Targeted designed variants of alpha-2-macroglobulin (A2M) attenuate cartilage degeneration in a rat model of osteoarthritis induced by anterior cruciate ligament transection.

BACKGROUND: The study was performed to evaluate whether targeted alpha-2-macroglobulin (A2M) variants have a similar or enhanced function at wild-type (wt)-A2M to attenuate cartilage degeneration in vivo. METHODS: In and ex-vivo experiment, bovine cartilage explants (BCE) were incubated with TNF-α and IL-1ß with or without wt-A2M or A2M variants. Cartilage catabolism was measured in culture supernatant by sulfated glycosaminoglycan (sGAG). In an in-vivo experiment, 2-month-old male Wistar rats (n = 77) were randomly divided into seven groups and treated with different doses of A2M or its variants by intra-articular injection at 24 hours and day 14 after anterior cruciate ligament transection (ACLT), receiving (1) ACLT/PBS; (2) ACLT/wt-A2M (0.153 mg); (3) ACLT/CYT-108 A2M (0.153 mg); (4) ACLT/CYT-108 A2M (0.077 mg); (5) ACLT/CYT-98 A2M (0.153 mg); (6) ACLT/CYT-98 A2M (0.077 mg); or (7) sham/PBS. The joints and synovial lavage were collected 8 weeks after surgery. Fluorescence molecular tomography was used to monitor inflammation in vivo using probes ProSense and MMPSense at 24 hours, and weeks 2, 4, and 6 after surgery. The cartilage damage was quantified using Osteoarthritis Research Society International score and matrix metalloproteinase (MMP)-3, -13, collagen (Col) X, Col 2, Runx2, and aggrecan (Acan) were detected by immunohistochemical analysis (IHC), ELISA, and RT-PCR. RESULTS: A2M variants inhibited catabolism in the BCE model by up to 200% compared with wt-A2M. ProSense and MMPSense were dramatically increased in all groups after surgery. Supplemental A2M or its variants reduced ProSense and MMPSense compared with the PBS treatment. Less cartilage damage, lower MMP-13 and Col 2 degraded product, and stronger Col 2 synthesis were detected in animals treated with A2M or its variants compared with PBS-treated animals. A2M and its variants enhanced Col 2 and Acan synthesis, and suppressed MMP-3, MMP-13, Runx2, and Col X production. A2M-108 variant demonstrated less cartilage damage compared with wt-A2M and A2M-98 variant. CONCLUSION: The targeted variants of A2M have a chondroprotective effect similar to wt-A2M. However, A2M-108 variant has enhanced function to attenuate cartilage degeneration compared with wt-A2M.

Epigenetic dominance of prion conformers.

Although they share certain biological properties with nucleic acid based infectious agents, prions, the causative agents of invariably fatal, transmissible neurodegenerative disorders such as bovine spongiform encephalopathy, sheep scrapie, and human Creutzfeldt Jakob disease, propagate by conformational templating of host encoded proteins. Once thought to be unique to these diseases, this mechanism is now recognized as a ubiquitous means of information transfer in biological systems, including other protein misfolding disorders such as those causing Alzheimer's and Parkinson's diseases. To address the poorly understood mechanism by which host prion protein (PrP) primary structures interact with distinct prion conformations to influence pathogenesis, we produced transgenic (Tg) mice expressing different sheep scrapie susceptibility alleles, varying only at a single amino acid at PrP residue 136. Tg mice expressing ovine PrP with alanine (A) at (OvPrP-A136) infected with SSBP/1 scrapie prions propagated a relatively stable (S) prion conformation, which accumulated as punctate aggregates in the brain, and produced prolonged incubation times. In contrast, Tg mice expressing OvPrP with valine (V) at 136 (OvPrP-V136) infected with the same prions developed disease rapidly, and the converted prion was comprised of an unstable (U), diffusely distributed conformer. Infected Tg mice co-expressing both alleles manifested properties consistent with the U conformer, suggesting a dominant effect resulting from exclusive conversion of OvPrP-V136 but not OvPrP-A136. Surprisingly, however, studies with monoclonal antibody (mAb) PRC5, which discriminates OvPrP-A136 from OvPrP-V136, revealed substantial conversion of OvPrP-A136. Moreover, the resulting OvPrP-A136 prion acquired the characteristics of the U conformer. These results, substantiated by in vitro analyses, indicated that co-expression of OvPrP-V136 altered the conversion potential of OvPrP-A136 from the S to the otherwise unfavorable U conformer. This epigenetic mechanism thus expands the range of selectable conformations that can be adopted by PrP, and therefore the variety of options for strain propagation.

