Citations

Pregnancy-associated plasma protein-A2 (PAPP-A2) is a novel homolog of PAPP-A in the metzincin superfamily. However, compared with the accumulating data on PAPP-A, very little is known about PAPP-A2. In this study, we determined the tissue expression pattern of PAPP-A2 mRNA in wild-type (WT) mice and characterized the phenotype of mice with global PAPP-A2 deficiency. Tissues expressing PAPP-A2 in WT mice were more limited than those expressing PAPP-A. The highest PAPP-A2 mRNA expression was found in the placenta, with abundant expression in fetal, skeletal, and reproductive tissues. Heterozygous breeding produced the expected Mendelian distribution for the pappa2 gene and viable homozygous PAPP-A2 knockout (KO) mice that were normal size at birth. The most striking phenotype of the PAPP-A2 KO mouse was postnatal growth retardation. Male and female PAPP-A2 KO mice had 10 and 25–30% lower body weight, respectively, than WT littermates. Adult femur and body length were also reduced in PAPP-A2 KO mice, but without significant effects on bone mineral density. PAPP-A2 KO mice were fertile, but with compromised fecundity. PAPP-A expression was not altered to compensate for the loss of PAPP-A2 expression, and proteolysis of PAPP-A2's primary substrate, IGF-binding protein-5, was not altered in fibroblasts from PAPP-A2 KO embryos. In conclusion, tissue expression patterns and biological consequences of gene KO indicate distinct physiological roles for PAPP-A2 and PAPP-A in mice.

The mechanisms of increased rectal stiffness in women with fecal incontinence (FI) and rectal urgency are not understood. Our hypothesis was that distention-induced activation of mechanosensitive L-type calcium channels in smooth muscle contributes to increased rectal stiffness in FI. Anal pressures, rectal distensibility (compliance, capacity, and contractile response to sinusoidal oscillation), and rectal sensation were assessed before and after oral nifedipine (30 + 10 mg) or placebo in 16 women with FI and 16 asymptomatic women. At baseline, FI patients had a lower anal pressure increment during squeeze (health, 66.9 ± 7.6: FI, 28.6 ± 5.9, mean ± SE, P ≤ 0.01), lower rectal capacity ( P = 0.052), and higher rectal pressures during sinusoidal oscillation (health, 13.7 ± 3.2: FI, 21.7 ± 1.4, mean ± SE, P = 0.02) than the healthy women, which suggests an exaggerated rectal contractile response to distention. Nifedipine decreased mean BP, increased heart rate ( P = 0.01 vs. placebo), and reduced anal resting pressure ( P ≤ 0.01) but did not significantly modify rectal distensibility in health or FI. Plasma nifedipine concentrations (health, 103 ± 21 ng/ml: FI, 162 ± 34 ng/ml) were correlated with increased rectal compliance ( r = 0.6, P = 0.02) in all study participants and, in healthy subjects, with decreased rectal pressures during sinusoidal oscillation ( r = 0.86, P = 0.01), indicative of reduced stiffness. No consistent effects on rectal perception were observed. These observations confirm that FI is associated with anal weakness and increased rectal stiffness. At therapeutic plasma concentrations, nifedipine reduced anal resting pressure but did not improve rectal distensibility in FI, outcomes that argue against a predominant contribution of myogenic L-type calcium channels to reduced rectal distensibility in FI.

