Vox Sang. 2003 Jul;85(1):20-4. Evaluation of depth filtration to remove prion challenge from an immune globulin preparation.
Van Holten RW, Autenrieth SM.
Immunohematology/RhoGAM Product Support, Ortho-Clinical Diagnostics, Raritan, NJ 08869, USA. rvanholcdus.jnj.com
BACKGROUND AND OBJECTIVES: Plasma-derived therapeutic proteins have the potential to contain transmissible spongiform encephalopathy (TSE) infectivity. This study evaluated the effectiveness and characterized the mechanism of abnormal prion protein removal during a depth-filtration step used in the manufacture of an immunoglobulin preparation. MATERIALS AND METHODS: Scrapie brain homogenate was treated with lysolecithin, sonicated and sequentially filtered through 0.45-, 0.22- and 0.1-microm membrane filters. The scrapie brain homogenate was then added (at a 1:51 dilution) to the Supernatant III fraction used in the manufacture of Rho(D) immune globulin (human). The spiked immunoglobulin preparation was then filtered through a depth filter under the same conditions used in full-scale production. After filtration, the depth filter was washed with hypertonic NaCl solutions to elute the abnormal prion protein (PrPSc) from the filter. A Western blot assay for PrPSc was used to quantify removal from the filtrate and recovery from the filter washes. A second run was performed whereby the PrPSc-spiked Supernatant III was filtered through a 0.22-microm membrane filter prior to depth filtration. A third run evaluated depth filtration of PrPSc in Tris-buffered saline (TBS). RESULTS: The depth filter removed greater than four logs of PrPSc from the Supernatant III filtrate. A significant portion of the PrPSc could be recovered from the depth filter by elution with high-molarity NaCl solutions. Prefiltration (through a 0.22-microm membrane filter) of the spiked Supernatant III prior to depth filtration removed all detectable PrPSc. Depth filtration removed less than one log of PrPSc from TBS. CONCLUSIONS: Depth filtration appears to remove PrPSc from the immunoglobulin preparation by mechanical straining rather than by adsorption to the filter matrix. The immunoglobulin preparation caused the PrPSc to aggregate from particles <0.1 microm in size to particles of >0.22 microm, probably as a result of the presence of methanol in the preparation. The depth filter failed to remove PrPSc from a purely aqueous environment.
PMID:_12823726 [PubMed - in process]
J Biol Chem. 2003 Sep 12;278(37):35592-6. Epub 2003 Jun 25. Influence of pH on NMR Structure and Stability of the Human Prion Protein Globular Domain.
The NMR structure of the globular domain of the human prion protein (hPrP) with residues 121-230 at pH 7.0 shows the same global fold as the previously published structure determined at pH 4.5. It contains three alpha-helices, comprising residues 144-156, 174-194, and 200-228, and a short anti-parallel beta-sheet, comprising residues 128-131 and 161-164. There are slight, strictly localized, conformational changes at neutral pH when compared with acidic solution conditions: helix alpha1 is elongated at the C-terminal end with residues 153-156 forming a 310-helix, and the population of helical structure in the C-terminal two turns of helix alpha2 is increased. The protonation of His155 and His187 presumably contributes to these structural changes. Thermal unfolding monitored by far UV CD indicates that hPrP-(121-230) is significantly more stable at neutral pH. Measurements of amide proton protection factors map local differences in protein stability within residues 154-157 at the C-terminal end of helix alpha1 and residues 161-164 of beta-strand 2. These two segments appear to form a separate domain that at acidic pH has a larger tendency to unfold than the overall protein structure. This domain could provide a "starting point" for pH-induced unfolding and thus may be implicated in endosomic PrPC to PrPSc conformational transition resulting in transmissible spongiform encephalopaties.
PMID:_12826672 [PubMed - in process]
Viral Immunol. 2003 Summer;16(2):123-39. Transmissible encephalopathies: speculations and realities.
Manuelidis L.
Yale Medical School, New Haven, Connecticut 06510, USA. laura.manuelidiale.edu
Virtually all transmissible encephalopathies (TSEs), such as scrapie, CJD, and BSE, are caused by a type of infectious particle that remains enigmatic. The language of prion theory supersedes the reality of what is, and what is not known. This review questions the predictive value, consistency and accuracy of this now dominant assumption. Many people believe the normal cellular prion protein (PrP) self-converts into an infectious amyloid protein or prion. Although the amyloidogenic capacity of proteins is well established, the concept of an infectious protein without nucleic acid was "revolutionary." Diverse experiments have repeatedly shown, however, that this protein alone, in any form, is incapable of reproducing transmissible infection. In contrast, the infectious agent copurifies with many other molecules, including nucleic acids, while it separates from the majority of PrP. The infectious particle has a homogeneous viral size of ~25 nm, and infectivity is markedly reduced by conditions that disrupt viral core components but do not disrupt multimers of PrP amyloid. Additionally, the infectious agent replicates to high levels before any PrP abnormalities can be detected. Hence, we initially proposed that PrP changes are part of the host's pathologic response to high levels of infectious agent, but not the agent itself. Newer data clarifying a role for myeloid cells in the spread of infection, the unique character of two different agent strains propagated in a single animal, and the demonstration of long nucleic acids in a variety of simplified high titer preparations continue to raise serious questions for the prion hypothesis. Moreover, the epidemic spread of TSEs, and the activation of host innate immune mechanisms by infection, further indicate these agents are recognizably foreign, and probably viral.
PMID:_12828865 [PubMed - in process]
Trends Mol Med. 2003 Jun;9(6):237-43. Is loss of function of the prion protein the cause of prion disorders?
Hetz C, Maundrell K, Soto C.
Serono Pharmaceutical Research Institute, 14 Chemin des Aulx, 1228 Plan les Ouates, Switzerland.
Transmissible spongiform encephalopathies are fatal neurodegenerative diseases that involve misfolding of the prion protein. Recent studies have provided evidence that normal prion protein might have a physiological function in neuroprotective signaling, suggesting that loss of prion protein activity might contribute to the pathogenesis of prion disease. However, studies using knockout animals do not support the loss-of-function hypothesis and argue that prion neurodegeneration might be associated with a gain of a toxic activity by the misfolded prion protein. Thus, the mechanism of neurodegeneration in spongiform encephalopathies remains enigmatic.
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