Lack of influence of prion protein gene expression on kainate-induced seizures in mice: studies using congenic, coisogenic and transgenic strains.

Prion protein (PrP) is a glycosylphosphatidylinositol (GPI) anchored cell surface protein expressed by many cells, including those of the mammalian nervous system. At present the physiologic functions of PrP remain unclear. Deletion of Prnp, the gene encoding PrP in mice, has been shown to alter normal synaptic and electrophysiologic activities, indicating a potential role in seizure susceptibility. However, published efforts to link PrP with seizures, using both in vivo and in vitro models, are conflicting and difficult to interpret due to use of various mouse backgrounds and seizure induction techniques. Here we investigated the role of PrP in kainic acid (KA)-induced seizure sensitivity, using three types of mice. In contrast to previous published results, Prnp-/- mice on the C57BL/10SnJ background had a significant decrease in KA-induced seizure susceptibility. In genetic complementation experiments using a PrP-expressing transgene, genes derived from strain 129/Ola, which flanked the Prnp-/- locus in C57BL/10SnJ mice, rather than Prnp itself, appeared to account for this effect. Furthermore, using coisogenic 129/Ola mice differing only at Prnp, this difference was not reproduced when comparing PrP-negative and PrP-positive mice. In contrast, substrains of PrP-expressing C57BL mice, showed large variations in KA-induced seizure sensitivity. The magnitude of these differences in susceptibility was larger than that associated with the presence of the Prnp gene, suggesting extensive influence of genes other than Prnp on seizure sensitivity in this system.

Non-amyloid and amyloid prion protein deposits in prion-infected mice differ in blockage of interstitial brain fluid.

AIMS: Prion diseases are characterized by brain deposits of misfolded aggregated protease-resistant prion protein (PrP), termed PrPres. In humans and animals, PrPres is found as either disorganized non-amyloid aggregates or organized amyloid fibrils. Both PrPres forms are found in extracellular spaces of the brain. Thus, both might block drainage of brain interstitial fluid (ISF). The present experiments studied whether ISF blockage occurred during amyloid and/or non-amyloid prion diseases. METHODS: Various-sized fluorescein-labelled ISF tracers were stereotactically inoculated into the striatum of adult mice. At times from 5 min to 77 h, uninfected and scrapie-infected mice were compared. C57BL/10 mice expressing wild-type anchored PrP, which develop non-amyloid PrPres similar to humans with sporadic Creutzfeldt-Jakob disease, were compared with Tg44+/+ mice (transgenic mice secreting anchorless PrP) expressing anchorless PrP, which develop amyloid PrPres similar to certain human familial prion diseases. RESULTS: In C57BL/10 mice, extensive non-amyloid PrPres aggregate deposition was not associated with abnormal clearance kinetics of tracers. In contrast, scrapie-infected Tg44+/+ mice showed blockage of tracer clearance and colocalization of tracer with perivascular PrPres amyloid. CONCLUSIONS: As tracer localization and clearance was normal in infected C57BL/10 mice, ISF blockage was not an important pathogenic mechanism in this model. Therefore, ISF blockage is unlikely to be a problem in non-amyloid human prion diseases such as sporadic Creutzfeldt-Jakob disease. In contrast, partial ISF blockage appeared to be a possible pathogenic mechanism in Tg44+/+ mice. Thus this mechanism might also influence human amyloid prion diseases where expression of anchorless or mutated PrP results in perivascular amyloid PrPres deposition and cerebral amyloid angiopathy.

Influence of proinflammatory cytokines and chemokines on the neuropathogenesis of oncornavirus and immunosuppressive lentivirus infections.

