Antibody to Alzheimer Amyloid
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Antibody treatment of Alzheimer amyloid
BSE: obstacles to testing
Infectivity survives boiling in SDS
Scrapie found throughout GI tract
27 new mammalian and 9 bird prions: high conservation of flexible region
Circadian regulation of prion mRNA: widespread synchronous rhythm.
Rescuing knockout mice symptoms with normal prion
Apoptotic cells found in CJD
Selective oxidation of prion methionine residues
Equilibrium folding properties of the yeast prion Ure2
Altered phospholipase release of prion mutants
Iatrogenic disease: all-time record claimed by hepatitus C

Circadian regulation of prion protein messenger RNA in the rat forebrain: a widespread and synchronous rhythm.

Neuroscience 1999;91(4):1201-4
Cagampang FR, Whatley SA, Mitchell AL, Powell JF, Campbell IC, Coen CW
Although the expression of the normal prion protein in the host is critical to the development of transmissible spongiform encephalopathies, the physiological role of this protein and the processes regulating its expression remain obscure. We now report that the messenger RNA for the prion protein is regulated in the rat brain in a marked circadian manner not only in the suprachiasmatic nuclei, the principal site for the generation of mammalian circadian rhythms, but also in other forebrain regions.

The data show a remarkable consistency in the concurrence of a single peak of prion protein messenger RNA at each of the sites early in the animal's phase of increased locomotor activity; behavioural arousal does not, however, appear to affect this expression. We believe this to be the first study demonstrating that the expression of prion protein messenger RNA can change over a relatively short period in vivo. The results are discussed with reference to the range of recently discovered "clock-related" transcripts which also have widespread tissue expression; these include the messenger RNAs for D-box binding protein and thyroid embryonic factor, transcription factors which bind to the prion protein promoter. [These have never been shown to bind prion promoter before and do not show up with online promoter analysis -- webmaster]

Prion protein: a role in sleep regulation?

J Sleep Res 1999 Jun;8 Suppl 1:30-6
Huber R, Deboer T, Tobler I
Since mutations in this protein lead to severe neurodegeneration and death in humans and animals, it is possible that the loss of its normal function contributes to the development of the pathology. Little is known about its normal function, but there are indications that it may play a role in circadian rhythm and sleep regulation in mice.

We explored further whether PrP plays a role in sleep regulation by comparing sleep and the effects of 6 h sleep deprivation in PrP knockout mice and isogenic wild-type mice of the 129/Ola strain. The mice did not differ in the amount and distribution of the vigilance states or in the power spectra.

The most remarkable difference was the larger and long-lasting increase of slow-wave activity (mean EEG power density 0.75-4.0 Hz) in non-rapid-eye-movement (NREM) sleep during recovery from sleep deprivation in the null mice. The results confirm our previous findings in mice with a mixed background. This observation applies also to slow-wave activity in NREM sleep episodes following spontaneous waking bouts of different duration. Sleep fragmentation in both genotypes was larger than in mice with the mixed background. A new aspect was revealed by the spectral analysis of the EEG, where the null mice had a lower peak frequency within the theta band in REM sleep and waking, and not in NREM sleep.

Behavioural observations concomitant with the EEG indicated that the EEG difference in waking may be attributed to the smaller amount of exploratory behaviour in the null mice. The difference between the genotypes in theta peak frequency was not an overall effect on the EEG, since it was absent in NREM sleep. PrP therefore may be affecting the theta-generating mechanisms in the hippocampus during waking and REM sleep. It remains unresolved whether PrP plays a role in sleep consolidation, nevertheless the data suggest that it is involved in sleep regulation. A passive avoidance test showed a difference between the genotypes. It is not probable that this was due to memory differences, since the genotypes reacted similarly in a delayed T-maze alternation procedure. The behavioural differences need to be pursued further.

Selective oxidation of prion methionine residues

Biochem Biophys Res Commun 1999 Jun 7;259(2):352-355
Wong BS, Wang H, Brown DR, Jones IM
Prion proteins are central to the pathogenesis of several neurodegenerative diseases through the postulated conversion of the endogenous cellular isoform (PrPc) into a pathogenic isoform (PrPSc). Although the cellular function of normal prion protein remains unresolved a number of studies have shown that prion proteins may be involved in the cellular response to oxidative stress. Here, using purified recombinant sources of mouse and chicken PrP refolded in the presence of copper (II) we show that the methionine residues of the protein are uniquely susceptible to oxidation. We suggest that Met residues may form an essential part of the mechanism of the antioxidant activity exhibited by normal prion protein.

Comment (webmaster): Methionine sulfoxide formation is no surprise here, given copper activation of molecular oxygen. Stadtman recently reviewed the role of methionine in proteins generally, concluding that its role is to adsorb oxidants and protect active sites, never being catalytically active itself (though it is commonly structurally important). Surface exposed methionine residues create an extremely high concentration of reactant, providing for efficient scavenging of oxidants. The methionine can be repaired by methionine sulfoxide reductase (MsrA, EC 1.8.4.6), providing a catalytic amplification of the antioxidant potential of each methionine residue. In some enzymes, susceptible residues are physically arranged in an array guarding the entrance to the active site.

Could prion protein function as a methionine sulfoxide reductase? The only sequenced mammalian MSR, from bovine, has 61% identity with the E. coli enzyme in a 199-amino acid overlap and no homology to prion sequence. In mouse, the gene maps to chromosome 14, in synteny with human 13 and 8p21. Conceivably, there could be separate families of Msr, analogously to superoxide dismutase. The normal source of reductant is thioredoxin (in conjunction with NADPH and thioredoxin reductase). It is not know constitutes the active site of the enzyme nor whether a metal might be involved; disulphides might be involved.

>sp P54149 bovine peptide methionine sulfoxide reductase 
MLSVTRRALQLFHSLFPIPRMGDSAAKIVSPQEALPGRKEPLVVAAKHHV
NGNRTVEPFPEGTQMAVFGMGCFWGAERKFWTLKGVYSTQVGFAGGYTPN
PTYKEVCSGKTGHAEVVRVVFQPEHISFEELLKVFWENHDPTQGMRQGND
HGSQYRSAIYPTSAEHVGAALKSKEDYQKVLSEHGFGLITTDIREGQTFY
YAEDYHQQYLSKDPDGYCGLGGTGVSCPLGIKK

If prion protein has an oxidative protective role for its normal function, as intimated in this paper, it itself might be exposed to severe oxidative stress. Thus methionine residues in prion protein could be oxidized accidently and be subject to MSR restoration. This could explain its reported 6 hour turnover and recycling to the surface. Additionally, surface methionines could point to an active site. Mature prion protein has 9 methionines, including 4 sandwiching AGAAAAGAVVGGLGG. None of these residues is suitable for a catalytic role.

