA in human tissues and serum. We found increased levels of demethylated insulin DNA in subjects with new-onset type 1 diabetes compared with age-matched control subjects. Our method provides a noninvasive approach for detecting β cell death in vivo that may be used to track the progression of diabetes and guide its treatment.

PMID: 22074781 [PubMed - indexed for MEDLINE]

In vivo imaging of radiation-induced tissue apoptosis by (99m)Tc(I)-his (6)-annexin A5.

Ann Nucl Med. 2012 Jan 26;

Authors: Lin KJ, Wu CC, Pan YH, Chen FH, Fu SY, Chiang CS, Hong JH, Lo JM

Abstract
OBJECTIVE: A recombinant annexin A5 with the N-terminal extension of six histidine residues was labeled with (99m)Tc(I)-tricarbonyl ion to produce the (99m)Tc-labeled annexin A5, referred to (99m)Tc(I)-his(6)-annexin A5. We have explored the agent as an effective imaging probe for in vivo detecting the apoptosis of internal tissue subjected with high radiation doses in a γ-irradiated mouse model. METHODS: [(99m)Tc(CO)(3)(OH(2))(3)](+) was prepared and taken to directly label his(6)-annexin A5. The radiochemical purity of (99m)Tc(I)-his(6)-annexin A5 after size-exclusion separation was measured by HPLC. The binding affinity of (99m)Tc(I)-his(6)-annexin A5 to apoptotic cells was assessed using 20 Gy-irradiated Jurkat T cells. The effectiveness of (99m)Tc(I)-his(6)-annexin A5 as an imaging probe to detect the internal tissue apoptosis was assessed by biodistribution study and nanoSPECT/CT using the animal model of C57BL/6J mice conducted with 10 Gy γ irradiation. RESULTS: The radiochemical purity of (99m)Tc(I)-his(6)-annexin A5 could attain ≥95%. The binding affinity of (99m)Tc(I)-his(6)-annexin A5 to the 20 Gy-irradiated Jurkat cells was found to be ca. 20-fold higher than that to the sham-irradiated cells. In the animal imaging study, the splenic uptake of (99m)Tc(I)-his(6)-annexin A5 for the 10 Gy-irradiated mice showed from ca. 3-fold to 5-fold higher than those of the sham-irradiated mice from 45 to 165 min postinjection. The corresponding intestinal uptake showed from ca. 2-fold to 3-fold higher during the same period of time postinjection. The biodistribution study demonstrated the organ uptakes comparable with the imaging results. The apoptotic extents of the spleen and the intestine from the SPECT/CT imaging were correlated with an immunohistochemical staining assay for caspase 3 active form fragment. CONCLUSION: This work is the first study to demonstrate that (99m)Tc(I)-his(6)-annexin A5 is a potential clinical imaging agent for detecting radiation-induced tissue apoptosis in an animal model.

PMID: 22278351 [PubMed - as supplied by publisher]

Dual modal in vivo imaging using upconversion luminescence and enhanced computed tomography properties.

Nanoscale. 2011 Oct 5;3(10):4365-71

Authors: Zhang G, Liu Y, Yuan Q, Zong C, Liu J, Lu L

Abstract
In vivo upconversion luminescence (UCL) imaging, exhibiting favorable characteristics such as high photostability, no blinking, sharp emission lines, and long lifetimes, is recognized as the excellent and significant photoluminescence imaging for the future. To develop the imaging system with high visual sensitivity and tissue penetration, the functional molecules with X-ray computed tomography (CT) contrast were grafted onto upconversion nanoparticles to obtain β-NaYF(4):18% Yb(3+),2%Er(3+)@SiO(2)-I/PEG (UCNPs@SiO(2)-I/PEG) nanoprobes. These nanoprobes are water-soluble, have low cytotoxicity, and possess excellent UCL and remarkable CT contrast. Of particular note is that, besides the element iodine, rare earth elements (Y, Yb, and Er) present in the nanoprobes also show CT contrast. Moreover, no background autofluorescence signal is found in in vivo UCL images. We believe that these nanoprobes with dual modal in vivo imaging of UCL and CT can serve as a promising platform for clinical diagnosis or biomedical studies.

PMID: 21904751 [PubMed - indexed for MEDLINE]

Orthodontic tooth movement causes decreased promoter expression of collagen type 1, bone sialoprotein and alpha-smooth muscle actin in the periodontal ligament.