Platelet-rich plasma increases matrix metalloproteinases in cultures of human synovial fibroblasts.

BACKGROUND: The effect of platelet-rich plasma on chondrocytes has been studied in cell and tissue culture. Less attention has been given to the effect of platelet-rich plasma on nonchondrocytic cell lineages within synovial joints, such as fibroblast-like synoviocytes, which produce cytokines and matrix metalloproteinases (MMPs) that mediate cartilage catabolism. The purpose of the present study was to determine the effect of platelet-rich plasma on cytokines and proteases produced by fibroblast-like synoviocytes. METHODS: Platelet-rich plasma and platelet-poor plasma from harvested autologous blood were prepared with a commercially available system. Fibroblast-like synoviocytes were treated with platelet-rich plasma, platelet-poor plasma, recombinant PDGFßß (platelet-derived growth factor ßß), or phosphate-buffered saline solution and incubated at 37°C for forty-eight hours. The concentrations of IL-1ß (interleukin-1ß), IL-1RA (IL-1 receptor antagonist), IL-6, IFN-γ (interferon-γ), IP-10 (interferon gamma-induced protein 10), MCP-1 (monocyte chemotactic protein-1), MIP-1ß (macrophage inflammatory protein-1ß), PDGFßß, RANTES, TNF-α (tumor necrosis factor-α), VEGF (vascular endothelial growth factor), MMP-1, MMP-3, and MMP-9 in the culture medium were determined by multiplex immunoassay. RESULTS: Platelet-rich plasma cultured in medium contained multiple catabolic mediators in substantial concentrations, including MMP-9 (15.8 ± 2.3 ng/mL) and MMP-1 (2.5 ± 0.8 ng/mL), as well as proinflammatory mediators IL-1ß, IL-6, IFN-γ, IP-10, MCP-1, MIP-1ß, RANTES, and TNF-α in concentrations between 20 pg/mL and 20 ng/mL. Platelet-poor plasma contained significantly lower concentrations of these compounds. Platelet-rich plasma was used to treat human fibroblast-like synoviocytes, and the resulting concentrations of mediators were corrected for the concentrations in the platelet-rich plasma alone. Compared with untreated fibroblast-like synoviocytes, synoviocytes treated with platelet-rich plasma exhibited significantly greater levels of MMP-1 (363 ± 94.0 ng/mL, p = 0.018) and MMP-3 (278 ± 90.0 ng/mL, p = 0.018). In contrast, platelet-poor plasma had little effect on mediators secreted by the synoviocytes. PDGFßß-treated fibroblast-like synoviocytes exhibited a broad proinflammatory cytokine response at four and forty-eight hours. CONCLUSIONS: Platelet-rich plasma was shown to contain a mixture of anabolic and catabolic mediators. Synoviocytes treated with platelet-rich plasma responded with substantial MMP secretion, which may increase cartilage catabolism. Synoviocytes responded to PDGF with a substantial proinflammatory response.

Prions on the move.

