Species-barrier-independent prion replication in apparently resistant species

Transmission of prions between mammalian species is thought to be limited by a “species barrier,” which depends on differences in the primary structure of prion proteins in the infecting inoculum and the host. Here we demonstrate that a strain of hamster prions thought to be nonpathogenic for conventional mice leads to prion replication to high levels in such mice but without causing clinical disease. Prions pathogenic in both mice and hamsters are produced. These results demonstrate the existence of subclinical forms of prion infection with important public health implications, both with respect to iatrogenic transmission from apparently healthy humans and dietary exposure to cattle and other species exposed to bovine spongiform encephalopathy prions. Current definitions of the species barrier, which have been based on clinical end-points, need to be fundamentally reassessed.

Both hypertrophic and dilated cardiomyopathies are caused by mutation of the same gene, δ-sarcoglycan, in hamster: An animal model of disrupted dystrophin-associated glycoprotein complex

Cardiomyopathy (CM) is a primary degenerative disease of myocardium and is traditionally categorized into hypertrophic and dilated CMs (HCM and DCM) according to its gross appearance. Cardiomyopathic hamster (CM hamster), a representative model of human hereditary CM, has HCM and DCM inbred sublines, both of which descend from the same ancestor. Herein we show that both HCM and DCM hamsters share a common defect in a gene for δ-sarcoglycan (δ-SG), the functional role of which is yet to be characterized. A breakpoint causing genomic deletion was found to be located at 6.1 kb 5′ upstream of the second exon of δ-SG gene, and its 5′ upstream region of more than 27.4 kb, including the authentic first exon of δ-SG gene, was deleted. This deletion included the major transcription initiation site, resulting in a deficiency of δ-SG transcripts with the consequent loss of δ-SG protein in all the CM hamsters, despite the fact that the protein coding region of δ-SG starting from the second exon was conserved in all the CM hamsters. We elucidated the molecular interaction of dystrophin-associated glycoproteins including δ-SG, by using an in vitro pull-down study and ligand overlay assay, which indicates the functional role of δ-SG in stabilizing sarcolemma. The present study not only identifies CM hamster as a valuable animal model for studying the function of δ-SG in vivo but also provides a genetic target for diagnosis and treatment of human CM.

Radioimmunotherapy with a 64Cu-labeled monoclonal antibody: a comparison with 67Cu.

67Cu (t1/2 = 62 h) has demonstrated potential as a radionuclide for radioimmunotherapy, but limited availability severely restricts its widespread use. 64Cu (t1/2 = 12.8 h) has been shown to have comparable effectiveness in vitro and in vivo. The present study was undertaken to examine the therapeutic potential of 64Cu- and 67Cu-bromoacetamidobenzyl-1,4,8,11-tetraazacyclotetradeca ne-N, N',N",N"'-tetraacetic acid (BAT)-2-iminothiolane (2IT)-1A3 (1A3 is a mouse anti-human colorectal cancer mAb) for treatment of GW39 human colon carcinoma carried in hamster thighs. Hamsters were injected with 64Cu- or 67Cu-BAT-2IT-1A3 or Cu-labeled nonspecific IgG (MOPC) or saline. Hamsters were killed 6-7 months after therapy or when tumors were > or = 10 g. Of the hamsters with small tumors (mean weight 0.43 +/- 0.25 g), 87.5% were disease-free 7 months after treatment with 2 mCi (1 Ci = 37 GBq) of 64Cu-BAT-2IT-1A3 or 0.4 MCi of 67Cu-BAT-2IT-1A3. The mean tumor doses at these activities of 64Cu- and 67Cu-BAT-2IT-1A3 were 586 and 1269 rad (1 rad = 0.01 Gy), respectively. In contrast, 76% of hamsters treated with 2 mCi of 64Cu-BAT-2IT-MOPC or 0.4 mCi of 67Cu-BAT-2IT-MOPC had to be killed before 6 months because of tumor regrowth. When hamsters with larger tumors (mean weight 0.66 +/- 0.11 g) were treated with 64Cu- or 67Cu-BAT-2IT-1A3, survival was extended compared with controls, but only one animal remained tumor-free to 6 months. These results demonstrate that 64Cu- and 67Cu-BAT-2IT-1A3 given in a single administered dose can eradicate small tumors without significant host toxicity, but additional strategies to deliver higher tumor doses will be needed for larger tumors.