Orthod Craniofac Res. 2012 Feb;15(1):52-61

Authors: Olson C, Uribe F, Kalajzic Z, Utreja A, Nanda R, Rowe D, Wadhwa S

Abstract
Olson C., Uribe F., Kalajzic Z., Utreja A., Nanda R., Rowe D., Wadhwa S. Orthodontic tooth movement causes decreased promoter expression of collagen type 1, bone sialoprotein and alpha-smooth muscle actin in the periodontal ligament. Orthod Craniofac Res 2012;15:52-61 © John Wiley & Sons A/S STRUCTURED ABSTRACT: Objective -  To evaluate the effects of orthodontic tooth movement on the promoter expression of collagen type 1 (3.6Col1), bone sialoprotein (BSP) and alpha-smooth muscle actin (αSMA) in the periodontal ligament (PDL) using transgenic mice containing transgenes of these promoters fused to green fluorescent proteins (GFP). Materials and Methods -  The maxillary first molars of 10-12 week-old transgenic mice were loaded with 10-12 g of force for 12, 48 h, or 7 days. Mice were transgenic for one of the following GFP-tagged bone markers of osteoblast lineage cells: 3.6-kb fragment of the rat collagen type 1 promoter (3.6Col1), BSP or α-smooth muscle actin (αSMA). Loaded molars under compression and tension were compared with contra-lateral unloaded controls. Results -  On the compression side of the PDL, orthodontic tooth movement caused a significant decrease in GFP expression of all the promoters at each time point. On the tension side, there was a significant increase in BSP-GFP expression, 12 h following loading compared to the contralateral unloaded controls. Conclusions -  An in vivo tooth movement model using transgenic mice with promoter-GFP constructs provides an efficient and effective way of investigating the cellular events underlying orthodontic tooth movement. PDL cells may undergo decreased differentiation in response to the compressive force.

PMID: 22264327 [PubMed - in process]

Essential roles for Pot1b in HSC self-renewal and survival.

Blood. 2011 Dec 1;118(23):6068-77

Authors: Wang Y, Shen MF, Chang S

Abstract
Maintenance of mammalian telomeres requires both the enzyme telomerase and shelterin, which protect telomeres from inappropriately activating DNA damage response checkpoints. Dyskeratosis congenita is an inherited BM failure syndrome disorder because of defects in telomere maintenance. We have previously shown that deletion of the shelterin component Pot1b in the setting of telomerase haploinsufficiency results in rapid telomere shortening and fatal BM failure in mice, eliciting phenotypes that strongly resemble human syskeratosis congenita. However, it was unclear why BM failure occurred in the setting of Pot1b deletion. In this study, we show that Pot1b plays an essential role in HSC survival. Deletion of Pot1b results in increased apoptosis, leading to severe depletion of the HSC reserve. BM from Pot1b(Δ/Δ) mice cannot compete with BM from wild-type mice to provide multilineage reconstitution, indicating that there is an intrinsic requirement for Pot1b the maintenance of HSC function in vivo. Elimination of the p53-dependent apoptotic function increased HSC survival and significantly extended the lifespan of Pot1b-null mice deficient in telomerase function. Our results document for the first time the essential role of a component of the shelterin complex in the maintenance of HSC and progenitor cell survival.

PMID: 21948176 [PubMed - indexed for MEDLINE]

Osteoblast specific overexpression of human interleukin-7 rescues the bone mass phenotype of interleukin-7 deficient female mice.

J Bone Miner Res. 2012 Jan 18;

Authors: Aguila HL, Mun SH, Kalinowski J, Adams DJ, Lorenzo JA, Lee SK

Abstract
Interleukin-7 is a critical cytokine for lymphoid development and a direct inhibitor of in vitro osteoclastogenesis in murine bone marrow cultures. To explore the role of IL-7 in bone, we generated transgenic mouse lines bearing the 2.3 Kb rat collagen 1A1 promoter driving the expression of human IL-7 specifically in osteoblasts. In addition we crossed these mice with IL-7 deficient mice to determine if the alterations in lymphopoiesis, bone mass and osteoclast formation observed in the IL-7 KO mice could be rescued by osteoblast-specific overexpression of IL-7. Here we show that mice overexpressing human IL-7 in the osteoblast lineage demonstrated increased trabecular bone volume in vivo by µCT and decreased osteoclast formation in vitro. Furthermore, targeted overexpression of IL-7 in osteoblasts rescued the osteopenic bone phenotype and B cell development of IL-7 KO mice but did not have an effect on T lymphopoiesis, which occurs in the periphery. The bone phenotypes in IL-7 KO mice and targeted IL-7 overexpressing mouse models were observed only in females. These results likely reflect both a direct inhibitory effects of IL-7 on osteoclastogenesis in vivo and gender specific differences in responses to IL-7. © 2012 American Society for Bone and Mineral Research.