Prions consist mainly, if not entirely, of PrP(Sc), an aggregated conformer of the host protein PrP(C). Prions come in different strains, all based on the same PrP(C) sequence, but differing in their conformations. The efficiency of prion transmission between species is usually low, but increases after serial transmission in the new host, suggesting a process involving mutation and selection. Even within the same species, the transfer of prions between cell types entails a selection of favoured 'substrains', and propagation of prions in the presence of an inhibitory drug can result in the appearance of drug-resistant prion populations. We propose that prion populations are comprised of a variety of conformers, constituting 'quasi-species', from which the one replicating most efficiently in a particular environment is selected.

Abrogation of complex glycosylation by swainsonine results in strain- and cell-specific inhibition of prion replication.

Neuroblastoma-derived N2a-PK1 cells, fibroblastic LD9 cells, and CNS-derived CAD5 cells can be infected efficiently and persistently by various prion strains, as measured by the standard scrapie cell assay. Swainsonine, an inhibitor of Golgi α-mannosidase II that causes abnormal N-glycosylation, strongly inhibits infection of PK1 cells by RML, 79A and 22F, less so by 139A, and not at all by 22L prions, and it does not diminish propagation of any of these strains in LD9 or CAD5 cells. Misglycosylated PrP(C) formed in the presence of swainsonine is a good substrate for conversion to PrP(Sc), and misglycosylated PrP(Sc) is fully able to trigger infection and seed the protein misfolding cyclic amplification reaction. Distinct subclones of PK1 cells mediate swainsonine inhibition to very different degrees, implicating misglycosylation of one or more host proteins in the inhibitory process. The use of swainsonine and other glycosylation inhibitors described herein enhances the ability of the cell panel assay to differentiate between prion strains. Moreover, as shown elsewhere, the susceptibility of prions to inhibition by swainsonine in PK1 cells is a mutable trait.

Transfer of a prion strain to different hosts leads to emergence of strain variants.

Prions consist mainly of PrP(Sc), a pathogenic conformer of host-encoded PrP(C). Prion populations with distinct phenotypes but associated with PrP(Sc), having the same amino acid sequence, constitute distinct strains. Strain identity is thought to be encoded by the conformation of PrP(Sc) and to be maintained by seeded conversion. Prion strains can be distinguished by the cell panel assay, which measures their ability to infect distinct cell lines. Brain-derived 22L prions characteristically are able to infect R33 cells (i.e., are "R33 competent"), as well as PK1 cells in the presence of the inhibitor swainsonine (i.e. are "swa resistant"). Here we report that 22L prions retained their characteristic cell tropism and swa resistance when transferred from brain to R33 cells. However, when transferred from the R33 cells to PK1 cells, they gradually became R33 incompetent and swa sensitive, unless the transfer was in the presence of swa, in which case swa resistance and R33 competence were retained. PrP(Sc) associated with swa-resistant/R33-competent and swa-sensitive/R33-incompetent prions had different conformational stabilities. When cloned R33-incompetent/swa-sensitive prions were again propagated in brain, their properties gradually reverted to those of the original brain-derived 22L prions. Our results support the view that 22L prion populations are heterogeneous and that distinct prion variants are selected in different cellular environments.

Prion strain mutation determined by prion protein conformational compatibility and primary structure.

Prions are infectious proteins composed of the abnormal disease-causing isoform PrPSc, which induces conformational conversion of the host-encoded normal cellular prion protein PrPC to additional PrPSc. The mechanism underlying prion strain mutation in the absence of nucleic acids remains unresolved. Additionally, the frequency of strains causing chronic wasting disease (CWD), a burgeoning prion epidemic of cervids, is unknown. Using susceptible transgenic mice, we identified two prevalent CWD strains with divergent biological properties but composed of PrPSc with indistinguishable biochemical characteristics. Although CWD transmissions indicated stable, independent strain propagation by elk PrPC, strain coexistence in the brains of deer and transgenic mice demonstrated unstable strain propagation by deer PrPC. The primary structures of deer and elk prion proteins differ at residue 226, which, in concert with PrPSc conformational compatibility, determines prion strain mutation in these cervids.

Darwinian evolution of prions in cell culture.

