Investigations of prion and virus safety of a new liquid IVIG product.

A highly purified, liquid, 10% immunoglobulin product stabilized with proline, referred to as IgPro10 has recently been developed. IgG was purified from human plasma by cold ethanol fractionation, octanoic acid precipitation and anion-exchange chromatography. The manufacturing process includes two distinctly different partitioning steps and virus filtration, which were also assessed for the removal of prions. Prion removal studies used different spike preparations (brain homogenate, microsomes, purified PrP(sc)) and three different detection methods (bioassay, Western blot, conformation-dependent immunoassay). All of the investigated production steps were shown to reduce significantly all different spike preparations, resulting in an overall reduction of >10log(10). Moreover, the biochemical assays proved equally effective to the bioassay for the demonstration of prion elimination. Four of the manufacturing steps cover three different mechanisms of virus clearance. These are: i) virus inactivation; ii) virus filtration; and iii) partitioning. These mechanisms were assessed for their virus reduction capacity. Virus validation studies demonstrated overall reduction factors of >18log(10) for enveloped and >7log(10) for non-enveloped model viruses. In conclusion, the IgPro10 manufacturing process has a very high reduction potential for prions and for a wide variety of viruses resulting in a state-of-the-art product concerning safety towards known and emerging pathogens.

Strengths and limitations of the model virus concept.

New plasma- or cell culture-based pharmaceutical manufacturing processes must be validated for their ability to eliminate potentially contaminating pathogens. To evaluate the virus elimination potential of such a process, current guidelines propose the use of model viruses. This approach is discussed based on two examples. These examples show the strengths of this approach but also its limitations. The blood processing industry was recently challenged by the emergence of a West Nile Virus (WNV) epidemic in the United States. The susceptibility of WNV and a frequently used model virus to commonly used inactivation methods is compared. Current data show a good correlation. Due to its physico-chemical properties and the high viremic titers, B19 virus (B19V), a small (diameter 18-26 nm), robust, non-enveloped parvovirus, is a considerable challenge for the plasma processing industry. Mice minute virus (MMV), an animal parvovirus, is used as a model for B19V. Data show that B19V is considerably more susceptible to some physico-chemical inactivation methods than MMV. The examples of WNV and B19V show that the model virus concept is a practicable tool to evaluate the safety of plasma- or cell culture-derived pharmaceuticals regarding known and emerging viruses. It also underlines the need for investigational studies of relevant viruses if they can be handled in a normal virology laboratory, under moderate biosafety conditions.

Two distinct proteins are associated with tetrameric acetylcholinesterase on the cell surface.

In mammalian brain, acetylcholinesterase (AChE) exists mostly as a tetramer of 70-kDa catalytic subunits that are linked through disulfide bonds to a hydrophobic subunit P of approximately 20 kDa. To characterize P, we reduced the disulfide bonds in purified bovine brain AChE and sequenced tryptic fragments from bands in the 20-kDa region. We obtained sequences belonging to at least two distinct proteins: the P protein and another protein that was not disulfide-linked to catalytic subunits. Both proteins were recognized in Western blots by antisera raised against specific peptides. We cloned cDNA encoding the second protein in a cDNA library from bovine substantia nigra and obtained rat and human homologs. We call this protein mCutA because of its homology to a bacterial protein (CutA). We could not demonstrate a direct interaction between mCutA and AChE in vitro in transfected cells. However, in a mouse neuroblastoma cell line that produced membrane-bound AChE as an amphiphilic tetramer, the expression of mCutA antisense mRNA eliminated cell surface AChE and decreased the level of amphiphilic tetramer in cell extracts. mCutA therefore appears necessary for the localization of AChE at the cell surface; it may be part of a multicomponent complex that anchors AChE in membranes, together with the hydrophobic P protein.

The mammalian gene of acetylcholinesterase-associated collagen.