PMID: 22258693 [PubMed - as supplied by publisher]

Inhibition of Pyk2 blocks lung inflammation and injury in a mouse model of acute lung injury.

Respir Res. 2012 Jan 18;13(1):4

Authors: Duan Y, Learoyd J, Meliton AY, Leff AR, Zhu X

Abstract
ABSTRACT: BACKGROUND: Proline-rich tyrosine kinase 2 (Pyk2) is essential in neutrophil degranulation and chemotaxis in vitro. However, its effect on the process of lung inflammation and edema formation during LPS induced acute lung injury (ALI) remain unknown. The goal of the present study was to determine the effect of inhibiting Pyk2 on LPS-induced acute lung inflammation and injury in vivo. METHODS: C57BL6 mice were given either 10 mg/kg LPS or saline intratracheally. Inhibition of Pyk2 was effected by intraperitoneal administration TAT-Pyk2-CT 1h before challenge. Bronchoalveolar lavage analysis of cell counts, lung histology and protein content in BAL were analyzed at 18 h after LPS treatment. KC and MIP-2 contents in BAL were measured by a mouse cytokine multiplex kit. The static lung compliance was determined by pressure-volume curve using a computer-controlled small animal ventilator. The extravasated Evans blue concentration in lung homogenate was determined spectrophotometrically. RESULTS: Intratracheal instillation of LPS induced significant neutrophil infiltration into the lung interstitium and alveolar space, which was attenuated by pre-treatment with TAT-Pyk2-CT. TAT-Pyk2-CT pretreatment also attenuated 1) myeloperoxidase content in lung tissues, 2) vascular leakage as measured by Evans blue dye extravasation in the lungs and the increase in protein content in bronchoalveolar lavage, and 3) the decrease in lung compliance. In each paradigm, treatment with control protein TAT-GFP had no blocking effect. By contrast, production of neutrophil chemokines MIP-2 and keratinocyte-derived chemokine in the bronchoalveolar lavage was not reduced by TAT-Pyk2-CT. Western blot analysis confirmed that tyrosine phosphorylation of Pyk2 in LPS-challenged lungs was reduced to control levels by TAT-Pyk2-CT pretreatment. CONCLUSIONS: These results suggest that Pyk2 plays an important role in the development of acute lung injury in mice and that pharmacological inhibition of Pyk2 might provide a potential therapeutic strategy in the pretreatment for patients at imminent risk of developing acute lung injury.

PMID: 22257498 [PubMed - as supplied by publisher]

L(1)-regularized Cerenkov luminescence tomography with a SP(3) method and CT fusion.

Conf Proc IEEE Eng Med Biol Soc. 2011 Aug;2011:6158-61

Authors: Zhong J, Tian J, Yang X, Qin C

Abstract
Imaging modality of radionuclides has been enriched by an optical approach, Cerenkov luminescence tomography (CLT). Referred to the traditional radionuclide imaging, such as positron emission tomography (PET) or single photon emission computed tomography (SPECT), any incremental improvement of CLT imaging is consistent with the application to information needs. In this contribution, the paper presents an l(1)-regularized imaging method for CLT problem. After utilizing the Vavilov-Cerenkov effect via third-order simplified spherical harmonics (SP(3)) approximation, we establish the large-scale linear equations in the CLT framework. The derived linear problem is seriously ill-posed, and transformed into an l(1)-regularized least squares program. The inverse solution to these equations is the three-dimensional radioisotope recovery data by an interior-point method. In the physical phantom and the in vivo mouse experiment, results demonstrate that the proposed technique produces better imaging quality and improves the reconstruction efficacy, compared with those from diffusion approximation with the Tikhonov regularization.

PMID: 22255745 [PubMed - in process]

Ectopic bone regeneration by human bone marrow mononucleated cells, undifferentiated and osteogenically differentiated bone marrow mesenchymal stem cells in beta-tricalcium phosphate scaffolds.