However, this paper may be another case of lemonade being made out of a lemon: a laudable effort to reconstitute apoprotein with copper ran afoul of an unwanted but not unexpected side reaction. The method of reconstitution does not mimic in vivo assembly of copper holoenzymes which often involve supplementary copper chaperones. The paper does not identify which specific methionines are oxidized, only bulk methionine.

The full 3D structure is not known so it is not possible to reliably identify surface methionines, because current surface might be masked once the repeat region is correctly positioned. To study holoprotein, it may be better to cleave off the GPI anchor and release native protein from a cell culture, as techniques for working with very small quantities of material are advancing rapidly. However, should prion protein prove to be an enzyme, an assay based on that activity would allow validation of holoenzyme formation from recombinant E. coli protein.

Analysis of 27 mammalian and 9 avian PrPs reveals high conservation of flexible regions of the prion protein.

J Mol Biol 1999 Jun 25;289(5):1163-1178
Wopfner F, Weidenhofer G, Schneider R, von Brunn A, Gilch S, Schwarz TF, Werner T, Schatzl HM
Prion diseases are fatal neurodegenerative disorders in man and animal associated with conformational conversion of a cellular prion protein (PrPc) into the pathologic isoform (PrPSc). The function of PrPc and the tertiary structure of PrPScare unclear. Various data indicate which parts of PrP might control the species barrier in prion diseases and the binding of putative factors to PrP. To elucidate these features, we analyzed the evolutionary conservation of the prion protein.

Here, we add the primary PrP structures of 20 ungulates, three rodents, three carnivores, one maritime mammal, and nine birds. Within mammals and birds we found a high level of amino acid sequence identity, whereas between birds and mammals the overall homology was low. Various structural elements were conserved between mammals and birds.

Using the CONRAD space-scale alignment, which predicts conserved and variable blocks, we observed similar patterns in avian and mammalian PrPs, although 130 million years of separate evolution lie in between. Our data support the suggestion that the repeat elements might have expanded differently within the various classes of vertebrates. Of note is the N-terminal part of PrP (amino acid residues 23-90), which harbors insertions and deletions, whereas in the C-terminal portion (91-231) mainly point mutations are found.

Strikingly, we found a high level of conservation of sequences that are not part of the structured segment 121-231 of PrPc and of the structural elements therein, e.g. the N-terminal region from amino acid residue 23-90 and the regions located upstream of alpha-helices 1 and 3.

Comment (webmaster):
These sequences represent a very important advance and place the prion gene near the top of the list of nuclear vertebrate genes with the most orthologues studied. Schatzl and coworkers account for 52 species whose prion sequences are posted at GenBank. There are many dividends from this data. The authors observe that if the N terminal domain were simply an unstructured random coil, its observed sequence conservation is paradoxical. It is reasonable to take the conservation of a given residue as a measure of its importance especially when so many sequences are in hand. The data simply does not fit the nmr stucture of (copperless) prion protein, suggesting the latter result is an apo-protein artefact.

They indicate fish and reptile prions might be sequenced shortly. This would be extremely valuable.

It is unfortunate that many of the sequences are incomplete in the coding region. This was a compromise between coverage of the mature protein in as many species as possible and effort/expense of sequencing species with varying problems. Some species, like ostrich and felids, simply did not respond to primers, even though it is doubtful that the sequences depart much from close relatives.

The bird sequences are not posted as of 17 July 99 despite the 25 Jun 99 print publication date, a 6 Jan 99 submission date, and explicit journal policies. The sequences in fact were largely completed in July 1997; while the authors have been generous in sharing sequences with other researchers who knew to put in requests, the international norm is to post to GenBank upon completion of sequencing. AF113937-45 (nine species) and AF117309-29 (21 species) only cover mammalian entries -- the journal and reviewers could see that there was no plan to post avian sequences.

The authors are very far from their strengths in discussing protein molecular evolution:

For example, they report rabbit and dolphin as sister taxa, saying "dolphin Prp did not reveal an unexpected clustering." Fossil and molecular evidence established years ago that dolphins and whales are modified artiodactyls, with hippopotamus as closest land relative. Rabbits branch off near the primate-rodent node. The correct topology for all species with sequenced prions was posted on this site in mid 1997.

Okada has identified SINEs common to whales and hippos but not present in the cud-chewing ruminants as well as other SINEs restricted to whales and artiodactyls, concluding whales and hippos split from the ruminants some 55 million years ago. (The results are in press at PNAS.) Conrad Matthee used 8200 nucleotide bases from eight genes in two whale and 24 ungulate species and reached the same conclusion (also in press, 1999).

Repeat regions in birds and mammals are obviously not homologous and should never be aligned for amino acid difference counts. More subtly, internal repeats are not homologous either because of contraction and expansion cycles. All this has been noted many times before here and in other tandem repeat proteins.

Bird sequences align conservatively in accord with the topology of the avian species tree. There is no support for convergent evolution as suggested by the authors (no data shown).

The authors find a poor correlation of residue conservation with secondary structure. This is no surprise: while higher order structure in proteins is indeed extremely conserved, it does not arise from conservation over time of individual amino acids making up alpha helices and beta sheets. Often individual residues can and do evolve rapidly. It is also important to realize that tertiary structure is far more conserved (much deeper) than secondary structure.

The prion literature is seriously confused over the relation of secondary structure in normal and rogue prion -- the cross beta in the latter arises from 106-126, not pre-existing helix or sheet which are basically irrelevent. (Recall that Y145- lacks all alpha helices and causes CJD; the repeat region is generally cleaved and so superfluous.)

Bird prion does not align at all well with mammalian in certain of the 5 secondary structure stretches. Within the globular domain of mammalian prion, homological identity does not exceed 45% or so, manually aligning reconstructed ancestral avian and mammalian prions. However, the two proteins have exactly the same micro-domain structure and are surely homologous.

Given that tertiary structure of proteins is extremely invariant, should not secondary structure in bird prion match that of mammal? The authors investigated this with secondary structure prediction algorthms (not described) and could not find a significant alignment with mammal helix and sheet. For inexplicable reasons, they have difficulty seeing that avian and mammal beta sheet stretches are in superb agreement given a 310 million year separation: YMLG to YAMG and VYYR to VYYR. Thus there is no real issue with conserved beta structure and this, along with the disulphide anchor, immediately constrains tertiary models of avian prion that might be built by energy threading or SwissPDBviewer.

