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ABSTRACTAbnormal expression of long non-coding RNAs (lncRNAs) is associated with the dysfunctions of human trophoblast cells and the occurrence of miscarriage (abnormal early embryo loss). BBC3/PUMA (BCL2 binding component 3) plays significant roles in regulation of cell apoptosis. However, whether specific lncRNAs might regulate BBC3 in trophoblast cells and further induce apoptosis and miscarriage remains completely unclear. Through screening, we identified a novel lnc-HZ12, which was significantly highly expressed in villous tissues of recurrent miscarriage (RM) patients relative to their healthy control (HC) group. Lnc-HZ12suppressed chaperone-mediated autophagy (CMA) degradation of BBC3, promoted trophoblast cell apoptosis, and was associated with miscarriage. In mechanism, lnc-HZ12downregulated the expression levels of chaperone molecules HSPA8 and LAMP2A in trophoblast cells. Meanwhile, lnc-HZ12(mainly lnc-HZ12-SO2 region in F2 fragment) and HSPA8 competitively bound with the 169RVLYNL174patch on BBC3, which prevented BBC3 from interactions with HSPA8 and impaired the formation of BBC3-HSPA8-LAMP2A complex for CMA degradation of BBC3. Thus, lnc-HZ12upregulated the BBC3-CASP9-CASP3 pathway and induced trophoblast cell apoptosis. In villous tissues, lnc-HZ12was highly expressed, CMA degradation of BBC3 was suppressed, and the apoptosis levels were higher in RM vs HC villous tissues, all of which were associated with miscarriage. Interestingly, knockdown of murine Bbc3could efficiently suppress placental apoptosis and alleviate miscarriage in a mouse miscarriage model. Taken together, our results indicated that lnc-HZ12and BBC3 played important roles in trophoblast cell apoptosis and miscarriage and might act as attractive targets for miscarriage treatment.Abbreviation: 7-AAD: 7-aminoactinomycin D; BaP: benzopyrene; BBC3/PUMA: BCL2 binding component 3; ChIP: chromatin immunoprecipitation; CHX: cycloheximide; CMA: chaperone-mediated autophagy; CQ: chloroquine; DMSO: dimethyl sulfoxide; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HC: healthy control; HSPA8: heat shock protein family A (Hsp70) member 8; IP: immunoprecipitation; LAMP2A: lysosomal associated membrane protein 2; LncRNA: long non-coding RNA; mRNA: messenger RNA; MT: mutant-type; NC: negative control; NSO: nonspecific oligonucleotide; PARP1: poly(ADP-ribose) polymerase 1; RIP: RNA immunoprecipitation; RM: recurrent miscarriage; TBP: TATA-box binding protein; WT: wild-type.

•Designed a micro-governance framework of multi-government environmental regulation (ER) synergy.•Developed a synergistic-based measure to quantify the degree of heterogeneous multi-government ER synergy.•Heterogeneous multi-government ER synergy has dissimilar impacts on corporate environmental responsibility (CER).•Financing constraints and executives’ attention to CER play mediating roles in the mechanism.•The impact of multi-government ER synergy is heterogeneous among different types of firms.

Nanoplastics (NPs), as emerging pollutants, have attracted global attention. Nevertheless, the adverse effects of NPs on female reproductive health, especially unexplained miscarriage, are poorly understood. Defects of trophoblast cell migration and invasion are associated with miscarriage. Migrasomes were identified as cellular organelles with largely unidentified functions. Whether NPs might affect migration, invasion, and migrasome formation and induce miscarriage has been completely unexplored. In this study, we selected polystyrene nanoplastics (PS-NPs, 50 nm) as a model of plastic particles and treated human trophoblast cells and pregnant mice with PS-NPs at doses near the actual environmental exposure doses of plastic particles in humans. We found that exposure to PS-NPs induced a pregnant mouse miscarriage. PS-NPs suppressed ROCK1-mediated migration/invasion and migrasome formation. SOX2 was identified as the transcription factor of ROCK1. PS-NPs activated autophagy and promoted the autophagy degradation of SOX2, thus suppressing SOX2-mediated ROCK1 transcription. Supplementing with murine SOX2 or ROCK1 could efficiently rescue migration/invasion and migrasome formation and alleviate miscarriage. Analysis of the protein levels of SOX2, ROCK1, TSPAN4, NDST1, P62, and LC-3BII/I in PS-NP-exposed trophoblast cells, villous tissues of unexplained miscarriage patients, and placental tissues of PS-NP-exposed mice gave consistent results. Collectively, this study revealed the reproductive toxicity of nanoplastics and their potential regulatory mechanism, indicating that NP exposure is a risk factor for female reproductive health.