Tissue Eng Part C Methods. 2012 Jan 18;

Authors: Ye X, Yin X, Yang D, Tan J, Liu G

Abstract
Tissue engineering approaches using the combination of porous ceramics and bone marrow mesenchymal stem cells (BMSCs) represent a promising bone substitute for repairing large bone defects. Nevertheless, optimal conditions for constructing tissue-engineered bone have yet to be determined. It remains unclear if transplantation of pre-differentiated BMSCs is superior to undifferentiated BMSCs or freshly isolated bone marrow mononucleated cells (BMNCs) in terms of new bone formation in vivo. The purpose of this study was to investigate the effect of in vitro osteogenic differentiation (β-glycerophosphate, dexamethasone and L-ascorbic acid) of human BMSCs on the capability to form tissue-engineered bone in unloaded conditions after subcutaneous implantation in nude mice. After isolation from human bone marrow aspirates, BMNCs were divided into three parts: one part was seeded onto porous beta-tricalcium phosphate (β-TCP) ceramics immediately and transplanted in a heterotopic nude mice model; two parts were expanded in vitro to passage 2 prior to cell seeding and in vivo transplantation, either under osteogenic conditions or not. Animals were sacrificed for micro-CT and histological evaluation at 4, 8, 12, 16 and 20 weeks post-implantation. The results showed that BMSCs differentiated into osteo-progenitor cells after induction, as evidenced by the altered cell morphology and elevated alkaline phosphatase (ALP) activity and calcium deposition, but their clonogenicity, proliferating rate and seeding efficacy were not significantly affected by osteogenic differentiation, compared with undifferentiated cells. Extensive new bone formed in the pores of all the scaffolds seeded with pre-differentiated BMSCs at 4 weeks after implantation, and maintained for 20 weeks. On the contrary, scaffolds containing undifferentiated BMSCs revealed limited bone formation only in 1 out of 6 cases at 8 weeks, and maintained for 4 weeks. For scaffolds with BMNCs, woven bone was observed sporadically only in one case at 8 weeks. Overall, this study suggests that ectopic osteogenesis of cell/scaffold composites is more dependent on the in vitro expansion condition, and osteo-differentiated BMSCs hold the highest potential concerning in vivo bone regeneration.

PMID: 22250840 [PubMed - as supplied by publisher]

Assessment of Axial Length Measurements in Mouse Eyes.

Optom Vis Sci. 2012 Jan 12;

Authors: Park HN, Qazi Y, Tan C, Jabbar SB, Cao Y, Schmid G, Pardue MT

Abstract
PURPOSE.: To compare measurements of murine ocular axial lengths (ALs) made with 780 nm partial coherence interferometry (PCI) and 1310 nm spectral domain-optical coherence tomography (SD-OCT). METHODS.: AL was measured at postnatal day (P) 58 in C57BL/6J mice. Repeated AL measurements were taken using a custom-made 780 nm PCI and a commercial 1310 nm SD-OCT. Intra- and interuser variability was assessed along the central optical axis and 2-degree off-axes angles with the SD-OCT. Data were collected and analyzed using Cronbach alpha (α), Bland-Altman coefficient of repeatability, agreement plots, and intraclass correlation coefficients (ICC). RESULTS.: AL measurements agreed well between the two instruments (3.262 ± 0.042 mm for PCI; 3.264 ± 0.047 mm for SD-OCT; n = 20 eyes). The ICC for PCI compared with SD-OCT was 0.92, confirming high agreement between the two instruments. Intrauser ICC for the PCI and SD-OCT were 0.814 and 0.995, respectively. Similarly, interuser ICC for PCI and SD-OCT were 0.970 and 0.943, respectively. Using SD-OCT, a 2-degree misalignment of the eye along the horizontal meridian produced mean differences in AL of -0.002 ± 0.017 mm relative to the centrally aligned images, whereas similar misalignment along the vertical meridian created 0.005 ± 0.018 mm differences in AL measurements. CONCLUSIONS.: AL measurements from the 780 nm PCI and 1310 nm SD-OCT correlate well. Multiple statistical indices indicate that both instruments have good precision and agreement for measuring murine ocular AL in vivo. Although the vertical meridian had the greater variability in AL in the small mouse eye; 2-degree off-axes differences were within the SD of centrally aligned AL.

PMID: 22246334 [PubMed - as supplied by publisher]

Pharmacological characterization of 2-methyl-N-((2'-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine (PF-04455242), a high-affinity antagonist selective for κ-opioid receptors.