Retroviral infection of the CNS can lead to severe debilitating neurological diseases in humans and other animals. Four general types of pathogenic effects with various retroviruses have been observed including: hemorrhage (TR1.3), spongiform encephalopathy (CasBrE, FrCasE, PVC211, NT40, Mol-ts1), demyelination with inflammatory lesions (HTLV-1, visna, CAEV), and encephalopathy with gliosis and proinflammatory chemokines and cytokines, usually with microglial giant cells and nodules [human immunodeficiencyvirus (HIV), feline immunodeficiencyvirus (FIV), simian immunodeficiency virus (SIV), Fr98]. This review focuses on this fourth group of retroviruses. In this latter group, proinflammatory cytokine and chemokine upregulation accompanies the disease process, and may influence pathogenesis by direct effects on resident CNS cells. The review first discusses the Fr98 murine polytropic virus system with particular reference to the roles of cytokines and chemokines in the pathogenic process. The Fr98 data are then compared and contrasted to the cytokine and chemokine data in the lentivirus systems, HIV, SIV, and FIV. Finally, various mechanisms are presented by which tumor necrosis factor (TNF) and several chemokines may alter the pathogenesis of retrovirus infection of the CNS.

A 6 week course of azithromycin treatment has no beneficial effect on atherosclerotic lesion development in apolipoprotein E-deficient mice chronically infected with Chlamydia pneumoniae.

OBJECTIVES: To evaluate whether antimicrobial chemotherapy prevents acceleration of atherosclerotic lesion development induced by infection with Chlamydia pneumoniae. METHODS: ApoE-deficient mice which develop hyperlipidaemia and atherosclerosis spontaneously were inoculated intranasally with C. pneumoniae. Animals were treated with azithromycin for 6 weeks after the third inoculation and the atherosclerotic lesion areas in the aortic sinus were measured by computer-assisted morphometry. RESULTS: At 12 weeks post-infection, infected untreated animals developed significantly larger lesion areas compared with sham-inoculated controls (8.7 x 10(4)+/-2.3 x 10(4) microm(2) versus 5.6 x 10(4)+/-2.4 x 10(4) microm(2)). However, there were no differences in lesion size of infected mice treated with azithromycin in comparison with untreated infected controls (11.0 x 10(4)+/-3.0 x 10(4) microm(2) versus 8.7 x 10(4)+/-2.3 x 10(4) microm(2)). CONCLUSIONS: Antibiotic treatment against C. pneumoniae has no beneficial effects on hyperlipidaemia-induced atherosclerosis accelerated by C. pneumoniae in a mouse model.

Long-term subclinical carrier state precedes scrapie replication and adaptation in a resistant species: analogies to bovine spongiform encephalopathy and variant Creutzfeldt-Jakob disease in humans.

Cattle infected with bovine spongiform encephalopathy (BSE) appear to be a reservoir for transmission of variant Creutzfeldt-Jakob disease (vCJD) to humans. Although just over 100 people have developed clinical vCJD, millions have probably been exposed to the infectivity by consumption of BSE-infected beef. It is currently not known whether some of these individuals will develop disease themselves or act as asymptomatic carriers of infectivity which might infect others in the future. We have studied agent persistence and adaptation after cross-species infection using a model of mice inoculated with hamster scrapie strain 263K. Although mice inoculated with hamster scrapie do not develop clinical disease after inoculation with 10 million hamster infectious doses, hamster scrapie infectivity persists in brain and spleen for the life span of the mice. In the present study, we were surprised to find a 1-year period postinfection with hamster scrapie where there was no evidence for replication of infectivity in mouse brain. In contrast, this period of inactive persistence was followed by a period of active replication of infectivity as well as adaptation of new strains of agent capable of causing disease in mice. In most mice, neither the early persistent phase nor the later replicative phase could be detected by immunoblot assay for protease-resistant prion protein (PrP). If similar asymptomatic carriers of infection arise after exposure of humans or animals to BSE, this could markedly increase the danger of additional spread of BSE or vCJD infection by contaminated blood, surgical instruments, or meat. If such subclinical carriers were negative for protease-resistant PrP, similar to our mice, then the recently proposed screening of brain, tonsils, or other tissues of animals and humans by present methods such as immunoblotting or immunohistochemistry might be too insensitive to identify these individuals.

N-terminal truncation of prion protein affects both formation and conformation of abnormal protease-resistant prion protein generated in vitro.

