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1.
Vox Sang ; 113(1): 13-20, 2018 Jan.
Article in English | MEDLINE | ID: mdl-28952159

ABSTRACT

BACKGROUND AND OBJECTIVES: Australia introduced bacterial contamination screening (BCS) for platelet components in April 2008. This study presents analysis performed to assess the efficacy of testing. MATERIALS AND METHODS: Seven-day aerobic and anaerobic culture is performed using the BacT/ALERT 3D system. Following an initial machine positive (IMP) flag, all associated components are recalled, and/or clinicians treating already transfused patients are notified. IMPs are categorized as 'machine false positive', 'confirmed positive' or 'indeterminate' depending on culture results of initial and repeat samples. RESULTS: Between 2010 and 2012, 1·1% of platelet donations tested IMP; since 2013, this rate has fallen to 0·6% through improved instrument management, reducing false-positive IMPs but maintaining sensitivity for cultures yielding bacterial growth. On average, 66% of confirmed positive and indeterminate platelet units had been transfused at the time of detection. The majority (95%) of these grew Propionibacterium sp., a slow-growing organism that rarely causes sepsis in the transfusion setting. The incidence of reported transfuion-transmitted bacterial infection (TTBI) has fallen since the introduction of BCS, with a 4·2-fold [0·5, 28·2] lower rate from platelets. CONCLUSION: BCS has been successful in detecting platelet units containing pathogenic bacteria. The incidence of TTBI from platelets has fallen since the introduction of BCS, but the risk has not been eliminated due to rare false-negative results. In the absence of a pathogen inactivation system for red blood cells, BCS provides 'surrogate' testing of red blood cells from which platelets have been manufactured.


Subject(s)
Bacterial Infections/prevention & control , Blood Platelets/microbiology , Australia/epidemiology , Bacterial Infections/epidemiology , Bacterial Infections/transmission , Blood Safety , Culture Techniques , Humans , Incidence , Platelet Transfusion/adverse effects
2.
Biologicals ; 35(3): 161-4, 2007 Jun.
Article in English | MEDLINE | ID: mdl-17074508

ABSTRACT

Sodium hydroxide (NaOH) has been shown to reduce the infectivity of transmissible spongiform encephalopathy (TSE) agents. This study investigated the efficacy of sodium hydroxide at 0.1M, 0.25M and 0.5M concentrations for the inactivation of mouse-adapted scrapie strain ME7. Times and temperatures modelled conditions used in an industrial plasma fractionation plant for sanitisation of ultrafilters, and the sodium hydroxide component of Clean In Place sanitisation. The concentration of scrapie ME7 brain homogenate in NaOH test solutions was 1% (w/v). At the end of incubation periods, the samples were adjusted to neutral pH prior to intracerebral inoculation into mice for bioassay. The conditions of 0.1M NaOH at 60 degrees C for 2min and 0.25M NaOH at 30 degrees C for 60min were found to inactivate 3.96 and 3.93logs of scrapie, respectively. Use of 0.5M NaOH at 30 degrees C for 60 or 75min was found to inactivate >or=4.23 and 4.15logs of scrapie. This indicates that the use of these conditions in an industrial process would substantially reduce prion infectivity.


Subject(s)
PrPSc Proteins/antagonists & inhibitors , Sodium Hydroxide/pharmacology , Adaptation, Physiological , Animals , Biological Assay/methods , Biological Products/adverse effects , Biological Products/isolation & purification , Cattle , Decontamination/methods , Mice , PrPSc Proteins/isolation & purification , Safety , Scrapie/prevention & control , Scrapie/transmission , Sheep , Temperature
3.
Vox Sang ; 91(4): 292-300, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17105604