Exceptions to YMLG in mammals:
-- YLLG elk allele
-- YVLG human allele

Exceptions to YAMG in birds:
-- YVMG ostrich
-- YAIG albatross

Exceptions to VYYR in mammals:
-- VYMR marsupial
-- VYYK camel, lama

Exceptions to VYYR in birds:
-- none (8 spp, 10 alleles)

There are a great many web tools that predict secondary structure, many sophisticated and well-tested on a great mass of crystalographic data. Some algorithms can exploit the situation where aligned sequences are available from many species, because only certain substitutions are appropriate assuming secondary structure is preserved, eg, proline does not belong inside alpha helices. The beta sheet is not likely to be predicted because it is so short and has unusual angles; the focus must be on alpha helix. The best strategy for predicting avian prion secondary structure consists of:

-- submitting only the globular domain of mature prion (the repeat region, signal, or post-GPI stretches can only confuse the software),

-- submitting a noise-reduced ancestral sequence to software wanting only one sequence; submitting all available aligned bird sequences (plus variable numbers of ancestral sequence) to software that can make use of alignments,

-- submitting avian sequences to all available prediction programs to assess the robustness of outcomes and define high probability consensus secondary structure, form a weighted 'average' favoring sophisticated well-maintained programs,

-- determining avian helix prior to mammalian comparison, software can only be confused by inclusion of non-homologuous mixed alignments,

-- recognizing that tertiary conservation is the driving force in alignment, not alpha helix alignment, and that the beta sheet, disulphides, and invariant residues severely constrain the possibilities for avian structure (glycosylation sites are of limited use as external hydrophilic substituents, birds have 3 sites of which only the first and third align with mammal, as can be seen by 3D cross-tie placement).

At the outset, one observes that mammal and bird align quite conservatively in the region spanning the beta strands (indeed out to the first cysteine and glycosylation site) indicating strongly not only that 9 residues of helix 1 and 6 residues of helix 2 will be found in birds but that this piece of tertiary structure will be essentially superimposable.

                         bbbb            aaaaaaaaaaa      bbbb                 aaaaaaaaaaaaaaaaaa          aaaaaaaaaaaaaaaaaaaaaaaaaaa
mammal KHVAGAAAAGAVVGGLGGYMLGSAMSRPLIHFGNDYEDRYYRENMYRYPNQVYYRPVDQYSS---QNNFVHDCVNITVKQHTVTTTTKGE""""""""NFTETDIKIME-RVV-----EQMCITQYQKEYEA
bird   KHVAGAAAAGAVVGGLGGYamG;;MS;;;;HF;;;;;;Rww;EN;;RYPNqVYYR--D-Yss"""Qd-FV-DCfNITV;;;;;;;;;;;;--------N.TEte---mE;kVV"""""E-MCIqQY-rEY--
The structure of bird prion is largely determined by invariant residues shown above (76/132 = 58%), which can be assumed to occupy the same coordinates as in mammal, and thus serve as cross-ties severely constraining more variable stretches. Noteworthy is that bird and mammal have the same number of residues between the beta strands and nearly the same between cysteines (41 vs 42) despite poor matching and gaps -- this allows for the 3D structures to match at cross-ties. The cross-stacked consecutive tyrosines probably correspond to cross-stacked consecutive tryptophans in birds. NxT/S, the glycan attachment signal, not so much a cross-bridge as a sequence alignment anchor, an external recognition site.

Most of the overall mismatch arises in the looped out distal portion of helix 2; by the time this returns to the main fold, agreement is good again. Helix 1 also agrees poorly where looped out but well as it approaches the highly invariant 'underpass' domain RYPNQ preceding the second beta strand. Internal hydrogen bonds also serve as cross-ties when one member is conserved in bird because internal unpartnered polar residues are not acceptable.

SwissProt provides links to a dozen or more methods of secondary structure prediction:

 
KHVAGAAAAGAVVGGLGGYamGrvMSgmhyHFdspdeyRwwnENsaRYPNqVYYRDYsspvsQdvFVaDCfNITVteynigpaakknvseaapaaNqTEtemEtkVVtkvirEMCIqQYrEYrl
---HHHHHHHHEE------EE-HEH--EE---------EEHH---------EEE----------EEE-----E---EE----------HHH--------HHHHHHHHHHHHHHHHHHH------ 
--HHHHHHHHHH------HHHHHH--HH--------------H---H-HH-------------HHHHH-HHHH---HHHH-HH--HHHHHHHHH----HHHHHHHHHHHHHHHHHHHHHH---- 
------HH-HHEE-----HHHHHHHHHH----------HHHH------------------------E-----------------HH----H--------HHHHHHHHHEEHHHHHHHHHHH--- 
        hhhhhh     eeeeee eeeeeeee   hhhhhh        eeee        eeeee    eeeee     hhhhhhhhh     hhhhhhhhhhhhhhhhhhhhh eeeee 
   ehhh hhhhh      hhhhhhh   e                     eeee         eeeh   eeeeee                     hhhhhhhhhhhhhhhhhhhhhhhh 
hhhhhhhhhhheee   heehhhhhh  eee                    eeee          ee     eee   eeee     hhhhhhhh     hhhhhhhhhhhhhhh      hhh 
KHVAGAAAAGAVVGGLGGYamGrvMSgmhyHFdspdeyRwwnENsaRYPNqVYYRDYsspvsQdvFVaDCfNITVteynigpaakknvseaapaaNqTEtemEtkVVtkvirEMCIqQYrEYrl
     HHHHHHHHHHHHHHHHHHHH                          EEE           EEEEEEEEEEEEE                        HHHHHHHHHHHHHHHHHH    
9852034553765432256641541375346899886322025531378730312478888788615441689986502124432330001233577632330667999999988752302379
   HHHHHHHHHHH HHHHHHHHHH   EEEE   HHHHHHHH        EEEE          EEE EEEEEEEEE          HHHHHH    HHHHHHHHHHHHHHHHHHHHHHH   

It may be seen from this that all methods agree that avian prion has a long alpha helix exactly where helix 3 occurs in mammals. Helix 1 is weaker but receives some support distally. Helix 2 is rarely predicted except distally, most programs see upstream areas as beta. At this point, the 3D structure of avian prion could be approximated within SwissPDBviewer by beginning with mouse prion and making point mutations, deletions, and insertions as needed while holding constraints fixed, then applying global energy minimization at the end.

A mouse prion protein transgene rescues purkinje cell degeneration and demyelination.

Lab Invest 1999 Jun;79(6):689-97
Nishida N, Tremblay P, Sugimoto T,...Torchia M, Sakaguchi S, DeArmond SJ, Prusiner SB, Katamine S
Disruption of both alleles of the prion protein gene, Prnp, renders mice resistant to prions; in a Prnp o/o line reported by some of us, mice progressively developed ataxia and Purkinje cell loss. Here we report torpedo-like axonal swellings associated with residual Purkinje cells in Prnp o/o mice, and we demonstrate abnormal myelination in the spinal cord and peripheral nerves in mice from two independently established Prnp o/o lines. Mice were successfully rescued from both demyelination and Purkinje cell degeneration by introduction of a transgene encoding wild-type mouse cellular prion protein. These findings suggest that cellular prion protein expression may be necessary to maintain the integrity of the nervous system.

