PRION CHARACTERIZATION USING CELL BASED APPROACHES

Prions are the causative agents of a group of lethal, neurodegenerative conditions that include sheep scrapie, bovine spongiform encephalopathy (BSE), and human Creutzfeldt-Jakob disease (CJD). Prions are derived from the conversion of a normal, primarily alpha-helical, cellular prion protein (PrPC), to an infectious, beta sheet-rich conformer (PrPSc). Many unresolved issues surround the process of PrP conversion, and we know very little about cellular responses to these unique pathogens. Our lack of knowledge relates, in part, to the difficulty of infecting cells in vitro with prions. While expression of PrPC is an absolute requirement for prion propagation, I show here that not all cells that express PrPC are capable of propagating PrPSc. The goal of this thesis is to understand the role that host factors play in sustaining prion infection and to develop systems in which the cellular response to prion infection can be assessed. We hypothesize that cellular permissiveness to prion infectivity is co-dependent on unidentified additional cellular factors. To study the role of PrPC expression in susceptibility to prion infectivity, and identify these cofactors in cell culture, we utilized cells which fail to express endogenous PrPC, but become susceptible to prions following stable expression of PrPC. Following transfection of a species specific PrP expression construct and isolation of single cell clones, we assessed PrP expression and susceptibility to prion infectivity by measuring accumulation of protease resistant PrPSc. Differential gene expression studies suggest significant transcriptional differences between susceptible and resistant clones. Using three independent gene expression databases our analyses suggest that the resistant transcriptional profile favors cell division/cycle and chromosomal regulation pathways, while the sensitive transcriptional profile is involved in protein homeostasis and quality control. The results of these studies will not only lead to a greater understanding of PrP cell biology and the mechanisms of prion pathogenesis, but should ultimately lead to sensitive and expedient methods for detecting and characterizing prion infectivity from a wide range of sources

Development of a New largely scalable in vitro prion propagation method for the production of infectious recombinant prions for high resolution structural studies.

The resolution of the three-dimensional structure of infectious prions at the atomic level is pivotal to understand the pathobiology of Transmissible Spongiform Encephalopathies (TSE), but has been long hindered due to certain particularities of these proteinaceous pathogens. Difficulties related to their purification from brain homogenates of disease-affected animals were resolved almost a decade ago by the development of in vitro recombinant prion propagation systems giving rise to highly infectious recombinant prions. However, lack of knowledge about the molecular mechanisms of the misfolding event and the complexity of systems such as the Protein Misfolding Cyclic Amplification (PMCA), have limited generating the large amounts of homogeneous recombinant prion preparations required for high-resolution techniques such as solid state Nuclear Magnetic Resonance (ssNMR) imaging. Herein, we present a novel recombinant prion propagation system based on PMCA that substitutes sonication with shaking thereby allowing the production of unprecedented amounts of multi-labeled, infectious recombinant prions. The use of specific cofactors, such as dextran sulfate, limit the structural heterogeneity of the in vitro propagated prions and makes possible, for the first time, the generation of infectious and likely homogeneous samples in sufficient quantities for studies with high-resolution structural techniques as demonstrated by the preliminary ssNMR spectrum presented here. Overall, we consider that this new method named Protein Misfolding Shaking Amplification (PMSA), opens new avenues to finally elucidate the three-dimensional structure of infectious prions

Abatacept increases T cell exhaustion in early RA individuals who carry HLA risk alleles

Exhausted CD8 T cells (TEX) are associated with worse outcome in cancer yet better outcome in autoimmunity. Building on our past findings of increased TIGIT+KLRG1+ TEX with teplizumab therapy in type 1 diabetes (T1D), in the absence of treatment we found that the frequency of TIGIT+KLRG1+ TEX is stable within an individual but differs across individuals in both T1D and healthy control (HC) cohorts. This TIGIT+KLRG1+ CD8 TEX population shares an exhaustion-associated EOMES gene signature in HC, T1D, rheumatoid arthritis (RA), and cancer subjects, expresses multiple inhibitory receptors, and is hyporesponsive in vitro, together suggesting co-expression of TIGIT and KLRG1 may broadly define human peripheral exhausted cells. In HC and RA subjects, lower levels of EOMES transcriptional modules and frequency of TIGIT+KLRG1+ TEX were associated with RA HLA risk alleles (DR0401, 0404, 0405, 0408, 1001) even when considering disease status and cytomegalovirus (CMV) seropositivity. Moreover, the frequency of TIGIT+KLRG1+ TEX was significantly increased in RA HLA risk but not non-risk subjects treated with abatacept (CTLA4Ig). The DR4 association and selective modulation with abatacept suggests that therapeutic modulation of TEX may be more effective in DR4 subjects and TEX may be indirectly influenced by cellular interactions that are blocked by abatacept

In Vitro Amplification of Misfolded Prion Protein Using Lysate of Cultured Cells

Protein misfolding cyclic amplification (PMCA) recapitulates the prion protein (PrP) conversion process under cell-free conditions. PMCA was initially established with brain material and then with further simplified constituents such as partially purified and recombinant PrP. However, availability of brain material from some species or brain material from animals with certain mutations or polymorphisms within the PrP gene is often limited. Moreover, preparation of native PrP from mammalian cells and tissues, as well as recombinant PrP from bacterial cells, involves time-consuming purification steps. To establish a convenient and versatile PMCA procedure unrestricted to the availability of substrate sources, we attempted to conduct PMCA with the lysate of cells that express cellular PrP (PrPC). PrPSc was efficiently amplified with lysate of rabbit kidney epithelial RK13 cells stably transfected with the mouse or Syrian hamster PrP gene. Furthermore, PMCA was also successful with lysate of other established cell lines of neuronal or non-neuronal origins. Together with the data showing that the abundance of PrPC in cell lysate was a critical factor to drive efficient PrPSc amplification, our results demonstrate that cell lysate in which PrPC is present abundantly serves as an excellent substrate source for PMCA