Eu3+-doped LiY(PO3)4(1-x)(SO4)4x(LYSO:Eu) phosphors are synthesized by high-temperature solid-phase method. The crystal structure, microscopic morphology and photo-spectrographic characteristics are analyzed respectively. The phosphor exhibits the strongest excitation peak at 397 nm, which indicates that the sample can be effectively excited by the near-ultraviolet/ultraviolet LED chip. The characteristic emission peaks of Eu3+ion 5D0→7FJ(J = 0, 1, 2, 3, 4) are observed from the PL spectrum, and the orange emission peak originating from the magnetic dipole transition 5D0→7F1of Eu3+is located near 597 nm. When the SO42−concentration exceeds 22.5 mol%, the quenching of the phosphor occurs. The Eu3+emission still retains 78.8% of the initial strength at 495 K, with excellent thermal stability. The emission intensity is decreased with the increase of SO42−concentration, indicating that the SO42−co-doping leads to an increase in the probability of non-radiative transitions. The best quantum efficiency of the LiY0.95(PO3)2.6(SO4)1.4: 0.05Eu3+sample is 86.65%, and the CIE chromaticity coordinates of this sample are (0.653, 0.347), which are close to the international standard red color coordinates (0.67, 0.33). These results indicate that LYSO:Eu has a good application in NUV/UV excited white LEDs. Influence of anionic substitutions of SO42−on the optical properties is discussed.

In polycythemia vera (PV), point mutation in JAK2 kinase (V617F) confers constitutive intracellular activity to JAK2 leading to a condition with excessive erythropoiesis that is independent of erythropoietin. Elevated hematocrit (HCT) and hyper-viscosity in the blood are risk factors for thrombosis and other symptoms. There is an increased iron demand required to support excessive erythropoiesis in the bone marrow. Hence, regulation of systemic iron in PV provides a mechanism for erythropoiesis control with potential disease modifying effects (Ginzburg YZ, Leukemia 2018).

Mattheakis: Protagonist Therapeutics: Current Employment, Current equity holder in private company. Zhao:Protagonist Therapeutics: Current Employment, Other: shareholder. Lee:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Tovera:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Zhao:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Cheng:Protagonist Therapeutics: Current Employment, Current equity holder in private company. Liu:Protagonist Therapeutics: Current Employment, Current equity holder in private company.

The RSpondin (Rspo) protein family is a recently described group of 4 distinct human secreted proteins. Reported activities for RSpo proteins include essential roles in vertebrate development and their ligand-type activities overlap substantially with those of the canonical Wnt ligands in that both RSpo and canonical Wnt signaling result in the activation of β-catenin. In a general functional screen for human secreted proteins using transgenic mouse models, we identified human Rspondin 1 (hRSpo1) protein as a potent and specific mitogen for the gastrointestinal epithelium and demonstrated potential therapeutic applications for the protein in mouse models of cancer therapy-induced mucositis. In contrast to previous studies, our data indicated only partial overlap between Wnt and RSpo ligand activities, suggesting that there may be independent receptor/signaling pathways for RSpo proteins that intersect those of Wnt at the level of β-catenin. Here we summarize the current reported data on the RSpo family and discuss these results in terms of alternate mechanisms of action. We have extended our observations on the potential therapeutic application of RSpo proteins by showing that all 4 human Rspo family members are capable of inducing epithelial proliferation and report the first non-vertebrate RSpo family member.

The proper development of Meckel's cartilage is critical for craniofacial skeletogenesis, because it serves as the primordium for the formation of mandible, malleus, incus, and sphenomandibular ligament. Cranial neural crest (CNC) cells contribute significantly to the formation of Meckel's cartilage. Members of the transforming growth factor beta (TGF-β) family control the proliferation and differentiation of CNC cells during craniofacial skeletogenesis. TGF-β signaling is transduced from the cell membrane to the nucleus by means of specific type I and type II receptors and phosphorylated Smad proteins. Here we demonstrate that application of TGF-β promotes chondrogenesis by specifically increasing proliferation of CNC-derived chondrocytes and production of extracellular matrix. To understand the molecular regulation of TGF-β signaling, we have examined the biological function of both TGF-β receptor-regulated and inhibitory Smads during Meckel's cartilage development. The expression patterns of Smad2, 3, and 7 are identical to the ones of endogenous TGF-β and its cognate receptors during Meckel's cartilage development, establishing the potential that these intracellular signaling Smads may regulate TGF-β-mediated chondrogenesis. Functional haploinsufficiency of Smad2 delays TGF-β–mediated Meckel's cartilage development. Overproduction of Smad7 severely inhibits Meckel's cartilage formation, indicating a negative feedback on TGF-β signaling by inhibitory Smad is critical in orchestrating TGF-β–mediated gene regulation during embryonic chondrogenesis. The effectiveness of TGF-β signaling is highly sensitive to the level of Smad gene expression. © 2002 Wiley-Liss, Inc.