Comment (webmaster): The effect of knocking out both copies of the prion gene, in some strains of mice, has some subtle effects. Here, such strain is revisited more convincingly, both in terms of pathology and specificity (rescueability). However, no real insight is gained into the normal function per se or how this translates into the observed effects. For example, if the normal role were to reclaim extra-cellular methinine sulfoxides formed by unwanted oxidative damage, then this would be only indirectly and weakly connected to Purkinje cells and the like, and not a neural or CNS-specific activity per se.

Vaccine for Alzheimer and CJD?

7 Jul 1999 By PETER SVENSSON The Associated Press
Raising hopes of someday preventing Alzheimer's, scientists have developed a vaccine that in mice appears to ward off and even reduce the brain-clogging deposits that are characteristic of the disease. It is uncertain whether the treatment will work in humans, but the San Francisco pharmaceutical company behind the research wants to test it on people soon.

Deposits in the brain of a sticky protein called amyloid are one of the characteristics of Alzheimer's. The vaccine appears to prevent the formation of these so-called plaques in mice that were genetically engineered to overproduce amyloid. Dr. Zaven Khachaturian, senior medical adviser to the Alzheimer's Association, called the study a landmark.

``If one can repeat the same phenomenon in humans, it will have a very important impact,'' he said. Even if it fails to produce a treatment, it introduces the idea of using a vaccination against protein deposits, which are associated with a variety of diseases, he said. There is no known cure for Alzheimer's, which is believed to affect more than 4 million Americans. The patients, most of them older than 60, progress from forgetfulness to dementia and usually die five to 10 years after diagnosis.

In the study, published in Thursday's issue of the journal Nature, a team of researchers led by Dale Schenk at Elan Corp. tried to trick the immune system of the mice to recognize amyloid as a foreign substance that should be attacked. The researchers injected nine 6-week-old mice with amyloid combined with substances that excite the immune system. Seventeen other mice of the same age did not get the vaccine. When the mouse brains were dissected after a year, the researchers were surprised to find no or very small plaques in the injected mice, while the unvaccinated mice had extensive deposits.

The researchers then tried a more ambitious experiment: injecting the vaccine into 24 year-old mice that already had plaques. Twenty-four similar mice did not get the vaccine. ``We saw that it completely stopped the further progression of the disease,'' Schenk said. ``It looks like it might have actually diminished the plaques.''

Elan wants to start trials with people later this year. Schenk said that the researchers hope to submit a vaccine to the Food and Drug Administration for approval in five years. But there are a number of reasons the method may not prevent or halt Alzheimer's in humans. Most important, the amyloid plaques may be a symptom of the disease, rather than the cause. Also, Alzheimer's patients have other changes in the brain that the mice do not fully exhibit, such as tangles of tau protein inside nerve cells, according to Dr. Blas Frangione, head of the Alzheimer's research unit at the New York University School of Medicine.

If the vaccine does work, scientists will face another challenge: determining who needs it. While detectable genetic flaws cause some Alzheimer's cases, most patients have no such telltale markers. ``If we are going to have the maximal effect of this, we need to find out who has the disease, who is at risk, much earlier,'' Khachaturian said.

Neurobiologist Peter St. George-Hyslop at the University of Toronto said the study raises the prospect of using immunization to treat or prevent other diseases associated with protein deposits. Creutzfeldt-Jakob disease, the fatal human brain disorder thought to be related to mad cow disease, would be a candidate. So would Parkinson's disease and myeloma, a form of cancer that overproduces protein in bone marrow.

Comment (webmaster): While this is a remarkable paper, its methods are not, so it is even more remarkable that this wasn't tried 10-15 years ago. Abeta 1-42 and serum amyloid P were used here as immunogens. Tau paired helical filaments are not addressed at this point.

With prion protein, it seems like more of a gamble to inject purified seed crystal. These antigens would hopefully not lead to autoimmune disease and knockout normal function as (in theory) they would not induce antibodies directed to normal protein but be more like a polyclonal Prionics, ie, be directed specifically to rogue without cross reaction to normal.

"Methods: For immunizations, 2 mg A42 (human A1-42; US Peptides) was added to 0.9 ml deionized water and the mixture was vortexed to generate a relatively uniform suspension. A 100-l aliquot of 0.15 M NaCl, 0.01 M sodium phosphate, pH 7.5 was added. Serum amyloid-P component immunogens were prepared using mouse SAP amino acids 77-85 and 164-173, each conjugated to sheep anti-mouse IgG.

A42 or SAP peptides (100 g antigen per injection) were emulsified 1:1 (v/v) with complete Freund's adjuvant for the first immunization, followed by a boost in incomplete Freund's adjuvant at 2 weeks, and monthly thereafter. A42 or SAP in PBS alone was injected from the fifth immunization onward. Detection used goat anti-mouse immunoglobulin conjugated to horseradish peroxidase and slow-TMB substrate. Titres were defined as the dilution yielding 50% of the maximal signal..."

"In summary, immunization with A42 greatly reduced the development of the AD-like pathology that otherwise occurs in the PDAPP mouse. Immunization preceding plaque development profoundly affected the occurrence of new lesions, as the progression of beta-amyloidosis and associated neuropathology was essentially wholly blocked, as seen both in the entire brain of the young animals and in at-risk brain regions of the older animals. Amyloid-immunization also significantly retarded the progression of existing pathology in affected regions of the older animals. Outcomes of A-plaque burden, neuritic dystrophy and gliosis were all significantly improved by A42 treatment in both young and old animals."

" In addition, the mechanism resulting in plaque reduction did not seem to produce any obvious signs of damage to the neuropil of A42-immunized animals. Histological examination of several organs, including brain and kidney, revealed no signs of immune-mediated complications, despite the high levels of human APP expressed in these tissues and the significant antibody titre to endogenous mouse A peptide (data not shown). "

"To our knowledge, this is the first report of a clinically relevant treatment that reduces the progression of AD-like neuropathology in a transgenic animal model of the disease. Although it remains unproven, it is not unreasonable to expect that a similar reduction of neuropathology in AD patients would be of clinical benefit. ...We have shown that A42 immunization results in the generation of anti-A antibodies and that A-immunoreactive monocytic/microglial cells appear in the regions of remaining plaques. Thus, one possible mechanism of action is that anti-A antibodies facilitate clearance of amyloid- either before deposition, or after plaque formation, by triggering monocytic/microglial cells to clear amyloid- using signals mediated by Fc receptors. "