Immunotargeting of antioxidant enzyme to the pulmonary endothelium.

Oxidative injury to the pulmonary endothelium has pathological significance for a spectrum of diseases. Administration of antioxidant enzymes, superoxide dismutase (SOD) and catalase (Cat), has been proposed as a method to protect endothelium. However, neither these enzymes nor their derivatives possess specific affinity to endothelium and do not accumulate in the lung. Previously we have described a monoclonal antibody to angiotensin-converting enzyme (ACE) that accumulates selectively in the lung after systemic injection in rats, hamsters, cats, monkeys, and humans. In the present work we describe a system for selective intrapulmonary delivery of CuZn-SOD and Cat conjugated with biotinylated anti-ACE antibody mAb 9B9 (b-mAb 9B9) by a streptavidin (SA)-biotin bridge. Both enzymes biotinylated with biotin ester at biotin/enzyme ratio 20 retain enzymatic activity and bind SA without loss of activity. We have constructed tri-molecular heteropolymer complexes consisting of b-mAb 9B9, SA, and biotinylated SOD or biotinylated Cat and have studied biodistribution and pulmonary uptake of these complexes in the rat after i.v. injection. Biodistribution of biotinylated enzymes was similar to that of nonmodified enzymes. Binding of SA markedly prolonged lifetime of biotinylated enzymes in the circulation. In contrast to enzymes conjugated with nonspecific IgG, other enzyme derivatives, and nonmodified enzymes, biotinylated enzymes conjugated with b-mAb 9B9 accumulated specifically in the rat lung (9% of injected SOD/g of lung tissue and 7.5% of injected Cat/g of lung tissue). Pulmonary uptake of nonmodified enzymes or derivatives with nonspecific IgG did not exceed 0.5% of injected dose/g. Both SOD and Cat conjugated with b-mAb 9B9 were retained in the rat lung for at least several hours. Trichloracetic acid-precipitable radiolabeled Cat was associated with microsomal and plasma membrane fractions of the lung tissue homogenate. Thus, modification of antioxidant enzymes with biotin and SA-mediated conjugation with b-mAb 9B9 prolongs the circulation of enzymes resulting in selective accumulation in the lung and intracellular delivery of enzymes to the pulmonary endothelium. These results provide the background for an approach to provide protection of pulmonary endothelium against oxidative insults.

Potentiation of the bioavailability of daidzin by an extract of Radix puerariae.

The dose effect of pure daidzin on the suppression of ethanol intake in Syrian golden hamsters was compared with that of crude daidzin contained in a methanol extract of Radix puerariae (RP). EC50 values estimated from the graded dose-response curves for pure daidzin and RP extract daidzin are 23 and 2.3 mg per hamster per day, respectively. Apparently the antidipsotropic activity of the RP extract cannot be accounted for solely by its daidzin content (22 mg/g). In addition to daidzin, six other isoflavones were identified in the RP extract and quantified--namely, puerarin (160 mg per g of extract), genistin (3.7 mg/g), daidzein (2.6 mg/g), daidzein-4',7-diglucoside (1.2 mg/g), genistein (0.2 mg/g), and formononetin (0.16 mg/g). None of these, administered either alone or combined, contributes in any significant way to the antidipsotropic activity of the extract. Plasma daidzin concentration-time curves determined in hamsters administered various doses of pure daidzin or RP extract by i.p.injection indicate that the crude extract daidzin has approximately 10 times greater bioavailability than the pure compound. Reconstruction of the dose-response effects for pure and crude daidzin using bioavailable daidzin rather than administered dose gives a single curve. Synthetic daidzin added to the RP extract acquires the bioavailability of the endogenous daidzin that exists naturally in the extract. These results show that (i) daidzin is the major active principle in methanol extracts of RP, and (ii) additional constituents in the methanol extract of RP assist uptake of daidzin in golden hamsters.