Prions are infectious proteins consisting mainly of PrP(Sc), a beta sheet-rich conformer of the normal host protein PrP(C), and occur in different strains. Strain identity is thought to be encoded by PrP(Sc) conformation. We found that biologically cloned prion populations gradually became heterogeneous by accumulating "mutants," and selective pressures resulted in the emergence of different mutants as major constituents of the evolving population. Thus, when transferred from brain to cultured cells, "cell-adapted" prions outcompeted their "brain-adapted" counterparts, and the opposite occurred when prions were returned from cells to brain. Similarly, the inhibitor swainsonine selected for a resistant substrain, whereas, in its absence, the susceptible substrain outgrew its resistant counterpart. Prions, albeit devoid of a nucleic acid genome, are thus subject to mutation and selective amplification.

The elk PRNP codon 132 polymorphism controls cervid and scrapie prion propagation.

The elk prion protein gene (PRNP) encodes either methionine (M) or leucine (L) at codon 132, the L132 allele apparently affording protection against chronic wasting disease (CWD). The corresponding human codon 129 polymorphism influences the host range of bovine spongiform encephalopathy (BSE) prions. To fully address the influence of this cervid polymorphism on CWD pathogenesis, we created transgenic (Tg) mice expressing cervid PrPC with L at residue 132, referred to as CerPrPC-L132, and compared the transmissibility of CWD prions from elk of defined PRNP genotypes, namely homozygous M/M or L/L or heterozygous M/L, in these Tg mice with previously described Tg mice expressing CerPrPC-M132, referred to as Tg(CerPrP) mice. While Tg(CerPrP) mice were consistently susceptible to CWD prions from elk of all three genotypes, Tg(CerPrP-L132) mice uniformly failed to develop disease following challenge with CWD prions. In contrast, SSBP/1 sheep scrapie prions transmitted efficiently to both Tg(CerPrP) and Tg(CerPrP-L132) mice. Our findings suggest that the elk 132 polymorphism controls prion susceptibility at the level of prion strain selection and that cervid PrP L132 severely restricts propagation of CWD prions. We speculate that the L132 polymorphism results in less efficient conversion of CerPrPC-L132 by CWD prions, an effect that is overcome by the SSBP/1 strain. Our studies show the accumulation of subclinical levels of CerPrPSc in aged asymptomatic CWD-inoculated Tg(CerPrP-L132) mice and also suggests the establishment of a latent infection state in apparently healthy elk expressing this seemingly protective allele.

Prions in skeletal muscles of deer with chronic wasting disease.

The emergence of chronic wasting disease (CWD) in deer and elk in an increasingly wide geographic area, as well as the interspecies transmission of bovine spongiform encephalopathy to humans in the form of variant Creutzfeldt Jakob disease, have raised concerns about the zoonotic potential of CWD. Because meat consumption is the most likely means of exposure, it is important to determine whether skeletal muscle of diseased cervids contains prion infectivity. Here bioassays in transgenic mice expressing cervid prion protein revealed the presence of infectious prions in skeletal muscles of CWD-infected deer, demonstrating that humans consuming or handling meat from CWD-infected deer are at risk to prion exposure.

Transmission of prions from mule deer and elk with chronic wasting disease to transgenic mice expressing cervid PrP.

We generated mice expressing cervid prion protein to produce a transgenic system simulating chronic wasting disease (CWD) in deer and elk. While normal mice were resistant to CWD, these transgenic mice uniformly developed signs of neurological dysfunction approximately 230 days following intracerebral inoculation with four CWD isolates. Inoculated transgenic mice homozygous for the transgene array developed disease after approximately 160 days. The brains of sick transgenic mice exhibited widespread spongiform degeneration and contained abnormal prion protein and abundant amyloid plaques, many of which were florid plaques. Transmission studies indicated that the same prion strain caused CWD in the analyzed mule deer and elk. These mice provide a new and reliable tool for detecting CWD prions.