Potential limitations to this approach

Nature 400, 116 - 117 (1999)  8 July 1999 News and Views commentary
Peter H. St George-Hyslop And David A. Westaway 
"People suffering from Alzheimer's disease develop a progressive dementia in adulthood, accompanied by three main structural changes in the brain: diffuse loss of neurons in the hippocampus and neocortex; accumulation of intracellular protein deposits termed neurofibrillary tangles; and accumulation of extracellular protein deposits termed amyloid or senile plaques, surrounded by misshapen nerve terminals (dystrophic neurites).... Although Alzheimer's disease can be treated, we can currently neither prevent nor cure it. Schenk et al. show that, in a mouse model of Alzheimer's disease, immunization with A inhibits the formation of amyloid plaques and the associated dystrophic neurites. "

"These results raise the possibility of vaccination with A against human Alzheimer's disease. But before this can be seriously entertained, several questions must be answered. Schenk and colleagues found that high levels of anti-human A antibody were necessary for the effect to be seen in mice. So, can injection with human A induce enough of the antibody? Will immune tolerance (whereby the immune system does not react against the body's own proteins) frustrate this, as it has often done with attempts to target cancers with antibodies? Conversely, is it safe to immunize people with high levels of a protein that is widely expressed outside the protective confines of the blood-brain barrier? ...They did not see any autoimmune responses. But then, the human Abeta antigen induced much lower levels of antibodies to endogenous mouse A or APP."

"The most critical question is whether depletion of the amyloid plaques is accompanied by an improvement in the behavioural/neurophysiological impairments, and a reduction in the nerve cell death of Alzheimer's disease? In other words, does immunization with A simply clear a neuropathological by-product or can it cure the disease?"

"This question may be difficult to answer because all of the current animal models (based on overexpression of human APP and/or presenilin-1 transgenes bearing missense mutations associated with Alzheimer's disease) provide only a partial model of the human condition. So, although these animals accumulate increased levels of A in the brain and have many amyloid-plaque deposits, they have only subtle behavioural and electrophysiological deficits. More problematically, these animals do not develop neurofibrillary tangles or show significant neurodegeneration."

"A second set of questions concerns the mechanism by which immunization with A blocks the formation of amyloid plaques. Antibodies against A might act as an artificial chaperone for extracellular A, possibly by binding to A and preventing it from aggregating or from changing into -pleated-sheet conformation. Alternatively, these antibodies could accelerate clearance of A from the central nervous system through one of several peripheral mechanisms (targeting A for destruction by the peripheral reticuloendothelial system, for example, or reducing the production of A in the periphery). Finally, A might affect immune modulation of inflammatory mechanisms that are thought to be activated in Alzheimer's disease."

"...There has long been a controversy as to whether extracellular deposition of fibrillar A in amyloid plaques is part of the biochemical processes that cause the neuronal dysfunction and death, or whether it is merely a by-product of this process. "

"One school of thought (the 'amyloid cascade' hypothesis) maintains that the processes that cause accumulation of A are central to the pathogenesis of Alzheimer's disease)....Opponents of this hypothesis argue that although deposition of extracellular A is an almost invariant event in the pathogenesis of Alzheimer's disease, it need not be the prime cause of the dementia.... " [It seems implausible that 22 unrelated genes forming amyloid could result in disease without the amyloid having a central pathogenic role. -- webmaster]

"The ability to modulate deposition of extracellular A in the central nervous system while monitoring behavioural and electrophysiological changes may now allow us to determine whether extracellular A deposits are the real villain in Alzheimer's disease. Moreover, if immunization with A is indeed useful against Alzheimer's disease, similar strategies might be applied in the other diseases where extracellular plaques are formed. These might include some forms of prion and transthyretin disease. This knowledge could even be useful in treating conditions where intracellular deposits are formed, such as Parkinson's disease (alpha-synuclein) and frontotemporal dementia (tau)." [How would IgG access intra-cellular substrates? And FTDP-17 is not an amyloid disease to begin with. -- webmaster]

Commentary in 8 Jul99 Science by Marcia Barinaga

Schenk's team injected the mutant mice with Ab at a young age, before plaque formation had begun, and found that those mice never developed plaque or neuron damage. When they immunized older mice that already had plaque in their brains, the plaque--and the signs of disease--largely went away. In the brains of these mice the team found evidence of an immune response: bits of remaining amyloid that were dotted with antibodies, and microglia, the scavenger immune cells of the brain, chock-full of amyloid protein they had cleared away.

The presence of antibodies on the remaining plaque means that the antibodies successfully crossed the blood-brain barrier, says neurologist Lawrence Steinman, who studies immune-brain interactions and amyloid at Stanford University Medical Center. Once there, he says, it's easy to see how they could block amyloid molecules from sticking together in plaques.

"If the amyloid protein is bound to an antibody, there is no way it can form these aggregations," he says. What's more, Sisodia notes that recent studies in mice showed that when amyloid deposition is halted by killing neurons that secrete Ab, existing deposits diminish over time. "The idea that you can ... get rid of [amyloid] is not inconceivable," he says. Researchers agree they'd like to see the immunization results repeated. They may not have long to wait, as at least one other group is rumored to have similar results.

But will the approach work in humans? Mice aren't a perfect mirror of human physiology, Steinman notes. In particular, he worries whether in humans "there is enough of a breach of the blood-brain barrier to allow this to happen." And St. George-Hyslop cautions that the protein precursor to Ab is found in many cell types, so immunization might induce a harmful autoimmune response in nonbrain tissues.

Allaying concerns about autoimmune reactions may require further animal testing. But by the end of the year, Elan hopes to start clinical trials of the therapy on Alzheimer's patients. Those trials could yield a verdict not only on this therapeutic approach but also on the importance of plaque in Alzheimer's disease. "The bottom line of this all," says St. George-Hyslop, is that "we will know quite clearly what the true role of extracellular Ab is in Alzheimer's disease. We will either get a brilliant treatment, or we will get some powerful insights that modify how we think about the disease."

Scrapie found throughout GI tract

 Wed, 23 Jun 1999 Medline
One very interesting aspect to me of this abstract is their observation that "little attention, however, has been paid to the gastro-intestinal tract in scrapie research." The same might be said for sporadic CJD. The Noelle Bons primate nvCJD paper is the only one I can recall where similar tissues were examined this thoroughly. But there it was not the ENS per se that was infected. What does this tell us about the role of dietary horizontal transmission in scrapie? Or is it simply infection spreading outward from the CNS of periferal neurons? t

Scrapie-associated Prion Protein in the Gastro-intestinal Tract of Sheep with Natural Scrapie.

J Comp Pathol 1999 Jul;121(1):55-63
van Keulen LJ, Schreuder BE, Vromans ME, Langeveld JP, Smits MA
The scrapie-associated prion protein (PrPSc), which is closely associated with scrapie infectivity, accumulates in the brain and lymphoid tissues of sheep with natural scrapie. The most probable portal of entry of the scrapie agent in sheep is the alimentary tract; little attention, however, has been paid to the gastro-intestinal tract in scrapie research.