ABSTRACT

BACKGROUND AND OBJECTIVES: Although there is no epidemiological evidence to suggest that classical Creutzfeldt-Jakob disease (CJD) is transmitted through blood or blood products, the variant form (vCJD) has been implicated in transmission via packed red blood cells. The potential threat of the infectious agent contaminating plasma pools has led to manufacturing processes being examined for capacity to remove prions. The objective of these studies was to examine the prion-removal potential of the chromatographic purification and ethanol precipitation steps used to fractionate immunoglobulins and albumin from human plasma. MATERIALS AND METHODS: Western blot assay was used to examine the partitioning of proteinase K-resistant scrapie prion protein (PrPsc) over DEAE Sepharose, CM Sepharose and Macro-Prep High Q chromatographic columns, utilizing microsomal scrapie 263K spiked into each scaled down feedstream and assayed after each chromatographic step. In further studies, bioassay in C57 black mice was used and spikes of 10 000 g clarified brain homogenate of scrapie ME7 were added to feedstreams before sequences of scaled down chromatographic or Cohn fractionation process steps. RESULTS: The microsomal spiking study with Western blot detection demonstrated substantial partitioning of PrPsc away from the target proteins in all ion exchange chromatographic steps examined. The log10 reduction factors (LRF) across DEAE Sepharose and CM Sepharose columns for albumin were > or = 4.0 and > or = 3.0 respectively. The reductions across DEAE Sepharose and Macro-Prep High Q for intravenous immunoglobulin were 3.3 and > or = 4.1 respectively. Bioassay demonstrated LRFs of >or = 5.6 across the combination of DEAE Sepharose and CM Sepharose columns in the albumin process and > or = 5.4 across the combination of DEAE Sepharose and Macro-Prep High Q columns in the intravenous immunoglobulin process. Bioassay studies also demonstrated a LRF of > or = 5.6 for immunoglobulin produced by Cohn fractionation. CONCLUSIONS: Using rodent-adapted scrapie as a model, the studies indicated that ion exchange chromatography, as well as Cohn immunoglobulin fractionation have the potential to effectively reduce the load of TSE agents should they be present in plasma pools.


Subject(s)
Chemistry, Pharmaceutical/methods , Immunoglobulins/isolation & purification , Prions/isolation & purification , Serum Albumin/isolation & purification , Animals , Brain , Chemical Fractionation , Chemical Precipitation , Chromatography, Ion Exchange , Consumer Product Safety , Cricetinae , Ethanol , Immunoglobulins/blood , Microsomes , Prions/blood
4.
Vox Sang ; 91(4): 301-8, 2006 Nov.
Article in English | MEDLINE | ID: mdl-17105605

ABSTRACT

BACKGROUND AND OBJECTIVES: Concerns about the potential for prions to be retained on chromatography gels during the manufacture of plasma products prompted development of an investigational strategy for detecting infectious prions bound to gels. The objective was to firstly examine methods of implanting gels intracerebrally (IC) in mice, then to examine prion cleaning from a scaled-down version of the DEAE Sepharose column used in a production process to fractionate immunoglobulins and albumin from human plasma. MATERIALS AND METHODS: The study consisted of two parts: (i) the pathophysiological impact by IC inoculation of ground gel beads was compared to whole gel beads; (ii) the feedstreams to two DEAE Sepharose columns were spiked with scrapie ME7. One column was subjected to the protein loading and elution portions of the chromatography cycle. The other column was subjected to the full cycle of protein loading and elution, followed by regeneration with 0.5 m NaCl, 1 m NaOH and solvent/detergent washes. The gels were unpacked and bioassayed by IC implantation in mice to quantify infectivity. RESULTS: IC inoculation of ground gel beads resulted in unacceptably high pathological impact in the mice whereas whole gel bead inoculation resulted in a reduced affect. Accordingly, the whole bead model system was used to assess prion removal/inactivation from chromatography gels at the pre- and postcleaning stage of the chromatography cycle. Infectious prions were detected on the DEAE Sepharose prior to the cleaning step; however, the gel cleaning cycle reduced infectivity by a log reduction factor (LRF) of > or = 2.75, thus reducing infectivity by bioassay to below detectable limits. CONCLUSIONS: A model system for assessment of prion inactivation/removal from chromatography gels has been established. Spiked prion infectivity does bind to DEAE Sepharose gel; however, the cleaning cycle removed infectivity to levels below that detectable by bioassay.