Members of the transforming growth factor-β (TGF-β) superfamily regulate cell proliferation, differentiation, and apoptosis, controlling the development and maintenance of most tissues. TGF-β signal is transmitted through the phosphorylation of Smad proteins by TGF-β receptor serine/threonine kinase. During early tooth development, TGF-β inhibits proliferation of enamel organ epithelial cells but the underlying molecular mechanisms are largely unknown. Here we tested the hypothesis that antagonistic effects between Smad2 and Smad7 regulate TGF-β signaling during tooth development. Attenuation of Smad2 gene expression resulted in significant advancement of embryonic tooth development with increased proliferation of enamel organ epithelial cells, while attenuation of Smad7 resulted in significant inhibition of embryonic tooth development with increased apoptotic activity within enamel organ epithelium. These findings suggest that different Smads may have differential activities in regulating TGF-β-mediated cell proliferation and death. Furthermore, functional haploinsufficiency of Smad2, but not Smad3, altered TGF-β-mediated tooth development. The results indicate that Smads are critical factors in orchestrating TGF-β-mediated gene regulation during embryonic tooth development. The effectiveness of TGF-β signaling is highly sensitive to the level of Smad gene expression.

Many membrane-bound protein precursors, including cytokines and growth factors, are proteolytically shed to yield soluble intercellular regulatory ligands. The responsible protease, tumor necrosis factor-α converting enzyme (TACE/ADAM-17), is a transmembrane metalloprotease-disintegrin that cleaves multiple cell surface proteins, although it was initially identified for the enzymatic release of tumor necrosis factor-α (TNF-α). Mammalian lung growth and development are tightly controlled by cytokines and peptide growth factors. However, the biological function of the cell shedding mechanism during lung organogenesis is not understood. We therefore evaluated the role of TACE as a “sheddase” during lung morphogenesis by analyzing the developmental phenotypes of lungs in mice with an inactive TACE gene in both in vivoand ex vivoorgan explant culture. Neonatal TACE-deficient mice had visible respiratory distress and their lungs failed to form normal saccular structures. These newborn mutant lungs had fewer peripheral epithelial sacs with deficient septation and thick-walled mesenchyme, resulting in reduced surface for gas exchange. At the canalicular stage of E16.5, the lungs of TACE mutant mice were impaired in branching morphogenesis, inhibited in epithelial cell proliferation and differentiation, and delayed in vasculogenesis. Embryonic TACE knockout mouse lungs (E12) branched poorly compared to wild-type lungs, when placed into serumless organ culture. Gene expression of both surfactant protein-C and aquaporin-5 were inhibited in cultured TACE-mutant embryonic lungs, indicating defects in both branching and peripheral epithelial cytodifferentiation in the absence of TACE protein. Furthermore, both the hypoplastic phenotype and the delayed cytodifferentiation in TACE-deficient lungs were rescued by exogenous addition of soluble stimulatory factors including either TNF-α or epidermal growth factor in embryonic lung culture. Thus, the impaired lung branching and maturation without TACE suggest a broad role for TACE in the processing of multiple membrane-anchored proteins, one or more of which is essential for normal lung morphogenesis. Taken together, our data indicate that the TACE-mediated proteolytic mechanism which enzymatically releases membrane-tethered proteins plays an indispensable role in lung morphogenesis, and its inactivation leads to abnormal lung development.

Transforming growth factors β (TGF-β) are known negative regulators of lung development, and excessive TGF-β production has been noted in pulmonary hypoplasia associated with lung fibrosis. Inhibitory Smad7 was recently identified to antagonize TGF-β family signaling by interfering with the activation of TGF-β signal-transducing Smad complexes. To investigate whether Smad7 can regulate TGF-β-induced inhibition of lung morphogenesis, ectopic overexpression of Smad7 was introduced into embryonic mouse lungs in culture using a recombinant adenovirus containing Smad7 cDNA. Although exogenous TGF-β efficiently reduced epithelial lung branching morphogenesis in control virus-infected lung culture, TGF-β-induced branching inhibition was abolished after epithelial transfer of the Smad7 gene into lungs in culture. Smad7 also prevented TGF-β-mediated down-regulation of surfactant protein C gene expression, a marker of bronchial epithelial differentiation, in cultured embryonic lungs. Moreover, we found that Smad7 transgene expression blocked Smad2 phosphorylation induced by exogenous TGF-β ligand in lung culture, indicating that Smad7 exerts its inhibitory effect on both lung growth and epithelial cell differentiation through modulation of TGF-β pathway-restricted Smad activity. However, the above anti-TGF-β signal transduction effects were not observed in cultured embryonic lungs with Smad6 adenoviral gene transfer, suggesting that Smad7 and Smad6 differentially regulate TGF-β signaling in developing lungs. Our data therefore provide direct evidence that Smad7, but not Smad6, prevents TGF-β-mediated inhibition of both lung branching morphogenesis and cytodifferentiation, establishing the mechanistic basis for Smad7 as a novel target to ameliorate aberrant TGF-β signaling during lung development, injury, and repair.