Targeted gene disruption of Hsp70-2 results in failed meiosis, germ cell apoptosis, and male infertility.

In addition to the five 70-kDa heat shock proteins (HSP70) common to germ cells and somatic tissues of mammals, spermatogenic cells synthesize HSP70-2 during meiosis. To determine if this unique stress protein has a critical role in meiosis, we used gene-targeting techniques to disrupt Hsp70-2 in mice. Male mice homozygous for the mutant allele (Hsp70-2 -/-) did not synthesize HSP70-2, lacked postmeiotic spermatids and mature sperm, and were infertile. However, neither meiosis nor fertility was affected in female Hsp70-2 -/- mice. We previously found that HSP70-2 is associated with synaptonemal complexes in the nucleus of meiotic spermatocytes from mice and hamsters. While synaptonemal complexes assembled in Hsp70-2 -/- spermatocytes, structural abnormalities became apparent in these cells by late prophase, and development rarely progressed to the meiotic divisions. Furthermore, analysis of nuclei and genomic DNA indicated that the failure of meiosis in Hsp70-2 -/- mice was coincident with a dramatic increase in spermatocyte apoptosis. These results suggest that HSP70-2 participates in synaptonemal complex function during meiosis in male germ cells and is linked to mechanisms that inhibit apoptosis.

Intrinsic changes in developing retinal neurons result in regenerative failure of their axons.

The failure of mature mammalian central nervous system axons to regenerate after transection is usually attributed to influences of the extraneuronal milieu. Using explant cocultures of retina and midbrain tectum from hamsters, we have found evidence that these influences account for failure of regrowth of only a small minority of retinal axons. For most of the axons, there is a programmed loss of ability to elongate in the central nervous system. We show that there is a precipitous decline in the ability of retinal axons to reinnervate tectal targets when the retina is derived from pups on or after postnatal day 2, even when the target is embryonic. By contrast, embryonic retinal axons can regrow into tectum of any age, overcoming growth-inhibiting influences of glial factors.

Species specificity in the cell-free conversion of prion protein to protease-resistant forms: a model for the scrapie species barrier.

Scrapie is a transmissible neurodegenerative disease that appears to result from an accumulation in the brain of an abnormal protease-resistant isoform of prion protein (PrP) called PrPsc. Conversion of the normal, protease-sensitive form of PrP (PrPc) to protease-resistant forms like PrPsc has been demonstrated in a cell-free reaction composed largely of hamster PrPc and PrPsc. We now report studies of the species specificity of this cell-free reaction using mouse, hamster, and chimeric PrP molecules. Combinations of hamster PrPc with hamster PrPsc and mouse PrPc with mouse PrPsc resulted in the conversion of PrPc to protease-resistant forms. Protease-resistant PrP species were also generated in the nonhomologous reaction of hamster PrPc with mouse PrPsc, but little conversion was observed in the reciprocal reaction. Glycosylation of the PrPc precursors was not required for species specificity in the conversion reaction. The relative conversion efficiencies correlated with the relative transmissibilities of these strains of scrapie between mice and hamsters. Conversion experiments performed with chimeric mouse/hamster PrPc precursors indicated that differences between PrPc and PrPsc at residues 139, 155, and 170 affected the conversion efficiency and the size of the resultant protease-resistant PrP species. We conclude that there is species specificity in the cell-free interactions that lead to the conversion of PrPc to protease-resistant forms. This specificity may be the molecular basis for the barriers to interspecies transmission of scrapie and other transmissible spongiform encephalopathies in vivo.