Subject(s)
Brain Chemistry , Chromatography, Gel/methods , Prion Diseases/therapy , Prions/isolation & purification , Sorption Detoxification/methods , Adsorption , Animals , Chromatography, DEAE-Cellulose , Disease Models, Animal , Mice , Prions/blood , Scrapie/therapy
5.
J Neurosci Res ; 65(6): 565-72, 2001 Sep 15.
Article in English | MEDLINE | ID: mdl-11550224

ABSTRACT

Transmissible spongiform encephalopathies are characterised by the transformation of the normal cellular prion protein (PrP(C)) into an abnormal isoform (PrP(TSE)). Previous studies have shown that N-methyl-D-aspartate (NMDA) receptor antagonists can inhibit glutathione depletion and neurotoxicity induced by PrP(TSE) and a toxic prion protein peptide, PrP106-126, in vitro. NMDA receptor activation is known to increase intracellular accumulation of Ca(2+), resulting in up-regulation of arachidonic acid (AA) metabolism. This can stimulate the lipoxygenase pathways that may generate a number of potentially neurotoxic metabolites. Because of the putative relationship between AA breakdown and PrP106-126 neurotoxicity, we investigated AA metabolism in primary cerebellar granule neuron cultures treated with PrP106-126. Our studies revealed that PrP106-126 exposure for 30 min significantly up-regulated AA release from cerebellar granule neurons. PrP106-126 neurotoxicity was mediated through the 5-lipoxygenase (5-LOX) pathway, as shown by abrogation of neuronal death with the 5-LOX inhibitors quinacrine, nordihydroguaiaretic acid, and caffeic acid. These inhibitors also prevented PrP106-126-induced caspase 3 activation and annexin V binding, indicating a central role for the 5-LOX pathway in PrP106-126-mediated proapoptosis. Interestingly, inhibitors of the 12-lipoxygenase pathway had no effect on PrP106-126 neurotoxicity or proapoptosis. These studies clearly demonstrate that AA metabolism through the 5-LOX pathway is an important early event in PrP106-126 neurotoxicity and consequently may have a critical role in PrP(TSE)-mediated cell loss in vivo. If this is so, therapeutic intervention with 5-LOX inhibitors may prove beneficial in the treatment of prion disorders.


Subject(s)
Arachidonate 5-Lipoxygenase/metabolism , Arachidonic Acid/metabolism , Neurons/drug effects , Neurotoxins/toxicity , Peptide Fragments/toxicity , Prion Diseases/enzymology , Prions/metabolism , Prions/toxicity , Animals , Annexin A5/drug effects , Annexin A5/metabolism , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Binding Sites/drug effects , Binding Sites/physiology , Caspase 3 , Caspases/drug effects , Caspases/metabolism , Cells, Cultured/drug effects , Cells, Cultured/enzymology , Cerebellar Cortex/drug effects , Cerebellar Cortex/enzymology , Enzyme Inhibitors/pharmacology , Excitatory Amino Acid Antagonists/pharmacology , Memantine/pharmacology , Mice , Mice, Inbred C57BL , Neurons/enzymology , Prion Diseases/physiopathology , Quinacrine/pharmacology
6.
Am J Pathol ; 155(5): 1723-30, 1999 Nov.
Article in English | MEDLINE | ID: mdl-10550328

ABSTRACT

The prion protein (PrP) has a central role in the pathogenesis of transmissible spongiform encephalopathies (TSE). Accumulating evidence suggests that normal cellular PrP (PrP(c)) may be involved in copper homeostasis and modulation of copper/zinc superoxide dismutase (Cu/ZnSOD) activity in neurons. Hydrogen peroxide (H(2)O(2)) is a toxic reactive oxygen species generated through normal cellular respiration, and neurons contain two important peroxide detoxifying systems (glutathione pathway and catalase). To determine whether PrP expression affects neuronal resistance to H(2)O(2), we exposed primary cerebellar granule neuron cultures derived from PrP knockout (PrP(-/-)) and wild-type (WT) mice to H(2)O(2) for 3, 6, and 24 hours. The PrP(-/-) neurons were significantly more susceptible to H(2)O(2) toxicity than WT neurons after 6 and 24 hours' exposure. The increased H(2)O(2) toxicity may be related to a significant decrease in glutathione reductase activity measured in PrP(-/-) neurons both in vitro and in vivo. This was supported by the finding that inhibition of GR activity with 1,3-bis(2-chloroethyl)-1-nitrosurea (BCNU) increased H(2)O(2) toxicity in WT neurons over the same exposure period. The PrP toxic peptide PrP106-126 significantly reduced neuronal glutathione reductase activity and increased susceptibility to H(2)O(2) toxicity in neuronal cultures suggesting that PrP toxicity in vivo may involve altered glutathione reductase activity. Our results suggest the pathophysiology of prion diseases may involve perturbed PrP(c) function with increased vulnerability to peroxidative stress.