Comparative Effectiveness of Abatacept vs. Tofacitinib in Rheumatoid Arthritis Patients who are CCP+

Abstract Introduction Currently there is limited data to drive clinical decision making regarding  the choice of biologic/targeted synthetic disease-modifying antirheumatic drugs (DMARD); thus, head-to-head comparisons are needed to help guide prescribing. In recent years, significant advancements have helped clarify the mechanistic basis of the clinical associations of autoantibodies in rheumatoid arthritis (RA). This study evaluated the effectiveness of abatacept versus tofacitinib in anti-cyclic citrullinated peptide (CCP+) patients with rheumatoid arthritis (RA). Methods CorEvitas (formerly known as CORRONA) Registry patients aged ≥ 18 years, who were CCP+ before initiating abatacept or tofacitinib (December 2012 onwards through October 2019), had 6-month follow-up data (baseline and 6-month Clinical Disease Activity Index [CDAI]), and were not in remission at index were included. Patients were frequency matched 1:1 by prior biologic use before propensity score matching (PSM). Primary (mean change [D] in CDAI) and secondary outcomes 6 months after index were compared using mixed-effects models adjusted for variables that remained unbalanced after PSM. Results Following PSM, most baseline characteristics for 291 patient pairs were well balanced between treatments, although fewer patients initiating abatacept versus tofacitinib received prior non-TNFi biologic DMARDs, and patients initiating abatacept versus tofacitinib had a higher physician global assessment score, patient-reported fatigue, and modified Health Assessment Questionnaire (mHAQ). In adjusted analyses, there were no significant differences in mean [D] from baseline in CDAI at 6 months with abatacept versus tofacitinib (P = 0.936). Patients naïve for b/tsDMARDs initiating abatacept had a numerically greater mean [D] in CDAI at 6 months versus tofacitinib, although this difference was not statistically significant (P = 0.662). There were no significant differences for any secondary outcomes. Conclusions In adjusted analyses, CCP+ patients with RA initiating treatment with abatacept versus tofacitinib did not show a statistically significant difference in reducing disease activity or improving patient-reported outcomes

In Silico Reconstruction of the Viral Evolutionary Lineage Yields a Potent Gene Therapy Vector

Adeno-associated virus (AAV) vectors have emerged as a gene-delivery platform with demonstrated safety and efficacy in a handful of clinical trials for monogenic disorders. However, limitations of the current generation vectors often prevent broader application of AAV gene therapy. Efforts to engineer AAV vectors have been hampered by a limited understanding of the structure-function relationship of the complex multimeric icosahedral architecture of the particle. To develop additional reagents pertinent to further our insight into AAVs, we inferred evolutionary intermediates of the viral capsid using ancestral sequence reconstruction. In-silico-derived sequences were synthesized de novo and characterized for biological properties relevant to clinical applications. This effort led to the generation of nine functional putative ancestral AAVs and the identification of Anc80, the predicted ancestor of the widely studied AAV serotypes 1, 2, 8, and 9, as a highly potent in vivo gene therapy vector for targeting liver, muscle, and retina

Additional file 1 of Comparative effectiveness of abatacept versus TNF inhibitors in rheumatoid arthritis patients who are ACPA and shared epitope positive

Supplementary Material

PMCA using cell lysate of RK13SHaPrP.

<p>(A) PK-resistant PrP^Sc amplification of two Syrian hamster-adapted TME prions (HY and DY) with cell lysate (CL) of RK13SHaPrP. The HY and DY seeds were diluted 100–2,500 fold for PMCA. The level of PrP^Sc in pre- (−) and post-PMCA (+) samples was analyzed by Western blotting. (B) Comparison of the PK-resistant PrP^Sc of HY and DY prions generated by PMCA. The HY and DY seeds were diluted 100–62,500 fold for PMCA. Ten % brain homogenate (BH) of HY- and DY-sick Syrian hamsters were used as controls. PrP^Sc in both panels A and B was detected by monoclonal anti-PrP 3F4 antibody.</p

Expression of PrP^C in a variety of cell lines.

<p>(A) Comparison of the PrP^C levels in cell lines and brain homogenate. Western blotting followed by densitometry demonstrated relative differences of the PrP^C levels. Extrapolation of multiplication factors to concentrate cell lysate was based on the amount of protein analyzed and the relative PrP^C levels. PrP^C was detected by D13 (left blot), 6H4 (middle blot), and 3F4 (right blot) antibodies. (B) Fluorescence images of RK13 cells expressing full length mouse PrP^C. Colocalization (yellow, overlay) of PrP^C (green) and GM1 (red) in the lipid rafts of the plasma membrane of RK13MoPrP was shown by confocal microscopy. The nuclei (blue) were stained by Hoechst 33258. Scale was shown by a 30 µm bar.</p

PMCA using lysates of a wide range of cell types.

<p>Cell lysate of neuronal (N2a), prion-free brain mesenchymal (SMB-PS), mixed cerebellar neuronal and glial (CRBL) or fibroblast (NIH 3T3) cells was concentrated to include the PrP^C level of wild brain homogenate. PMCA was performed by seeding with RML-sick (RML) and normal (NBH) brain homogenate. The seed dilution fold was 100–24,000. The PrP^Sc level of PMCA before (−) and after (+) was compared. Monoclonal anti-PrP 6H4 antibody was used for Western blotting.</p