Nutraceutical approach to the modulation of cholesterol metabolism: in vitro and in vivo studies

Negli ultimi anni, si sono diffusi nuove strategie per il trattamento delle malattie cardiovascolari, che possano supportare una terapia medica, o in alcuni casi, sostituirla. Infatti, l’abbandono delle terapie è il più importante problema di salute pubblica del mondo occidentale, soprattutto per le malattie croniche. Ciò è dovuto alla complessità delle terapie farmacologiche e ai numerosi e in alcuni casi gravi effetti collaterali dei farmaci somministrati. Di conseguenza, una riduzione di questi effetti migliorerebbe le condizioni di vita del paziente e quindi diminuirebbe il rischio di abbandono della terapia. Per ottenere ciò, è possibile affiancare al trattamento farmacologico una terapia nutraceutica, consistente nella somministrazione di complessi molecolari o microorganismi, provenienti da piante, latte o cibi funzionali. Lo scopo generale di questo studio è indagare le attività ipolipidemizzanti di un composto nutraceutico e di un ceppo batterio specifico nel modello animale che presenta elevati alti livelli plasmatici di colesterolo. Inoltre, sono stati analizzati gli effetti del trattamento nutraceutico sui meccanismi fisiologici che contrastano la creazione della placca aterosclerotica come l’efflusso di colesterolo dalle “foam cells” presenti nell’ateroma, o la riduzione dell’assorbimento intestinale di colesterolo. La presente tesi è divisa in due parti. Nella prima parte, abbiamo analizzato la capacità dei Bifidobacteria di ridurre i livelli di colesterolo nel medium di crescita. Dall’analisi, si è osservato che vari ceppi del genere Bifidobacteria presentano un’ampia capacità di assimilazione del colesterolo all’interno della cellula batterica, in particolare il Bifidobacterium bifidum PRL2010. Le analisi di trascrittomica del Bb PRL2010 incubato in presenza di colesterolo, hanno rivelato un significativo aumento dei livelli di trascrizione di geni codificanti trasportatori e riduttasi, responsabili del meccanismo di accumulo all’interno della cellula batterica e della conversione del colesterolo in coprostanolo. L’attività ipolipidemizzante del Bb PRL2010 è stata poi valutata nel modello murino, mostrando la modificazione del microbiota dei topi trattati dopo somministrazione del batterio in questione. Nella seconda parte del progetto di ricerca, abbiamo indagato sugli effetti di un composto coperto da brevetto, chiamato “Ola”, sull’efflusso di colesterolo di criceti trattati con questo composto nutraceutico. L’efflusso di colesterolo è il primo step del meccanismo fisiologico noto come Trasporto Inverso del Colesterolo, che consente l’eliminazione del colesterolo dalle placche aterosclerotiche, attraverso l’interazione fra le HDL, presenti nella circolazione sanguigna, e specifici trasportatori delle foam cells, come ABCA1/G1 e SR-BI. In seguito, le lipoproteine rilasciano il colesterolo alle cellule epatiche, dove è metabolizzato ed escreto attraverso le feci. Per valutare l’effetto dell’Ola sul profilo lipidico dei criceti, sono state condotte analisi in vitro. I risultati mostrano un aumento dell’efflusso di colesterolo in cellule che esprimono il trasportatore ABCA1, comparato con il gruppo controllo. Questi due studi mostrano come l’approccio nutraceutico può essere un importante modo per contrastare l’aterosclerosi. Come mostrato in letteratura, gli effetti dei composti nutraceutici sull’aterosclerosi e su altre malattie croniche, hanno portato a un ampio uso come supporto alle terapie farmacologiche, ed in alcuni casi hanno rimpiazzato la terapia farmacologica stessa.

Clinical anatomy of the European hamster Cricetus cricetus, L. /

Mode of access: Internet.

Successful hamster raising.

Mode of access: Internet.