Subject(s)
Cerebellum/metabolism , Cerebellum/pathology , Glutathione Reductase/metabolism , Hydrogen Peroxide/metabolism , Neurons/metabolism , Neurons/pathology , Oxidative Stress , Prions/metabolism , Animals , Cell Survival/drug effects , Cells, Cultured , Hydrogen Peroxide/toxicity , Mice , Mice, Knockout , Prions/genetics , Reactive Oxygen Species
7.
Biochemistry ; 38(11): 3280-4, 1999 Mar 16.
Article in English | MEDLINE | ID: mdl-10079070

ABSTRACT

A considerable body of data supports the model that the infectious agent (called a prion) which causes the transmissible spongiform encephalopathies is a replicating polypeptide devoid of nucleic acid. Prions are believed to propagate by changing the conformation of the normal cellular prion protein (PrPc) into an infectious isoform without altering the primary sequence. Proteins equivalent to the mature form of the wild-type mouse prion protein (residues 23-231) or with a mutation equivalent to that associated with Gerstmann-Straüssler-Scheinker disease (proline to leucine at codon 102 in human; 101 in mouse) were expressed in E. coli. The mutation did not alter the relative proteinase K susceptibility properties of the mouse prion proteins. The wild-type and mutant proteins were analyzed by circular dichroism under different pH and temperature conditions. The mutation was associated with a decrease in alpha-helical content, while the beta-sheet content of the two proteins was unchanged. This suggests the mutation, while altering the secondary structure of PrP, is not sufficient to induce proteinase K resistance and could therefore represent an intermediate isoform along the pathway toward prion formation.


Subject(s)
Prion Diseases/genetics , Prion Diseases/metabolism , Prions/chemistry , Prions/genetics , Recombinant Proteins/chemistry , Amino Acid Substitution/genetics , Animals , Circular Dichroism , Drug Resistance/genetics , Endopeptidase K/metabolism , Escherichia coli/enzymology , Escherichia coli/genetics , Leucine/genetics , Mice , Mice, Inbred BALB C , Prion Diseases/enzymology , Prions/biosynthesis , Proline/genetics , Protein Structure, Secondary , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism
8.
Clin Mater ; 9(3-4): 201-9, 1992.
Article in English | MEDLINE | ID: mdl-10149971

ABSTRACT

Periodontal bone defects were established in four dogs, with one proximal lesion and one furcation lesion in each quadrant. These defects were treated with the implantation of collagen membranes, collagen sponge or a combination of membrane and sponge, inserted between the mucoperiosteal flaps and the bone defects. Control sites were treated in a similar surgical manner to the experimental sites, but no collagen was inserted. Substantial amounts of new bone formed in those cases treated with the collagen products, especially those treated with the membrane either with or without the sponge. The membranes limited the infiltration of small round cells, whereas in the control sites, inflammatory cells infiltrated to the bone surface. New connective tissue attachment was established in experimental situations, especially with the use of the membranes alone or in conjunction with sponge.


Subject(s)
Collagen , Osseointegration/drug effects , Osteogenesis/drug effects , Periodontal Diseases/surgery , Periodontal Dressings/pharmacology , Animals , Collagen/pharmacology , Dogs , Materials Testing , Microscopy, Electron, Scanning , Periodontal Diseases/drug therapy , Periodontal Diseases/pathology
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