ABSTRACTEosinophils and neutrophils contribute to larval killing during the primary immune response, and neutrophils are effector cells in the secondary response toStrongyloides stercoralisin mice. The objective of this study was to determine the molecular mechanisms used by eosinophils and neutrophils to control infections withS. stercoralis. Using mice deficient in the eosinophil granule products major basic protein (MBP) and eosinophil peroxidase (EPO), it was determined that eosinophils kill the larvae through an MBP-dependent mechanism in the primary immune response if other effector cells are absent. InfectingPHILmice, which are eosinophil deficient, withS. stercoralisresulted in development of primary and secondary immune responses that were similar to those of wild-type mice, suggesting that eosinophils are not an absolute requirement for larval killing or development of secondary immunity. TreatingPHILmice with a neutrophil-depleting antibody resulted in a significant impairment in larval killing. Naïve and immunized mice with neutrophils deficient in myeloperoxidase (MPO) infected withS. stercoralishad significantly decreased larval killing. It was concluded that there is redundancy in the primary immune response, with eosinophils killing the larvae through an MBP-dependent mechanism and neutrophils killing the worms through an MPO-dependent mechanism. Eosinophils are not required for the development or function of secondary immunity, but MPO from neutrophils is required for protective secondary immunity.

Abstract Background: Cytokine stimulated signaling pathways contribute to multiple myeloma (MM) disease progression and in acquired resistance to current treatment options making MM an incurable malignancy. The PI3K/Akt/mTOR pathway is up-regulated in MM and appears to be critical for cell proliferation and blocking this pathway might have profound impact on disease progression. In addition, this pathway also has been found to cross talk with other signal transduction pathways preventing drug-induced apoptosis. Methods: MK2206 was obtained from Merck Inc. MTT assay was performed to study drug induced cytotoxicity and thymidine uptake was used as a measure to study differences in proliferation. Flow cytometry using Annexin V-FITC and propidium iodide (PI) was used to measure drug induced apoptosis in cell lines and patient cells. In addition, apo-2.7 was also used to measure apoptosis in patient cells. Results: MK2206 treatment led to dose and time dependent decrease in cell viability as observed by MTT assay with an IC50 range of 1-5µM on most MM cell lines tested. Most of the cytotoxicity was observed by 48hours with minimal increase in cytotoxicity beyond that point. The drug was able to induce cytotoxicity in myeloma cell lines when cells were cultured with IGF, IL6, or VEGF with minimal increase in IC50 values. MK2206 also inhibited MM cell proliferation as observed by reduced thymidine uptake. This reduction in proliferation was also observed when MM cells were co-cultured with bone marrow stromal cells. When myeloma cell lines were treated with the drug for various time points, percentage of apoptotic cells increased with time of incubation with the drug as observed by flow cytometry and annexin V-FITC/PI staining. There was also a time dependent increase in caspase cleavage observed indicating that MK2206 might elicit its functions by inducing caspase dependent pathways. Further studies are being carried out to understand the importance of caspase dependent pathways for the success of anti-MM effects of MK2206. Also, MK2206 was able to induce apoptosis in patient derived primary cells, though with varying degrees of response observed between patients. Examining signaling pathways by immunoblotting demonstrated clear down regulation of pAkt and Bcl-xl levels post treatment with the drug with minimal decrease in proteins associated with other signaling pathways, like pSTAT3 and pERK. Conclusions: Taken together, our studies demonstrate significant anti-MM activity of MK2206 in-vitro. We are currently testing MK2206 in combination with known inhibitors of the other important signaling pathways implicated in MM disease biology as well as in-vivo experiments in mouse models. Performing these experiments will further validate the efficacy of MK2206 as an anti-MM agent and form the basis for it to be taken up for clinical evaluation either as a single agent or in combination with other agent(s). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1796.