Histological analysis of gallbladder diseases in relation to opisthorchiasis in endemic areas of Thailand

Chronic gallbladder disease frequently accompanies infection with the liver fluke, Opisthorchis viverrini, in Northeast Thailand. However, the pathology and pathogenesis of the gallbladder disease have not been described. Accordingly, gallbladder specimens from 187 consecutive patients who had undergone cholecystectomy at a referral hospital in an endemic area in Thailand were histologically characterized in relation to O. viverrini infection. The infection was assessed by the presence of parasite eggs in the bile and/or antibody response to the liver fluke. The average level of parasite-specific IgG was significantly higher in patients with Opisthorchis eggs in the bile than those without (P < 0.001). The main histopathologic features of the gallbladder included inflammation, mucosal atrophy/or hyperplasia, goblet cell metaplasia, mucous gland hyperplasia, Rokitansky-Aschoff sinus formation, dysplasia and fibrosis. The fibrosis was strongly associated with elevated levels of Opisthorchis-specific antibody (P < 0.001) but not with the presence of parasite eggs. Other pathologic features did not vary in frequency or severity with parasitological status. Our results show that severe fibrosis of the gallbladder is a more common histologic feature of cholecystitis among those with O. viverrini infection compared to those without infection. The close relationship between parasite-specific IgG and severe fibrosis suggests that specific immune response to the parasite play an important role in the pathogenesis of the fibrotic change. (C) 2003 Elsevier B.V. All rights reserved.

NS1 protein secretion during the acute phase of West Nile virus infection

The West Nile virus (WNV) nonstructural protein NS1 is a protein of unknown function that is found within, associated with, and secreted from infected cells. We systematically investigated the kinetics of NS1 secretion in vitro and in vivo to determine the potential use of this protein as a diagnostic marker and to analyze NS1 secretion in relation to the infection cycle. A sensitive antigen capture enzyme-linked immunosorbent assay (ELISA) for detection of WNW NS1 (polyclonal-ACE) was developed, as well as a capture ELISA for the specific detection of NS1 multimers (4G4-ACE). The 4G4-ACE detected native NS1 antigens at high sensitivity, whereas the polyclonal-ACE had a higher specificity for recombinant forms of the protein. Applying these assays we found that only a small fraction of intracellular NS1 is secreted and that secretion of NS1 in tissue culture is delayed compared to the release of virus particles. In experimentally infected hamsters, NS1 was detected in the serum between days 3 and 8 postinfection, peaking on day 5, the day prior to the onset of clinical disease; immunoglobulin M (IgM) antibodies were detected at low levels on day 5 postinfection. Although real-time PCR gave the earliest indication of infection (day 1), the diagnostic performance of the 4G4-ACE was comparable to that of real-time PCR during the time period when NS1 was secreted. Moreover, the 4G4-ACE was found to be superior in performance to both the IgM and plaque assays during this time period, suggesting that NS1 is a viable early diagnostic marker of WNV infection.

Berberine - a novel approach to cholesterol lowering

Although low-density lipoprotein (LDL)-cholesterol lowering with the statins reduces the mortality and morbidity associated with coronary artery disease, considerable mortality and morbidity remains. Berberine upregulates the LDL receptor (LDLR) by a mechanism distinct from that of the statins, which involves stabilising the LDLR mRNA. In hamsters fed a high-fat and high-cholesterol diet for 2 weeks, the oral administration of berberine 100 mg/kg for 10 days reduced total serum cholesterol from &SIM; 4.8 to 2.7 mmol/l, and LDL-cholesterol from &SIM; 2.5 to 1.4 mmol/l. In subjects with hypercholesterolaemia, berberine hydrochloride (0.5 g b.i.d. for 3 months) reduced LDL-cholesterol (from 3.2 to 2.4 mmol/l) without any effect on high-density lipoprotein-cholesterol. Berberine also caused a reduction in triglyceride levels from 2.3 to 1.5 mmol/l. As berberine and statins both upregulate LDLR, their lipid-lowering profiles are similar. Thus, this mechanism is unlikely to make berberine an attractive alternative to statins for lipid lowering in most circumstances. However, the other effects of berberine (anti hypertensive, inotropic and class III antiarrhythmic properties) may make it a useful agent in the treatment of cardiovascular disease.

Effets de l'administration chronique de l'hormone de croissance sur l'expression et les concentrations sériques des peptides natriurétiques chez le hamster cardiomyopathique UM-X7.1

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.