Smad7 was recently shown to antagonize TGF-β-induced activation of signal-transducing Smad2 and Smad3 proteins. However, the biological function of Smad7 in the process of lung organogenesis is not known. Since Smad2/3-mediated TGF-β signaling is known to inhibit embryonic lung branching morphogenesis, we tested the hypothesis that Smad7 regulates early lung development by modulating TGF-β signal transduction. An antisense oligodeoxynucleotide (ODN) was designed to specifically block endogenous Smad7 gene expression at both transcriptional and translational levels in embryonic mouse lungs in culture. TGF-β-mediated inhibition of lung branching morphogenesis was significantly potentiated in cultured embryonic lungs in the absence of Smad7 gene expression: abrogation of Smad7 potentiated TGF-β-mediated inhibition of lung branching morphogenesis from 76 to 52% of the basal level in lungs cultured in the presence of 5 ng/ml TGF-β1 ligand. Likewise, TGF-β1 EC50(concentration of TGF-β1 that induced half maximal branching inhibition) was reduced from 5 to 1 ng/ml when Smad7 gene expression was abrogated in lung culture, indicating an enhanced level of TGF-β signaling in lung tissue with abolished Smad7 gene expression. By immunocytochemistry, Smad7 protein was co-localized with both Smad2 and Smad3 in distal bronchial epithelial cells, supporting the concept that Smad7 inhibits TGF-β signaling by competing locally with Smad2 and Smad3 for TGF-β receptor complex binding during lung morphogenesis. Furthermore, antisense Smad7 ODN increased the negative effect of TGF-β1 on epithelial cell growth in developing lungs in culture. We also demonstrated that Smad7 mRNA levels were rapidly and potently induced upon TGF-β1 stimulation of lungs in culture, suggesting that Smad7 regulates TGF-β responses in a negative feedback loop. These studies define a novel function for Smad7 as an intracellular antagonist of TGF-β-induced, Smad2/3-mediated inhibition of murine embryonic lung growth and branching morphogenesis in culture. The optimization of TGF-β signaling during early lung development therefore requires a finely-regulated competitive balance between both permissive and inhibitory members of the Smad family.

Many of the signaling pathways regulating fetal lung mesenchymal cell proliferation are mediated by the Shc intracellular signaling proteins. Shc is expressed as three isoforms: 52 kD and 46 kD proteins (Shc 52 and Shc 46, respectively) translated from the same mRNA, and a 66 kD form (Shc 66) translated from a separate mRNA. Shc 52 is an activator of Ras and mitogen-activated protein kinase, whereas Shc 66 antagonizes Ras activation. The function of Shc 46 is unclear. We hypothesized that the Shc isoforms are differentially regulated during fetal mouse lung morphogenesis. Relative Shc 66 and Shc 46 protein expression are high until parturition (term = 18.5 d), when a dramatic decrease begins; by postconceptual d 20, relative Shc 66 and Shc 46 expression have fallen by 75 and 69%, respectively. A similar pattern of decreasing Shc 66 mRNA expression in the peripartum period was detected by reverse transcription and competitive polymerase chain reaction during the same period. By isoform-specific immunohistochemistry, Shc 66 is widely distributed in the embryonic lung but becomes restricted to the bronchial smooth muscle and overlying epithelia, periarterial smooth muscle, and the interlobar pleura late in gestation. After parturition, Shc 66 is virtually absent from the lung. All three Shc isoforms are phosphorylated by epidermal growth factor stimulation in fetal lung mesenchymal cells, indicating that Shc 66 is functional in these cells. These data indicate that Shc isoforms are differentially regulated during lung development.

Many of the signaling pathways regulating fetal lung mesenchymal cell proliferation are mediated by the Shc intracellular signaling proteins. Shc is expressed as three isoforms: 52 kD and 46 kD proteins (Shc 52 and Shc 46, respectively) translated from the same mRNA, and a 66 kD form (Shc 66) translated from a separate mRNA. Shc 52 is an activator of Ras and mitogen-activated protein kinase, whereas Shc 66 antagonizes Ras activation. The function of Shc 46 is unclear. We hypothesized that the Shc isoforms are differentially regulated during fetal mouse lung morphogenesis. Relative Shc 66 and Shc 46 protein expression are high until parturition (term = 18.5 d), when a dramatic decrease begins; by postconceptual d 20, relative Shc 66 and Shc 46 expression have fallen by 75 and 69%, respectively. A similar pattern of decreasing Shc 66 mRNA expression in the peripartum period was detected by reverse transcription and competitive polymerase chain reaction during the same period. By isoform-specific immunohistochemistry, Shc 66 is widely distributed in the embryonic lung but becomes restricted to the bronchial smooth muscle and overlying epithelia, periarterial smooth muscle, and the interlobar pleura late in gestation. After parturition, Shc 66 is virtually absent from the lung. All three Shc isoforms are phosphorylated by epidermal growth factor stimulation in fetal lung mesenchymal cells, indicating that Shc 66 is functional in these cells. These data indicate that Shc isoforms are differentially regulated during lung development.