The collagen-tailed or asymmetric forms (A) represent a major component of acetylcholinesterase (AChE) in the neuromuscular junction of higher vertebrates. They are hetero-oligomeric molecules, in which tetramers of catalytic subunits of type T (AChET) are attached to the subunits of a triple-stranded collagen "tail." We report the cloning of a rat AChE-associated collagen subunit, Q. We show that collagen tails are encoded by a single gene, COLQ. The ColQ subunits form homotrimers and readily form collagen-tailed AChE, when coexpressed with rat AChET. We found that the same ColQ subunits are incorporated, in vivo, in asymmetric forms of both AChE and butyrylcholinesterase. A splice variant from the COLQ gene encodes a proline- rich AChE attachment domain without the collagen domain but does not represent the membrane anchor of the brain tetramer. The COLQ gene is expressed in cholinergic tissues, brain, muscle, and heart, and also in noncholinergic tissues such as lung and testis.

The membrane anchor of mammalian brain acetylcholinesterase consists of a single glycosylated protein of 22 kDa.

Mammalian brain acetylcholinesterase (AChE; EC 3.1.1.7) is membrane-bound through a structural subunit of about 20 kDa. So far little is known about this anchor because it is only detectable after hydrophobic labelling. In the present study we demonstrate that the two bands migrating around 20 kDa on SDS-PAGE derive from the same protein containing the same N-terminal amino acid sequence. The difference in their mobility is due to different N-glycosidation. Radioalkylation of cysteine residues reveals that the anchor contains just the two cysteine residues involved in binding the catalytic subunits.

Monoclonal antibodies against a C-terminal peptide of human brain acetylcholinesterase distinguish between erythrocyte and brain acetylcholinesterases.

Monoclonal antibodies (mAbs) were raised against a peptide of the 10 C-terminal amino acids of human brain acetylcholinesterase (AChE): H-Tyr-Ser-Lys-Gln-Asp-Arg-Cys-Ser-Asp-Leu-OH. Two positive clones (mAbs 190-1 and 190-2) were selected and tested for their ability to distinguish between mammalian brain and erythrocyte AChEs. In a solid-phase enzyme antigen immunoassay as well as by Western- and dot-blot analysis, both antibodies showed clear binding to AChE from human and bovine brain but not to AChE from erythrocytes. MAbs 190-1 and 190-2 reacted with neither AChE from electric eel nor butyrylcholinesterase from human serum. Both antibodies were used in a quantitative assay for AChE in amniotic fluids, where AChE activity could be found only in samples from open neural tube-defect pregnancies, but not in fluids from normal pregnancies or in artificially blood-contaminated samples.

Characterization of salt-soluble forms of acetylcholinesterase from bovine brain.

The hydrophilic, salt-soluble (SS) form of acetylcholinesterase (AChE) from bovine brain caudate nucleus exists mainly as a tetramer sedimenting at 10.3S (approximately 40%), and a monomer sedimenting at 3.4S (approximately 60%). The enzyme is N-glycosylated and contains similar HNK-1 carbohydrates as detergent-soluble (DS) AChE. No O-linked carbohydrates could be detected. Amino acid sequencing showed that the N terminus of SS-AChE is identical to that of DS-AChE. In tetrameric SS-AChE, two pairs of disulfide-linked dimers are associated by hydrophobic forces located in the C terminus. Antibodies were raised against a peptide identical to the last 10 amino acid residues of bovine brain DS-AChE. The peptide included the sequence of residues 574-583 (H-Tyr-Ser-Lys-Gln-Asp-Arg-Cys-Ser- Asp-Leu-OH) of the enzyme. The antibodies cross-reacted with tetrameric, but not with monomeric, SS-AChE, showing that in the latter form, the C terminus is truncated. Limited proteolysis of tetrameric SS-AChE at the C terminus led to the formation of an enzymatically active monomer, which did not react with anti-C-terminal antibody. Although the DS form of AChE contains a structural subunit that serves as membrane anchor, no anchor was detected in SS-AChE. Enzyme antigen immunoassays showed that SS-AChE reacted with all monoclonal antibodies directed against the catalytic subunit of DS-AChE, but not with monoclonal antibodies targeting the membrane-anchored subunits. From our results, we conclude that SS-AChE utilizes the same alternative splicing pattern as DS-AChE, leading to tetrameric SS-AChE devoid of the membrane anchor.(ABSTRACT TRUNCATED AT 250 WORDS)

The membrane form of acetylcholinesterase from rat brain contains a 20 kDa hydrophobic anchor.

