Role of prion protein glycosylation in prion propagation, The

2018 Spring.Includes bibliographical references.Transmissible Spongiform Encephalopathies (TSEs) are a group of neurodegenerative diseases that affect humans and animals alike. TSEs are caused by the accumulation of a disease producing isoform referred to as PrPSc that results from the misfolding of the normal cellular prion protein PrPC. The pathological outcomes of TSEs include amyloid plaque build-up and spongiform degeneration in the brain of infected hosts. Clinical signs of prion disease can vary between TSEs, but often include neurologic impairment that is subtle in onset and tends to progress slowly. Prion diseases are relatively recently discovered and have sparked much controversy due to the scientific findings that directly challenge some of the most well established scientific dogmas. Among these is that the infectious agent responsible for the transmission of TSEs is proteinacious in nature and devoid of the nucleic acids present in pathogens like viruses and bacteria. As a result of this hypothesis, both PrPC and PrPSc share the same amino acid sequence in the host. Therefore, central to our understanding of the prion hypothesis is to recognize the structural differences between PrPC and PrPSc. PrPC has been proven to include three α-helices and two, short β-pleated sheets whereas PrPSc consists of high β-sheet content, aggregates in the presence of detergents, and is resistant to protease treatment. These characteristics of PrPSc have inhibited researchers to successfully examine the abnormal isoform in high-resolution structural studies. Therefore, an alternative means of distinguishing PrPC and PrPSc is necessary. Since then, several groups have created monoclonal antibodies (mAbs) that differentiate between infectious prion protein (PrP) aggregates. Two such mAbs, PRC5 and PRC7 were the first mAbs discovered in which the involvement of individual residues in functional, discontinuous, and conformationaly dependent epitopes was studied. Of these antibodies, PRC7, is dependent on N-linked glycosylation at mono-1 of the prion protein and specifically binds to the infected isoform of PrP. Therefore, we hypothesized that an underglycosylated form of PrP is preferentially generated during prion replication in the infected host. In this body of work, we have systemically ablated mono-1, one of the two N-linked glycan attachment sites on the murine prion protein to address the role of underglycosylation in prion propagation at N180 and at S/T182 of the consensus sequence by mutating N or S/T to each of the other 19 amino acids individually. Here we present novel evidence showing the effects of underglycosylation in prion propagation of prion isolates RML, 22L, 139A, and mCWD. These preliminary data demonstrate the importance of post-translational differences between PrPC and PrPSc which represent a fundamental, unresolved aspect of the prion hypothesis

Recombinant antibodies derived from laser captured single plasma cells of multiple sclerosis brain identified phage peptides which may be used as tools for characterizing intrathecal IgG response

Oligoclonal bands and increased IgG antibody levels can be detected in the cerebrospinal fluid in vast majority of patients with Multiple Sclerosis (MS). However, the antigenic specificity of oligoclonal IgG has yet to be determined. Using laser capture microdissection, we isolated single CD38+ plasma cells from lesion areas in two autopsy MS brains, and generated three recombinant antibodies (rAbs) from clonally expanded plasma cells. Panning phage-displayed random peptide libraries was carried out to determine peptide antigen specificities of these MS brain rAbs. We identified 25 high affinity phage peptides from which 5 peptides are unique. Database searches revealed that they shared sequence homologies with Epstein-Barr nuclear antigens 4 and 6, as well as with other viral proteins. Significantly, these peptides were recognized by intrathecal IgG and oligoclonal IgG bands in other MS patients. Our results demonstrate that functional recombinant antibodies can be generated from clonally expanded plasma cells in MS brain lesions by laser capture microdissection, and that these MS brain rAbs have the potential for determining the targets of intrathecal IgG and oligoclonal bands

Cadherin-11 contributes to the heterogenous and dynamic Wnt-Wnt-β-catenin pathway activation in Ewing sarcoma.

Ewing sarcoma is the second most common bone cancer in children, and while patients who present with metastatic disease at the time of diagnosis have a dismal prognosis. Ewing sarcoma tumors are driven by the fusion gene EWS/Fli1, and while these tumors are genetically homogenous, the transcriptional heterogeneity can lead to a variety of cellular processes including metastasis. In this study, we demonstrate that in Ewing sarcoma cells, the canonical Wnt/β-Catenin signaling pathway is heterogeneously activated in vitro and in vivo, correlating with hypoxia and EWS/Fli1 activity. Ewing sarcoma cells predominantly express β-Catenin on the cell membrane bound to CDH11, which can respond to exogenous Wnt ligands leading to the immediate activation of Wnt/β-Catenin signaling within a tumor. Knockdown of CDH11 leads to delayed and decreased response to exogenous Wnt ligand stimulation, and ultimately decreased metastatic propensity. Our findings strongly indicate that CDH11 is a key component of regulating Wnt//β-Catenin signaling heterogeneity within Ewing sarcoma tumors, and is a promising molecular target to alter Wnt//β-Catenin signaling in Ewing sarcoma patients

Eicosanoid profiling in an orthotopic model of lung cancer progression by mass spectrometry demonstrates selective production of leukotrienes by inflammatory cells of the microenvironment.