The precise spatial-temporal role that expression and activation of transforming growth factor (TGF)-β plays in mammalian organ morphogenesis remains incompletely understood. Using replication deficient adenoviral vectors containing engineered TGF-β1 cDNAs, we studied the spatial effects of locally over-expressing either latent or mutated, constitutively active TGF-β1 protein during embryonic mouse lung branching morphogenesis in culture. Transfer of exogenous genes into lung epithelium was achieved by intra-tracheal microinjection of recombinant adenovirus, while submerging lungs in virus resulted in gene transfer into the pleura and subjacent mesenchymal cells, as revealed by cytochemical staining for β-galactosidase. Only lungs transfected with active, but not latent TGF-β1 gene, showed elevated levels of active TGF-β. Epithelial over-expression of active, but not latent TGF-β1, via intra-tracheal micro-injection inhibited lung branching morphogenesis by 36%. In contrast, lungs submerged with either active or latent TGF-β1 recombinant virus did not demonstrate an inhibitory effect upon branching. Pulmonary gene regulation was assayed by competitive polymerase chain reaction coupled with reverse transcription. Direct respiratory tract micro-injection of adenovirus over-expressing active TGF-β1 resulted in a dose-dependent inhibition of epithelial surfactant protein (SP)-C and SP-B mRNA levels by up to 76% and 70%, respectively, while in contrast, fibronectin and matrix Gla protein (MGP) mRNA levels remained stable.

TGF-β1 is a known inhibitor of branching morphogenesis when added exogenously to mouse embryonic lungs in culture. However, the issue of whether endogenous TGF-β signaling has a function in the process of lung organogenesis is not completely resolved. We utilized immunoperturbation and antisense oligodeoxynucleotide inhibitory strategies to abrogate TGF-β type II receptor function in embryonic mouse lungs undergoing branching morphogenesis in serumless explant culture. Antisera directed against a TGF-β type II receptor N-terminal peptide that perturbs TGF-β ligand–receptor binding increased branching by 70%. Similarly, antisense TGF-β type II receptor oligodeoxynucleotides (40 μM) resulted in a 58% increase in branching, compared to scrambled and mismatched sequence controls, while TGF-β type II receptor mRNA and its protein expression levels were suppressed by 95 and 84%, respectively. Addition of exogenous TGF-β1 did not overcome the stimulatory effects either of TGF-β type II receptor immunoperturbation or of antisense oligodeoxynucleotide treatment on lung branching morphogenesis. Usingin situhybridization and immunohistochemistry, both TGF-β type II receptor mRNA and protein were localized to the epithelium lining the developing airways, and to the surrounding mesenchyme, indicating that TGF-β type II receptor is an important regulator of epithelial–mesenchymal interaction. Exogenous TGF-β1 decreased cyclin A mRNA levels in control embryonic lung explants, while TGF-β type II receptor antisense oligodeoxynucleotides prevented the downregulation of cyclin A mRNA expression by exogenous TGF-β1. In addition, PCNA immunostaining of the primitive bronchial epithelium was increased in the presence of TGF-β type II receptor antisense oligodeoxynucleotides either alone or together with exogenous TGF-β1, whereas TGF-β1 alone decreased PCNA staining. Thus, abrogation of TGF-β type II receptor expression prevented TGF-β1-induced epithelial cell G1arrest. These results demonstrate, for the first time, that abrogation of the TGF-β type II receptor stimulates embryonic lung organogenesis in culture and reverses the negative influence of endogenous TGF-β signaling upon epithelial cell cycle progression.

Smad genes are recently identified intracellular effectors for receptor signaling in the BMP/activin/TGF-β pathway. Since TGF-β ligands are known to inhibit embryonic lung branching morphogenesis, we tested the hypothesis that Smad genes negatively regulate lung organogenesis. Antisense oligodeoxynucleotides were designed to attenuate Smad3 and Smad2 gene expression in embryonic (E11) mouse lungs over 4 days in culture. Endogenous Smad3 and Smad2 mRNA levels were suppressed by 97 and 91%, respectively, in cultured embryonic lungs when antisense oligodeoxynucleotide (40 μM) to Smad was added, compared to scrambled and sense sequence controls. The corresponding Smad3 and Smad2 protein amounts were also decreased respectively by 86 and 90% in lungs treated with Smad3 and Smad2 antisense oligodeoxynucleotide. Phenotypically, Smad antisense oligodeoxynucleotides resulted in a concentration-dependent increase in lung branching: embryonic lung branching was stimulated by up to 53% in culture with 40 μM antisense oligodeoxynucleotide, whereas both scrambled and sense controls showed no stimulatory effect. Thus, inhibition of endogenous Smad3 and Smad2 gene expression resulted in stimulation of embryonic lung branching similar to that caused by inhibition of TGF-β type II receptor expression and signaling (J. Zhaoet al.,1996,Dev. Biol.180, 242–257). Abrogation of Smad4 (DPC4), the downstream mediator of Smad3 and Smad2 proteins, with antisense oligodeoxynucleotide, also resulted in increased branching morphogenesis. Furthermore, while TGF-β alone inhibited lung branching morphogenesis in culture, addition of exogenous TGF-β1 could not overcome the stimulatory effect on lung branching of Smad antisense oligodeoxynucleotide treatment. By immunohistochemistry, Smad proteins were localized mainly to the epithelial cells lining the branching distal airways, indicating that Smad genes could regulate lung morphogenesis through mesoderm–endoderm interaction. Our results demonstrate, for the first time, that abrogation of Smad2 and Smad3 or of Smad4 gene expression stimulated early mouse embryonic lung branching morphogenesis in culture, possibly through reversing the negative influence of endogenous TGF-β signaling upon lung branching morphogenesis.