In this study, we examined the presence and distribution of PrPSc within the gastro-intestinal tract of sheep with natural scrapie and scrapie-negative sheep. It was found that PrPSc accumulated in the enteric nervous system (ENS) of all scrapie-infected sheep but not in scrapie-negative sheep. The distribution of PrPSc within the ENS was then studied along the entire gastro-intestinal tract in seven scrapie-infected sheep carrying various PrP genotypes.

In sheep with the highest genetically determined susceptibility to scrapie, PrPScwas detected in the ENS from the oesophagus to the rectum. In sheep with a lower genetic susceptibility to scrapie, PrPScwas present in the ENS of the forestomachs, small intestine and large intestine but not in the oesophagus. In a scrapie-negative sheep with a PrP genotype associated with scrapie resistance, no PrPSc was seen in the ENS at any site along the gastro-intestinal tract. The presence of PrPSc within the ENS of scrapie-infected sheep indicates a possible role of the ENS in the pathogenesis of natural scrapie as a portal of entry to the central nervous system.

Prion Proteins Carrying Pathogenic Mutations Are Resistant to Phospholipase Cleavage of Their Glycolipid Anchors.

Biochemistry 1999 Jul 6;38(27):8770-8777
Narwa R, Harris DA 
Familial prion diseases are linked to mutations in the gene encoding PrP, a protein of unknown function that is attached to the plasma membrane of neurons and several other cell types by a phosphatidylinositol-containing, glycolipid anchor. We have previously found that PrP molecules carrying disease-associated mutations display several biochemical attributes of PrPSc, the pathogenic isoform of PrP, when expressed in cultured Chinese hamster ovary cells.

One of the distinctive properties of these mutant PrPs is their abnormal association with cell membranes, as revealed by their retention on the cell surface after treatment with a bacterial phospholipase that normally cleaves the glycolipid anchor. We demonstrate here that mutant PrP molecules, either expressed on intact cells or solubilized in nondenaturing detergents, are partially resistant to phospholipase cleavage. The anchor becomes fully susceptible to the enzyme when the proteins are denatured in SDS.

These results suggest that the mutant PrP conformation, state of aggregation, or association with other molecules renders the glycolipid anchor physically inaccessible to cleavage. This conclusion stands in contrast to our previous suggestion that mutant PrP molecules are poorly released from the cell surface because they possess a secondary mechanism of membrane attachment in addition to the glycolipid anchor. Since PrPSc from scrapie-infected brain and cultured cells is also inefficiently released from membranes by phospholipase, resistance to this enzyme may be a molecular marker of the scrapie state.

Survival of scrapie agent after exposure to sodium dodecyl sulphate and heat.

Vet Microbiol 1999 Jun 1;67(1):13-6 
Taylor DM, Fernie K, McConnell I, Steele PJ
Fifty mg aliquots of macerated mouse-brain infected with the 22A strain of scrapie agent were treated by exposing them without mechanical mixing to (a) distilled water for 2 h, (b) 5% sodium dodecyl sulphate (SDS) for 2 h, (c) autoclaving at 121 degrees C for 15 min in distilled water, (d) autoclaving at 121 degrees C for 15 min in 5% SDS, or (e) boiling in 5% SDS for 15 min. Prior to injection into mice, all samples were washed by a procedure that is described and was shown not to reduce infectivity titres.

Although the infectivity titre of the sample that was autoclaved in SDS was reduced considerably, infectivity was present in all of the samples exposed to cold or hot SDS.

Comment (webmaster):
Boiling and autoclaving in SDS are fairly extreme processes for a protein to survive (or recover from). The abstract does not state that disulphide reducing agent was present. Since there is an intramolecular disulphide in prion protein, that would have greatly hindered renaturation after return to normal conditions. On the other hand, prion intermolecular binding is non-covalent. Note that rogue prion protein displays normally for its size on SDS-PAGE, eg, MJ Schmeer's capillary electrophoresis method boils prion aggregate for 10 minutes in 1% SDS and 5% 2-mercaptoethanol. [J Chromatogr B Biomed Sci Appl 1997 Sep 12;697(1-2):223-9 ]

Reading quickly through the 234 Medline abstracts under 'boil* SDS' turns up other proteins not particularly affected by these conditions. The most interesting case concerns paired helical filaments (PHFs) in Alzheimer's disease and corticobasal degeneration (CBD), Acta Neuropathol (Berl) 1998 Nov;96(5):520-6. Recall tau filaments in CBD are wider and twisted at longer intervals than those in AD, and also display less ultrastructural stability. [J Neurochem 1999 Mar;72(3):1243-9 explains why.] SDS-insoluble filaments were prepared from PHFs by boiling in the presence of SDS and 2-mercaptoethanol and collected by sedimentation. In both disorders, the pellets contained highly aggregated and bundled filaments.

Tau filaments have an unknown structural basis quite distinct from cross beta polymers and have never been tested for infectivity; note tauopathies are also autosomal dominant and pronase resistant. Tau filaments can be dissociated into monomers if formic acid is additionally present; only 3 tandem repeats can be shielded in the core. [EMBO J 1991 Oct;10(10):2725-9 ]. Tau that cannot bind well to microtubules is 'over-produced' in the sense that it is missing its former sink.

Boiling in SDS is actually exploited in Pick's disease to quickly purify native filaments on a sucrose gradient. This would be worth trying in CJD given Taylor's results to determine more systematically what the termini are found in each variant. If normal prion monomer but not aggregate is affected, then this treatment might have value in diagnostic assays that required complete separation.

See also: Comp Biochem Physiol B Biochem Mol Biol 1998 Oct;121(2):153-60 lectin
Mol Microbiol 1998 Jul;29(1):139-50 cell wall porin

Obstacles to BSE testing

Thu, 8 Jul 1999 Markus Moser 
[The webmaster had commented adversely on 25 June 99, writing:
"I don't like the sound of this. Here the EU has validated 3 lab tests for detecting BSE but they have made no effort to determine how well the tests work in preclinical cows and they have no intent of deploying them anytime soon in random testing even though the tests are inexpensive.

In other words, testing now might disrupt marketplaces by finding quite a few preclinical animals; better to wait a few years until these have been consumed, hold off on testing until the disease has (hopefully) played itself out."

]

Markus Moser of Prionics responds:


"I basically agree with your concerns; however, the most efficient evaluation of tests with preclinical BSE cows would only be doable with (lots) of material from a pathogenesis study including material from cattle which were infected with low doses and high doses of BSE and sacrificed at various time points during incubation. Such material, however, is not available at the moment, nor will it be in large quantities in the future."

"So what's the next best thing? I'm afraid the next best thing is an extremely tedious study: You have to screen thousends of cattle for subclinical animals with both your test and a reference method, and compare the results. We have done so in our validation with the Swiss authorities. The reference methods were histological examination and immunohistochemistry. "

"We have shown that with our test we can detect subclinical BSE in cattle thet looked hisologically normal but were positive by immunohistochemistry (to our knowledge this has only been shown for our test so far). These results were already discussed on this list, and will be published shortly in Acta Neuropathologica (We prefer to publish peer reviewed articles rather than just releasing reports or non-reviewed communications, the disadvantage of course being that this process takes time)."