Eicosanoids are bioactive lipid mediators derived from arachidonic acid(1) (AA), which is released by cytosolic phospholipase A2 (cPLA2). AA is metabolized through three major pathways, cyclooxygenase (COX), lipoxygenase (LO) and cytochrome P450, to produce a family of eicosanoids, which individually have been shown to have pro- or anti-tumorigenic activities in cancer. However, cancer progression likely depends on complex changes in multiple eicosanoids produced by cancer cells and by tumor microenvironment and a systematic examination of the spectrum of eicosanoids in cancer has not been performed. We used liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to quantitate eicosanoids produced during lung tumor progression in an orthotopic immunocompetent mouse model of lung cancer, in which Lewis lung carcinoma (LLC) cells are injected into lungs of syngeneic mice. The presence of tumor increased products of both the cyclooxygenase and the lipoxygenase pathways in a time-dependent fashion. Comparing tumors grown in cPLA2 knockout vs wild-type mice, we demonstrated that prostaglandins (PGE2, PGD2 and PGF2a) were produced by both cancer cells and the tumor microenvironment (TME), but leukotriene (LTB4, LTC4, LTD4, LTE4) production required cPLA2 expression in the TME. Using flow cytometry, we recovered tumor-associated neutrophils and 2 types of tumor-associated macrophages from tumor-bearing lungs and we defined their distinct eicosanoid profiles by LC/MS/MS. The combination of flow cytometry and LC/MS/MS unravels the complexity of eicosanoid production in lung cancer and provides a rationale to develop therapeutic strategies that target select cell populations to inhibit specific classes of eicosanoids

Moderate postnatal hyperoxia accelerates lung growth and attenuates pulmonary hypertension in infant rats after exposure to intra-amniotic endotoxin

To determine the separate and interactive effects of fetal inflammation and neonatal hyperoxia on the developing lung, we hypothesized that: 1) antenatal endotoxin (ETX) causes sustained abnormalities of infant lung structure; and 2) postnatal hyperoxia augments the adverse effects of antenatal ETX on infant lung growth. Escherichia coli ETX or saline (SA) was injected into amniotic sacs in pregnant Sprague-Dawley rats at 20 days of gestation. Pups were delivered 2 days later and raised in room air (RA) or moderate hyperoxia (O2, 80% O2 at Denver's altitude, ∼65% O2 at sea level) from birth through 14 days of age. Heart and lung tissues were harvested for measurements. Intra-amniotic ETX caused right ventricular hypertrophy (RVH) and decreased lung vascular endothelial growth factor (VEGF) and VEGF receptor-2 (VEGFR-2) protein contents at birth. In ETX-exposed rats (ETX-RA), alveolarization and vessel density were decreased, pulmonary vascular wall thickness percentage was increased, and RVH was persistent throughout the study period compared with controls (SA-RA). After antenatal ETX, moderate hyperoxia increased lung VEGF and VEGFR-2 protein contents in ETX-O2 rats and improved their alveolar and vascular structure and RVH compared with ETX-RA rats. In contrast, severe hyperoxia (≥95% O2 at Denver's altitude) further reduced lung vessel density after intra-amniotic ETX exposure. We conclude that intra-amniotic ETX induces fetal pulmonary hypertension and causes persistent abnormalities of lung structure with sustained pulmonary hypertension in infant rats. Moreover, moderate postnatal hyperoxia after antenatal ETX restores lung growth and prevents pulmonary hypertension during infancy

Eicosanoid levels in tumor-bearing lungs of WT and cPLA2-KO mice.

<p>LLC-Luc cells or matrigel (control) were injected into left lung lobes of wild-type or cPLA₂-KO mice. Left lobes with tumor were harvested 2 or 3 weeks after injection, eicosanoids were analyzed by LC/MS/MS and normalized to protein content of the sample. Top numbers indicate median levels of each eicosanoid. Numbers in parentheses indicate the interquartile ranges. Eicosanoid levels are expressed as pg/mg protein, except for DHA and AA, which are measured in ng/mg protein. *FDR value <0.05</p

Increase in eicosanoid levels during lung tumor progression in orthotopic lung cancer model in mice.

<p>Wild-type C57B/L mice were injected in left lung lobe with LLC-Luc cells. Whole left lung lobes containing tumors were harvested at 2 and at 3 weeks after injection, along with left lobes from control mice injected with PBS/Matrigel. Eicosanoids extracted from the harvested lungs were analyzed by LC/MS/MS and normalized to the protein content of the sample. Numbers in the table indicate fold increase in 3-week tumor bearing lung vs. matrigel-injected lung. ND – fold increase was not determined for LTB4, since in control mice this analyte was not detectable. * FDR<0.05</p

Multiple eicosanoids are increased in tumor bearing mice.

<p>LLC-Luc cells or matrigel (control) were injected into left lung lobes of WT C57Bl/6 mice and tumor-bearing and control lungs were harvested 2 or 3 weeks after injection. Eicosanoid levels were analyzed by LC/MS/MS and normalized to the protein content of the sample. Levels on graphs are expressed as fold over control. A. Early eicosanoids – induced >3 fold at 2 weeks, and further increasing at 3 weeks. B. Late eicosanoids – not upregulated (<2-fold) at 2 weeks, but increased at 3 weeks. C. Unchanged eicosanoids.</p

Recovery of inflammatory cells from TME by flow cytometry.

<p>LLC-Luc cells were injected into left lung lobes of WT or cPLA₂-KO mice and tumor-bearing lungs were harvested 2.5 weeks later. Tissues from 4 mice were pooled, and inflammatory cells were recovered by flow cytometry. A. Sequential flow cytometry gating strategy used to recover inflammatory cells: Neu: neutrophils (CD11b+/Ly6G+), MacA macrophages (SigF+/CD11c+/Ly6G-) and MacB macrophages (F480+/CD11b+/Ly6G-/SigF-). B. Numbers of cells recovered by flow cytometry from uninjected or tumor-bearing left lung lobes of WT and cPLA₂-KO mice. Data show average from 3 separate experiments, with each sorting performed on a pool of 4 mice. Error bars show S.E.M.</p