Matrix Gla protein (MGP) is a vitamin K-dependent extracellular matrix protein with a wide tissue distribution. Developmental expression of the MGP gene is characterized by competitive RT-PCR in kidney and calvaria. High levels of MGP mRNA were observed in kidneys and calvaria from 19-day-old embryos to 1-month-old rats. There was a peak in MGP mRNA at 7 days in both tissues. MGP mRNA expression was very low or undetectable in 3-, 5- and 7-month-old kidneys. Similar observations were seen in lung, heart and spleen. However, in connective tissues like calvaria, tibia and trachea, low levels of MGP mRNA are maintained throughout life. Kidney MGP protein was present from birth to 15 days, with the highest MGP protein level at 7 days. Calvarial MGP protein was present throughout development and maturation but peaked at 7 days. The highest MGP protein levels were coincident with peak levels of MGP mRNA. Thus, MGP protein level correlated with mRNA level during rat development. In situ hybridization revealed that MGP staining was most intense in the straight tubules of the developing kidney medulla at 7 days. Staining was absent in stromal cells and in mature nephrons. Taken together, our finding of high MGP mRNA and its intense in situ staining during the postnatal growth phase prove that increased MGP synthesis occurs at a specific time and place during development and maturation.

CLL-1 (C-type Lectin-Like Molecule-1) is an inhibitory receptor expressed on myeloid cells which was previously shown to be expressed on AML cancer stem cells. To further validate the potential therapeutic against CLL-1, we generated a series of monoclonal antibodies (mAbs) against CLL-1 and assessed their cytotoxic and anti-cancer activities. While expression of CLL-1 was restricted to myeloid cells, it was expressed in 80% (37/46) of AML blasts but not in ALL blasts (n=5). Expression on AML CD34+/CD38- stem cells was detected in 71% (12/17) of cases. Selected anti-CLL-1 mAbs mediated dose-dependent. Complement Dependent Cytotoxicity (CDC) against various AML-derived cell lines with no detectable cytotoxic activity against lymphoid derived cell lines. Moreover, human embryonic kidney 293 cells became susceptible to anti-CLL-1 mAb mediated killing only after transfection with CLL-1, demonstrating that cytotoxic activity is mediated through a specific interaction with CLL-1. Furthermore, anti-CLL-1 mAbs showed CDC activity against all AML blasts tested in ex vivoassays (n=13), while no activity was observed against ALL blasts. CLL-1 efficiently internalizes upon antibody binding in both 293 transfected cells as well as AML cell lines, demonstrating the potential therapeutic opportunity for toxin-conjugation of anti-CLL-1 mAbs. In vivoanti-cancer activity of the lead chimeric mAb against CLL-1 was tested in an HL-60 xenograft model. In this model, growth of established tumors was reduced by 38% at 5 mg/kg twice weekly dosing. Our results demonstrate CLL-1 as an attractive target for AML with restricted expression on cells from myeloid origin, AML blasts and leukemic stem cells, as well as specific cytotoxic activity in in vitro, ex vivoand in vivoassays. We are currently undertaking additional xenograft models to evaluate the therapeutic potential of these mAbs against primary AML engrafted cells and in combination with chemotherapy in vivo.

CLL-1 (C-type Lectin-Like Molecule-1) is an inhibitory receptor expressed on myeloid cells which was previously shown to be expressed on AML cancer stem cells. To further validate the potential therapeutic against CLL-1, we generated a series of monoclonal antibodies (mAbs) against CLL-1 and assessed their cytotoxic and anti-cancer activities. While expression of CLL-1 was restricted to myeloid cells, it was expressed in 80% (37/46) of AML blasts but not in ALL blasts (n=5). Expression on AML CD34+/CD38- stem cells was detected in 71% (12/17) of cases. Selected anti-CLL-1 mAbs mediated dose-dependent. Complement Dependent Cytotoxicity (CDC) against various AML-derived cell lines with no detectable cytotoxic activity against lymphoid derived cell lines. Moreover, human embryonic kidney 293 cells became susceptible to anti-CLL-1 mAb mediated killing only after transfection with CLL-1, demonstrating that cytotoxic activity is mediated through a specific interaction with CLL-1. Furthermore, anti-CLL-1 mAbs showed CDC activity against all AML blasts tested in ex vivo assays (n=13), while no activity was observed against ALL blasts. CLL-1 efficiently internalizes upon antibody binding in both 293 transfected cells as well as AML cell lines, demonstrating the potential therapeutic opportunity for toxin-conjugation of anti-CLL-1 mAbs. In vivo anti-cancer activity of the lead chimeric mAb against CLL-1 was tested in an HL-60 xenograft model. In this model, growth of established tumors was reduced by 38% at 5 mg/kg twice weekly dosing. Our results demonstrate CLL-1 as an attractive target for AML with restricted expression on cells from myeloid origin, AML blasts and leukemic stem cells, as well as specific cytotoxic activity in in vitro, ex vivo and in vivo assays. We are currently undertaking additional xenograft models to evaluate the therapeutic potential of these mAbs against primary AML engrafted cells and in combination with chemotherapy in vivo.