" Of course, it was an immense financial effort to carry out these tests, considering that the whole procedure took over one year with several months of actual working time. The tests for the EU-evaluation, for comparison, were completed in one week and represented less than half of the tests carried out in total in our lab during that week. "

"Switzerland has now set up a surveillance program for BSE using the Prionics test with an anticipated number of approximately 13,000-14,000 tests carried out this year. The results of the first few months can be seen in the Swiss BSE statistics published on the website of the Swiss Veterinary Office (Statistics on animal diseases). While only 14 cases of BSE were reported in Switzerland in 1998; in 1999, 22 cases were already reported until the beginning of June."

"What other methods are there to address subclinical BSE? The EU group prepared a dilution series of homogenates as an alternative approach. However, when testing these samples with our test, in all the samples the normal PrP was incompletely digested, so that the results were not really conclusive. This probably reflects the fact that we are using homogenates directly for testing without intermediate steps. For such a protocol to work it is obviously essential to follow exactly the sample-preparation / homogenization procedure optimized for our test. "

"In the case of a suboptimal result as above, it is also possible to follow a protease-incubation calibration procedure outlined in our test-protocol to tune the system for the respective source material. However, as Prionics was not allowed to repeat tests, we had to leave it with the one shot."

"Since the Western Blot identifies positive probes based on both presence of signal and size-shift of full-size PrP to the protease resistant PrP fragment, we were still able to identify most of the lower dilutions, based on the fact that the signal originating from the 27-30kd bands were much stronger than the signal from above 30kd."

"We were a bit surprised to learn from the EC-report on the BSE test results that the other candidates were obviously allowed to repeat tests, while Prionics was not. In fact, the 100% sensitivity and specificity of two other tests in identifying clinical BSE was only reached by repeating tests."

"We still think that the exercise of testing 1,400 samples from animals with clinical symptoms is a valuable exercise to document the reproducibility and reliability of diagnostic tests. It provided an opportunity for us to prove the reliability of our test to officials who have received the results of our Swiss Validation but who have chosen to ignore them. The Prionics test was the only test in this exercise to reach a 100% sensitivity and 100% specificity. Two other tests have also correctly identified all probes after partial repeated testing (which, however, in the strict scientific sense cannot be interpreted as 100% sensitivity/specificity)."

In addition to testing freshly frozen tissue of good quality, it would also be necessary to include partly autolyzed tissue in an evaluation exercise (as done in the Swiss validation), to mimick bad probe quality, which is often seen in probes from fallen stock. As fallen stock has a higher incidence of BSE than the general cattle population, it is one of the target populations for active surveillance programs."

"The EC evaluation report is published on the EC website."

Tests for BSE evaluated

Correspondence section : Nature 400, 105 8 July 1999 Four rapid tests for the diagnosis of bovine spongiform encephalopathy (BSE) in bovines were evaluated for the EC . Of ten tests submitted, four were accepted for evaluation on grounds such as their ability to be scaled up quickly for rapid evaluation on the timescale required. Tests A and D take less than 24 hours to perform, whereas tests B and C take 8 and 4 hours, respectively, and also have the highest throughput. The tests were:

Test A (E. G. & G. Wallac): a two-site non-competitive immunometric procedure using two different monoclonal antibodies. DELFIA technology is used to generate the reading signal.

Test B (Prionics): an immunoblotting test based on a western blotting procedure for the detection of the protease-resistant fragment PrPSc using a monoclonal antibody.

Test C (Enfer): a chemiluminescent ELISA, using a polyclonal anti-PrP antibody for detection.

Test D (CEA): a sandwich immunoassay for PrPSc carried out after denaturation and concentration steps. Two monoclonal antibodies are used.

We evaluated sensitivity and specificity in relation to samples from true positive and true negative animals. We obtained positive samples from cows showing clinical signs of BSE and in which the disease was confirmed by histopathological examination. Negative samples were obtained from healthy cows of similar age slaughtered in New Zealand. The tissues used in the evaluation were from the same animals but were those tissues for which each test was developed: brain stem for three tests and anterior cervical spinal cord for the fourth, with a total of 1,000 negative and 300 positive samples for each test. Samples that weighed approximately 1g each were prepared, taking precautions to avoid any cross-contamination. In addition, positive brain homogenate of known infectivity titre was tested at dilutions in negative brain of up to 10-5 to estimate the detection limits of the tests.

We carried out the evaluation under supervision at the participants' laboratories over a one-month period. Testing was done with all involved blinded, including the supervisor being unaware of the identity of the samples. We interpreted the results using a cut-off point proposed by the participants. Inconclusive categories were established in advance and it was decided that, in the case of a retest of these samples, the second result would be the valid result. Results are summarized in Tables 1 and 2.
The results indicate that tests B, C and D have excellent potential for detecting or confirming clinical BSE for diagnostic purposes or for screening dead or slaughtered animals for such cases, particularly casualty animals or carcasses sent for rendering. Even though BSE is a rare disease, the high specificity indicates that these tests may be useful for general post-mortem screening of older bovines...

Jim Moynagh, Heinz Schimmel 
Directorate General XXIV, Consumer Policy and Health Protection, 
European Commission 

Equilibrium folding properties of the yeast prion protein determinant Ure2.

J Mol Biol 1999 Jul 2;290(1):331-345 
Perrett S, Freeman SJ, Butler PJ, Fersht AR
The yeast non-Mendelian factor [URE3] propagates by a prion-like mechanism, involving aggregation of the chromosomally encoded protein Ure2. The [URE3] phenotype is equivalent to loss of function of Ure2, a protein involved in regulation of nitrogen metabolism. The prion-like behaviour of Ure2 in vivo is dependent on the first 65 amino acid residues of its N-terminal region which contains a highly repetitive sequence rich in asparagine. This region has been termed the prion-determining domain (PrD).

Removal of as little as residues 2-20 of the protein is sufficient to prevent occurrence of the [URE3] phenotype. Removal of the PrD does not affect the regulatory activity of Ure2. The C-terminal portion of the protein has homology to glutathione S -transferases, which are dimeric proteins. We have produced the Ure2 protein to high yield in Escherichia coli from a synthetic gene. The recombinant purified protein is shown to be a dimer. The stability, folding and oligomeric state of Ure2 and a series of N-terminally truncated or deleted variants were studied and compared. The stability of Ure2, DeltaGD-N, H2O, determined by chemical denaturation and monitored by fluorescence, is 12.1(+/-0.4) kcal mol-1at 25 degrees C and pH 8.4.