Rat brain acetylcholinesterase (AChE, EC 3.1.1.7) consists of about 80% amphiphilic detergent-soluble (DS-) AChE and 20% hydrophilic salt-soluble (SS-) AChE. DS-AChE contains about 65% tetrameric, 20% dimeric and 10% monomeric, SS-AChE about 40% tetrameric and 60% monomeric forms. N-terminal sequencing of DS- and SS-AChE gave identical N-termini corresponding to the published cDNA sequence of the mature enzyme. The band pattern on SDS-gels is similar to that of AChE from human and bovine brain. SDS-PAGE of hydrophobically labeled DS-AChE revealed the presence of a disulfide bonded hydrophobic membrane anchor of about 20 kDa. Monoclonal antibodies (mAbs) recognizing the anchor-containing subunits of mammalian brain DS-AChE, crossreacted with rat brain DS-AChE but not with SS-AChE. DS- and SS-AChE also reacted with antibodies raised against a peptide comprising the last 10 amino acids of the sequence of bovine brain AChE. Our results led us to conclude that both DS- and SS-AChE from rat brain contain T-type catalytic subunits, and DS-AChE in addition a P-type hydrophobic anchor similar to other mammalian brain DS-AChE.

Fluorescein angiography of optociliary shunt vessels.

Progressive loss of vision, optic disc pallor, and optociliary shunt vessels (the Hoyt-Spencer sign) constitute the clinical triad of optic nerve sheath meningiomas. However, optociliary shunt vessels may also follow central vein occlusions, and less commonly occur with a few other conditions. This report presents a comparative study of fluorescein angiograms performed on eight patients with optociliary shunt vessels. Four patients had optic nerve sheath meningiomas, and four patients had central retinal vein occlusions. The following differences in the fluorescein angiograms were noted in the two groups. In the optic nerve sheath meningioma group, the shunt vessels fill earlier (in the arteriovenous phase), the flow drains to central venous tributaries, and the late staining is hyperfluorescent to other veins. However, in the central vein occlusion group, the shunts fill later (in the venous phase), show a flow draining to the outer disc margin, and late staining is eufluorescent with other veins. Although optic nerve sheath meningiomas and old vein occlusions are usually rather easily differentiated by a complete examination, the fluorescein angiographic patterns are not only pertinent with regards to the pathogenesis of optociliary vessels, but in certain cases may be clinically helpful in making an important clinical differentiation.

Primary and secondary heart tumors in mice maintained on various diets.

Two mice of the TM strain, maintained on Purina chow supplemented with refined coin oil plus free fatty acids, in one case, and with refined corn oil without free fatty acids, in the other case, developed primary heart rhabdomyosarcomas. The tumors metastasized to the lungs and the kidney, in the first case, and to the lungs alone in the second case. One mouse of the TM strain, on the diet supplemented with corn oil plus free fatty acids, developed a fibrosarcoma behind the right anterior extremity. Metastases from the tumor were found in the liver, lungs and myocardium. Another mouse of the C57 Bl. strain, injected subcutaneously, in the interscapular region, at the age of 7 days, with a single dose of 30 mug of Delestrogen, developed, at the point of injection, a large rhabdomyosarcoma. At the age of 5333 days, when the animal was killed, metastases from the tumor were found in the intercostal muscles and in the heart. Three mice, one of the TM strain, on a diet supplemented with corn oil and cholesterol, and two of the C57 Br. strain, on a diet supplemented with raw egg yolk (rich in cholesterol) had organized blood clots in the atria (in two mice) and one extending into the ventricle.