NTB-A is a CD2-related cell surface protein expressed on lymphoid cells including B-lymphocytes from Chronic Lymphocytic Leukemia (CLL) and lymphoma patients. We have generated a series of mAbs against NTB-A and assessed their therapeutic potential in preclinical models. Selected mAbs to NTB-A were further tested in functional Complement Dependent Cytotoxicity (CDC) and Antibody Dependent Cellular Cytotoxicty (ADCC) assays in cell lines and B lymphocytes freshly isolated from CLL and lymphoma patients. Potent cytotoxic activity was demonstrated against B cells isolated from CLL patients and B lymphoma cell lines. Chimeric anti-NTB-A mAbs demonstrated anti-tumor activity equal to rituximab against CA46 human lymphoma xenografts in nude mice at a low dose. In a chimpanzee safety study, a single dose of lead anti-NTB-A mAb 994.1 resulted in near-complete depletion of peripheral lymphocytes while having a minimal effect on T cell activation. Taken together, these results demonstrate NTB-A as a promising target with an acceptable safety profile for immunotherapy of leukemia and lymphomas.

NTB-A is a CD2-related cell surface protein expressed on lymphoid cells including B-lymphocytes from Chronic Lymphocytic Leukemia (CLL) and lymphoma patients. We have generated a series of mAbs against NTB-A and assessed their therapeutic potential in preclinical models. Selected mAbs to NTB-A were further tested in functional Complement Dependent Cytotoxicity (CDC) and Antibody Dependent Cellular Cytotoxicty (ADCC) assays in cell lines and B lymphocytes freshly isolated from CLL and lymphoma patients. Potent cytotoxic activity was demonstrated against B cells isolated from CLL patients and B lymphoma cell lines. Chimeric anti-NTB-A mAbs demonstrated anti-tumor activity equal to rituximab against CA46 human lymphoma xenografts in nude mice at a low dose. In a chimpanzee safety study, a single dose of lead anti-NTB-A mAb 994.1 resulted in near-complete depletion of peripheral lymphocytes while having a minimal effect on T cell activation. Taken together, these results demonstrate NTB-A as a promising target with an acceptable safety profile for immunotherapy of leukemia and lymphomas.

Recombinant nematode anticoagulant protein c2 (rNAPc2) is a specific inhibitor of tissue factor (TF)/factor VIIa complex with novel anti-metastatic, anti-angiogenic, and anti-thrombotic activities. TF is highly expressed in human colorectal tumors and the level of TF expression positively correlates with disease stage and inversely correlates with survival. To explore the therapeutic potential of rNAPc2 during tumor growth and metastasis, we tested rNAPc2 efficacy in experimental colorectal cancers in mice. Administration of rNAPc2 inhibited pulmonary metastasis in mice systemically disseminated with CT26 murine colon carcinoma cells in a dose-dependent fashion, as measured by either number of lung surface metastases or lung mass. While rNAPc2 treatment alone moderately reduced primary tumor growth, combining rNAPc2 with the cytotoxic agent 5-fluorouracil (5-FU) resulted in synergistic growth inhibition of HCT116 human colorectal tumor xenografts in nude mice. Likewise, rNAPc2 further reduced tumor growth in HCT116 human colorectal tumor xenograft-bearing mice receiving bevacizumab (humanized anti-vascular endothelial growth factor monoclonal antibody). Using CD31 and Ki67 immunohistochemisty, we found that rNAPc2 synergized with either 5-FU or bevacizumab in inhibiting microvessel density and tumor cell proliferation in HCT116 human colorectal tumor xenografts. Furthermore, rNAPc2 synergized with CPT-11 in inhibiting hepatic metastasis in nude mice with portal vein injection of HCT116 human colorectal tumor cells. Long-term administration of rNAPc2 also significantly suppressed formation of intestinal adenomas and adenocarcinomas in ApcMin/+ mice. The dosing regimens of rNAPc2 used in these studies were well tolerated up to a three-month period by recipient mice without major hemorrhage or other adverse effects. In conclusion, the synergistic tumor inhibitory activity of rNAPc2 in pre-clinical colorectal cancer models suggests that rNAPc2 may be an effective anti-tumor agent in human colorectal cancer patients to potentiate chemo- or anti-angiogenic therapies.