A range of structural probes show a single, coincident unfolding transition, which is invariant over a 550-fold change in protein concentration. The stability is the same within error for Ure2 variants lacking all or part of the prion-determining domain. The data indicate that in the folded protein the PrD is in an unstructured conformation and does not form specific intra- or intermolecular interactions at micromolar protein concentrations. This suggests that the C-terminal domain may stabilise the PrD against prion formation by steric means, and implies that the PrD does not induce prion formation by altering the thermodynamic stability of the folded protein.

Apoptotic cells found inCJD

Bull Acad Natl Med 1999;183(2):305-20; discussion 320-1 [Article in French]
Gray F, Adle-Biassette H, Chretien F, Ereau T, Delisle MB, Vital C
Neuronal loss is a salient feature of prion diseases; however, its causes and mechanisms are unclear. The possibility that it could occur through an apoptotic process has been postulated and this is consistent with the lack of inflammation in prion disorders as supported by experimental studies. In order to test this hypothesis in humans, we examined samples of frontal and temporal cerebral cortex, striatum, thalamus and cerebellum from 26 patients who died from prion diseases.

They included 16 cases of Creutzfeldt-Jakob disease (5 sporadic cases, 5 familial, 3 iatrogenic, and 3 cases with the new variant), and 10 cases of fatal familial insomnia including 8 homozygotes methionine/methionone at codon 129 of the prion protein gene and 2 heterozygotes. These were compared with age and sex matched controls.

Using in situ end labelling, we identified apoptotic neurons in all the cases of Creutzfeldt-Jakob disease. A single labelled neuron was found in the eldest control. Apoptotic neurons were mostly found in damaged regions and their presence and abundance seemed to correlate closely with neuronal loss. This supports the view that apoptosis of neurons is a feature of prion diseases and may contribute to the neuronal loss which is one of the main characteristics of these conditions.

Neuronal apoptosis also correlated well with microglial activation as demonstrated by the expression of major histocompatibility complex class II antigens and axonal damage as identified by beta-amyloid protein precursor immunostaining. In contrast, we found no obvious relationship between the topography and severity of neuronal apoptosis and the type, topography and abundance of prion protein deposits as demonstrated by immunohistochemistry.

Public Health Effort Unwittingly Spread Hepatitus C

2 July 99 Science and sidebar Jon Cohen
A virus that infects some 170 million people worldwide is causing rising rates of liver disease. For years, clinicians knew that something in the blood supply was causing a small fraction of transfusion recipients to suffer short-lived flulike symptoms followed in some cases by liver disease years later. Hepatitis C has little in common with A and B, (though all inflame the liver). HCV is a single strand of RNA and just a single transcriptional unit, coding for a polyprotein that is subsequently spliced into at least 10 functional proteins.

No compelling clues point to where or when HCV first infected humans, and no other species appears to serve as a natural host to the virus. Yet studies clearly have shown that the main routes of transmission are by tainted blood transfusions and dirty needles used by injecting drug users, practitioners of folk medicine, and even public health campaigns. Aside from direct blood contact, HCV is a very difficult to transmit. Maternal-to-fetal transmission is less than 6%. Whether HCV can be transmitted sexually is still debated. A study by NIDDK looked at 8,568 blood samples stored by the U.S. Air Force between 1948 and 1954 and found that 17 tested positive for HCV antibodies; only one infected individuals (5.8%) died from liver disease.

Treatment is expensive -- a 48-week treatment with interferon and ribavirin costs nearly $20,000--and cause debilitating flulike symptoms in most people, leading about 20% of the patients in both trials to stop treatment early. "Interferon is a difficult drug to take, and ribavirin makes it worse," says NIH. These results have sparked a sharp debate over whether to treat asymptomatic patients.

"Roughly 24% of the people in Egypt are estimated to carry hepatitis C virus (HCV), making it the hardest hit country in the world. Tibet, Bolivia, and 4 African countries are also over 20% infected. The US has 1.8% infectred. Researchers have long suspected that the culprit might be a decades-old strategy to combat a parasitic disease known as schistosomiasis. Now a study headed by Thomas Strickland, an epidemiologist at the University of Maryland, Baltimore, takes this idea from the realm of the possible to the probable. Maryland's Christina Frank, who presented the data at the Sixth International Symposium on Hepatitis C and Related Viruses for Strickland's lab and their collaborators at Ain Shams University in Cairo, Egypt, says the schisto-HCV link "may very well be the world's largest iatrogenic transmission scenario known to date."

"Schistosomiasis in Egypt dates back at least to the time of the Pharaohs. Caused by a flatworm that propagates in water snails, the disease attacks the intestines, bladder, liver, and other organs. In 1918, Frank explained, physicians began popularizing various injectable treatments, called parenteral antischistosomal therapy, or PAT, which typically required 10 to 12 injections and was usually given with reusable syringes. The campaigns began tapering off in the 1970s when oral schisto drugs became available."

"By studying schistosomiasis archives at the World Health Organization, PAT records at the Egyptian Ministry of Health, census records, and HCV infection surveys of 10,000 Egyptians, the researchers pieced together the link between the two diseases. Age and demographic data indicate that people who were most exposed to PAT had the highest HCV prevalence. "That's the first time the data convincingly show this," says Robert Purcell, a virologist at the U.S. National Institute of Allergy and Infectious Diseases, whose laboratory has played central roles in the study of every hepatitis-causing virus."

Potent Neuroprotective Properties against the Alzheimer -Amyloid by an Endogenous Melatonin-related Indole Structure, Indole-3-propionic Acid

J Biol Chem, Vol. 274, Issue 31, 21937-21942, July 30, 1999 
Yau-Jan Chyan, Burkhard Poeggeler, ...Miguel A. Pappolla 
Widespread cerebral deposition of a 40-43-amino acid peptide called the amyloid -protein (Abeta) in the form of amyloid fibrils is one of the most prominent neuropathologic features of Alzheimer's disease. Numerous studies suggest that A is toxic to neurons by free radical-mediated mechanisms. We have previously reported that melatonin prevents oxidative stress and death of neurons exposed to A. In the process of screening indole compounds for neuroprotection against A, potent neuroprotective properties were uncovered for an endogenous related species, indole-3-propionic acid (IPA). This compound has previously been identified in the plasma and cerebrospinal fluid of humans, but its functions are not known.

IPA completely protected primary neurons and neuroblastoma cells against oxidative damage and death caused by exposure to Abeta, by inhibition of superoxide dismutase, or by treatment with hydrogen peroxide. In kinetic competition experiments using free radical-trapping agents, the capacity of IPA to scavenge hydroxyl radicals exceeded that of melatonin, an indoleamine considered to be the most potent naturally occurring scavenger of free radicals. In contrast with other antioxidants, IPA was not converted to reactive intermediates with pro-oxidant activity. These findings may have therapeutic applications in a broad range of clinical situations.

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