Abstract The number of non-coding transcripts (NCTs) exceeds the number of transcripts that code for protein. Their diverse and important functions are now being identified and utilized in the lab as well as in the clinic. Utilizing the Affymetrix Gene Chip Human Tiling 1.0R Array Set, we analyzed the transcription across the entire genome in both normal human bronchial epithelial cells (NHBE) alone and cells exposed to the tobacco carcinogen (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone), NNK. We focused our efforts on the identification and function of novel long non-coding transcripts that had increased expression after exposure to NNK. We report on the identification of 12 long NNK-induced non-coding transcripts (NiTs) that are 300 nucleotides and larger in length. We first validated that these transcripts were indeed stress responsive using quantitative real-time RT-PCR (qPCR) and by Northern blot analysis revealed that these transcripts were between two and four kilobases in length. We also show using qPCR that these transcripts are more abundantly expressed in normal rapidly growing tissues or tissues that are under cellular stress. We show that there is altered expression of NiTs in a panel of lung cancer cell lines and many of these transcripts had increased expression in many breast cancer cell lines. We further analyzed one of these transcripts, NiT5 and identified it to be a 2.5 kb antisense, polyadenylated transcript by RLM-RACE and by Northern Blot analysis. Isolating nuclear and cytoplasmic fractions in BT474 cells, we identified Nit5 to be localized in the nucleus and found it to be transcribed by RNA polymerase III. NiT5 also has increased expression in breast, ovarian and cervical cancer-derived cell lines relative to normals from these three sites, but not increased expression in lung cancer cell lines. In summary, our results suggest that these novel long non-coding transcripts play an important role in stress also may play a role in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4083.

Abstract The molecular target(s) cooperating with proteasome inhibition in multiple myeloma (MM) remain unknown. We therefore measured proliferation in MM cells transfected with 13 984 small interfering RNAs in the absence or presence of increasing concentrations of bortezomib. We identified 37 genes, which when silenced, are not directly cytotoxic but do synergistically potentiate the growth inhibitory effects of bortezomib. To focus on bortezomib sensitizers, genes that also sensitized MM to melphalan were excluded. When suppressed, the strongest bortezomib sensitizers were the proteasome subunits PSMA5, PSMB2, PSMB3, and PSMB7 providing internal validation, but others included BAZ1B, CDK5, CDC42SE2, MDM4, NME7, RAB8B, TFE3, TNFAIP3, TNK1, TOP1, VAMP2, and YY1. The strongest hit CDK5 also featured prominently in pathway analysis of primary screen data. Cyclin-dependent kinase 5 (CDK5) is expressed at high levels in MM and neural tissues with relatively low expression in other organs. Viral shRNA knockdown of CDK5 consistently sensitized 5 genetically variable MM cell lines to proteasome inhibitors (bortezomib and carfilzomib). Small-molecule CDK5 inhibitors were demonstrated to synergize with bortezomib to induce cytotoxicity of primary myeloma cells and myeloma cell lines. CDK5 regulation of proteasome subunit PSMB5 was identified as a probable route to sensitization.

Background— Giant cell arteritis is a granulomatous vasculitis of the aorta and its branches that causes blindness, stroke, and aortic aneurysm. CD4 T cells are key pathogenic regulators, instructed by vessel wall dendritic cells to differentiate into vasculitic T cells. The unique pathways driving this dendritic cell–T-cell interaction are incompletely understood, but may provide novel therapeutic targets for a disease in which the only established therapy is long-term treatment with high doses of corticosteroids. Methods and Results— Immunohistochemical and gene expression analyses of giant cell arteritis-affected temporal arteries revealed abundant expression of the NOTCH receptor and its ligands, Jagged1 and Delta1. Cleavage of the NOTCH intracellular domain in wall-infiltrating T cells indicated ongoing NOTCH pathway activation in large-vessel vasculitis. NOTCH activation did not occur in small-vessel vasculitis affecting branches of the vasa vasorum tree. We devised 2 strategies to block NOTCH pathway activation: γ-secretase inhibitor treatment, preventing nuclear translocation of the NOTCH intracellular domain, and competing for receptor-ligand interactions through excess soluble ligand, Jagged1-Fc. In a humanized mouse model, NOTCH pathway disruption had strong immunosuppressive effects, inhibiting T-cell activation in the early and established phases of vascular inflammation. NOTCH inhibition was particularly effective in downregulating Th17 responses, but also markedly suppressed Th1 responses. Conclusions— Blocking NOTCH signaling depleted T cells from the vascular infiltrates, implicating NOTCH- NOTCH ligand interactions in regulating T-cell retention and survival in vessel wall inflammation. Modulating the NOTCH signaling cascade emerges as a promising new strategy for immunosuppressive therapy of large-vessel vasculitis.