Recombinant nematode anticoagulant protein c2 (rNAPc2) is a specific inhibitor of tissue factor (TF)/factor VIIa complex with novel anti-metastatic, anti-angiogenic, and anti-thrombotic activities. TF is highly expressed in human colorectal tumors and the level of TF expression positively correlates with disease stage and inversely correlates with survival. To explore the therapeutic potential of rNAPc2 during tumor growth and metastasis, we tested rNAPc2 efficacy in experimental colorectal cancers in mice. Administration of rNAPc2 inhibited pulmonary metastasis in mice systemically disseminated with CT26 murine colon carcinoma cells in a dose-dependent fashion, as measured by either number of lung surface metastases or lung mass. While rNAPc2 treatment alone moderately reduced primary tumor growth, combining rNAPc2 with the cytotoxic agent 5-fluorouracil (5-FU) resulted in synergistic growth inhibition of HCT116 human colorectal tumor xenografts in nude mice. Likewise, rNAPc2 further reduced tumor growth in HCT116 human colorectal tumor xenograft-bearing mice receiving bevacizumab (humanized anti-vascular endothelial growth factor monoclonal antibody). Using CD31 and Ki67 immunohistochemisty, we found that rNAPc2 synergized with either 5-FU or bevacizumab in inhibiting microvessel density and tumor cell proliferation in HCT116 human colorectal tumor xenografts. Furthermore, rNAPc2 synergized with CPT-11 in inhibiting hepatic metastasis in nude mice with portal vein injection of HCT116 human colorectal tumor cells. Long-term administration of rNAPc2 also significantly suppressed formation of intestinal adenomas and adenocarcinomas in ApcMin/+mice. The dosing regimens of rNAPc2 used in these studies were well tolerated up to a three-month period by recipient mice without major hemorrhage or other adverse effects. In conclusion, the synergistic tumor inhibitory activity of rNAPc2 in pre-clinical colorectal cancer models suggests that rNAPc2 may be an effective anti-tumor agent in human colorectal cancer patients to potentiate chemo- or anti-angiogenic therapies.

Recombinant nematode anticoagulant protein c2 (rNAPc2) is a specific inhibitor of tissue factor (TF)/factor VIIa complex with novel anti-metastasis, anti-angiogenesis, and anti-thrombosis activities. rNAPc2 has been previously shown to inhibit both the primary growth and metastasis of murine B16 melanoma and Lewis lung carcinoma in mice. TF is highly expressed in human colorectal tumors and the level of TF expression positively correlates with the progression of malignancy. To explore the therapeutic potential of rNAPc2 during tumor growth and progression, we tested rNAPc2 efficacy in experimental colorectal cancer in mice. Both primary and metastatic colorectal tumor models were used in the current study and rNAPc2 was given to mice via daily intraperitoneal injections. Administration of rNAPc2 inhibited pulmonary metastasis in mice systemically disseminated with CT26 murine colon carcinoma cells in a dose-dependent fashion, as measured by either number of lung surface metastases or lung mass. While rNAPc2 treatment alone moderately reduced primary tumor growth, combining rNAPc2 with the cytotoxic agent 5-fluorouracil (5-FU) resulted in synergistic growth inhibition of HCT116 human colorectal tumor xenografts in nude mice. Likewise, rNAPc2 further reduced tumor growth in HCT116 human colorectal tumor xenograft-bearing mice receiving bevacizumab (humanized anti-vascular endothelial growth factor monoclonal antibody). The doses and dosing regimens of rNAPc2 used in these murine models of colorectal cancer were well tolerated by recipient mice without major complications of hemorrhage or any other adverse effects. In conclusion, the synergistic tumor inhibitory activity of rNAPc2 in pre-clinical colorectal cancer models suggests that rNAPc2 may be an effective anti-tumor agent in human colorectal cancer patients to potentiate chemo- or anti-angiogenic therapies.

Recombinant nematode anticoagulant protein c2 (rNAPc2) is a specific inhibitor of tissue factor (TF)/factor VIIa complex with novel anti-metastasis, anti-angiogenesis, and anti-thrombosis activities. rNAPc2 has been previously shown to inhibit both the primary growth and metastasis of murine B16 melanoma and Lewis lung carcinoma in mice. TF is highly expressed in human colorectal tumors and the level of TF expression positively correlates with the progression of malignancy. To explore the therapeutic potential of rNAPc2 during tumor growth and progression, we tested rNAPc2 efficacy in experimental colorectal cancer in mice. Both primary and metastatic colorectal tumor models were used in the current study and rNAPc2 was given to mice via daily intraperitoneal injections. Administration of rNAPc2 inhibited pulmonary metastasis in mice systemically disseminated with CT26 murine colon carcinoma cells in a dose-dependent fashion, as measured by either number of lung surface metastases or lung mass. While rNAPc2 treatment alone moderately reduced primary tumor growth, combining rNAPc2 with the cytotoxic agent 5-fluorouracil (5-FU) resulted in synergistic growth inhibition of HCT116 human colorectal tumor xenografts in nude mice. Likewise, rNAPc2 further reduced tumor growth in HCT116 human colorectal tumor xenograft-bearing mice receiving bevacizumab (humanized anti-vascular endothelial growth factor monoclonal antibody). The doses and dosing regimens of rNAPc2 used in these murine models of colorectal cancer were well tolerated by recipient mice without major complications of hemorrhage or any other adverse effects. In conclusion, the synergistic tumor inhibitory activity of rNAPc2 in pre-clinical colorectal cancer models suggests that rNAPc2 may be an effective anti-tumor agent in human colorectal cancer patients to potentiate chemo- or anti-angiogenic therapies.