J Pharmacol Exp Ther. 2011 Nov;339(2):555-66

Authors: Grimwood S, Lu Y, Schmidt AW, Vanase-Frawley MA, Sawant-Basak A, Miller E, McLean S, Freeman J, Wong S, McLaughlin JP, Verhoest PR

Abstract
2-Methyl-N-((2'-(pyrrolidin-1-ylsulfonyl)biphenyl-4-yl)methyl)propan-1-amine (PF-04455242) is a novel κ-opioid receptor (KOR) antagonist with high affinity for human (3 nM), rat (21 nM), and mouse (22 nM) KOR, a ∼ 20-fold reduced affinity for human μ-opioid receptors (MORs; K(i) = 64 nM), and negligible affinity for δ-opioid receptors (K(i) > 4 μM). PF-04455242 also showed selectivity for KORs in vivo. In rats, PF-04455242 blocked KOR and MOR agonist-induced analgesia with ID(50) values of 1.5 and 9.8 mg/kg, respectively, and inhibited ex vivo [(3)H](2-(benzofuran-4-yl)-N-methyl-N-((5S,7R,8R)-7-(pyrrolidin-1-yl)-1-oxaspiro[4.5]decan-8-yl)acetamide ([(3)H]CI977) and [(3)H](2S)-2-[[2-[[(2R)-2-[[(2S)-2-amino-3-(4-hydroxyphenyl) propanoyl]amino]propanoyl]amino]acetyl]-methylamino]-N-(2-hydroxyethyl)-3-phenylpropanamide ([(3)H]DAMGO) binding to KOR and MOR receptors with ID(50) values of 2.0 and 8.6 mg/kg, respectively. An in vivo binding assay was developed using (-)-4-[(3)H]methoxycarbonyl-2-[(1-pyrrolidinylmethyl]-1-[(3,4-dichlorophenyl)acetyl]-piperidine ([(3)H]PF-04767135), a tritiated version of the KOR positron emission tomography ligand (-)-4-[(11)C]methoxycarbonyl-2-[(1-pyrrolidinylmethyl]-1-[(3,4-dichlorophenyl)acetyl]-piperidine ([(11)C]GR103545) in which PF-04455242 had an ID(50) of 5.2 mg/kg. PF-04455242 demonstrated antidepressant-like efficacy (mouse forced-swim test), attenuated the behavioral effects of stress (mouse social defeat stress assay), and showed therapeutic potential in treating reinstatement of extinguished cocaine-seeking behavior (mouse conditioned place preference). KOR agonist-induced plasma prolactin was investigated as a translatable mechanism biomarker. Spiradoline (0.32 mg/kg) significantly increased rat plasma prolactin levels from 1.9 ± 0.4 to 41.9 ± 4.9 ng/ml. PF-04455242 dose-dependently reduced the elevation of spiradoline-induced plasma prolactin with an ID(50) of 2.3 ± 0.1 mg/kg, which aligned well with the ED(50) values obtained from the rat in vivo binding and efficacy assays. These data provide further evidence that KOR antagonists have potential for the treatment of depression and addiction disorders.

PMID: 21821697 [PubMed - indexed for MEDLINE]

Age-dependent disruption in hippocampal theta oscillation in amyloid-β overproducing transgenic mice.

Neurobiol Aging. 2012 Jan 6;

Authors: Scott L, Feng J, Kiss T, Needle E, Atchison K, Kawabe TT, Milici AJ, Hajós-Korcsok E, Riddell D, Hajós M

Abstract
Transgenic mice are used to model increased brain amyloid-β (Aβ) and amyloid plaque formation reflecting Alzheimer's disease pathology. In our study hippocampal network oscillations, population spikes, and long-term potentiation (LTP) were recorded in APPswe/PS1dE9 (APP/PS1) and presenilin1 (PS1) transgenic and wild type mice at 2, 4, and 8 months of age under urethane anesthesia. Hippocampal theta oscillations elicited by brainstem stimulation were similar in wild type and PS1 mice at all age groups. In contrast, APP/PS1 mice showed an age-dependent decrease in hippocampal activity, characterized by a significant decline in elicited theta power and frequency at 4 and 8 months. Magnitudes of population spikes and long-term potentiation in the dentate gyrus were similar across groups at both 4 and 8 months. In APP/PS1 mice, soluble and insoluble Aβ, and hippocampal and cortical plaque load increased with age, and the disruption in hippocampal theta oscillation showed a significant correlation with plaque load. Our study shows that, using in vivo electrophysiological methods, early Aβ-related functional deficits can be robustly detected in the brainstem-hippocampus multisynaptic network.

PMID: 22227005 [PubMed - as supplied by publisher]