Transmissible spongiform encephalopathy diseases are characterized by conversion of the normal protease-sensitive host prion protein, PrP-sen, to an abnormal protease-resistant form, PrP-res. In the current study, deletions were introduced into the flexible tail of PrP-sen (23) to determine if this region was required for formation of PrP-res in a cell-free assay. PrP-res formation was significantly reduced by deletion of residues 34-94 relative to full-length hamster PrP. Deletion of another nineteen amino acids to residue 113 further reduced the amount of PrP-res formed. Furthermore, the presence of additional proteinase K cleavage sites indicated that deletion to residue 113 generated a protease-resistant product with an altered conformation. Conversion of PrP deletion mutants was also affected by post-translational modifications to PrP-sen. Conversion of unglycosylated PrP-sen appeared to alter both the amount and the conformation of protease-resistant PrP-res produced from N-terminally truncated PrP-sen. The N-terminal region also affected the ability of hamster PrP to block mouse PrP-res formation in scrapie-infected mouse neuroblastoma cells. Thus, regions within the flexible N-terminal tail of PrP influenced interactions required for both generating and disrupting PrP-res formation.

Differences in cytokine and chemokine responses during neurological disease induced by polytropic murine retroviruses Map to separate regions of the viral envelope gene.

Infection of the central nervous system (CNS) by several viruses can lead to upregulation of proinflammatory cytokines and chemokines. In immunocompetent adults, these molecules induce prominent inflammatory infiltrates. However, with immunosuppressive retroviruses, such as human immunodeficiency virus (HIV), little CNS inflammation is observed yet proinflammatory cytokines and chemokines are still upregulated in some patients and may mediate pathogenesis. The present study examined expression of cytokines and chemokines in brain tissue of neonatal mice infected with virulent (Fr98) and avirulent (Fr54) polytropic murine retroviruses. While both viruses infect microglia and endothelia primarily in the white matter areas of the CNS, only Fr98 induces clinical CNS disease. The pathology consists of gliosis with minimal morphological changes and no inflammation, similar to HIV. In the present experiments, mice infected with Fr98 had increased cerebellar mRNA levels of proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), TNF-beta, and interleukin-1 alpha and chemokines macrophage inflammatory protein-1 alpha (MIP-1 alpha), MIP-1 beta, monocyte chemoattractant protein 1 (MCP-1), gamma-interferon-inducible protein 10 (IP-10), and RANTES compared to mice infected with Fr54 or mock-infected controls. The increased expression of these genes occurred prior to the development of clinical symptoms, suggesting that these cytokines and chemokines might be involved in induction of neuropathogenesis. Two separate regions of the Fr98 envelope gene are associated with neurovirulence. CNS disease associated with the N-terminal portion of the Fr98 env gene was preceded by upregulation of cytokines and chemokines. In contrast, disease associated with the central region of the Fr98 env gene showed no upregulation of cytokines or chemokines and thus did not require increased expression of these genes for disease induction.

Sulfated glycans and elevated temperature stimulate PrP(Sc)-dependent cell-free formation of protease-resistant prion protein.

A conformational conversion of the normal, protease- sensitive prion protein (PrP-sen or PrP(C)) to a protease-resistant form (PrP-res or PrP(Sc)) is commonly thought to be required in transmissible spongiform encephalopathies (TSEs). Endogenous sulfated glycosaminoglycans are associated with PrP-res deposits in vivo, suggesting that they may facilitate PrP-res formation. On the other hand, certain exogenous sulfated glycans can profoundly inhibit PrP-res accumulation and serve as prophylactic anti-TSE compounds in vivo. To investigate the seemingly paradoxical effects of sulfated glycans on PrP-res formation, we have assayed their direct effects on PrP conversion under physiologically compatible cell-free conditions. Heparan sulfate and pentosan polysulfate stimulated PrP-res formation. Conversion was stimulated further by increased temperature. Both elevated temperature and pentosan polysulfate promoted interspecies PrP conversion. Circular dichroism spectropolarimetry measurements showed that pentosan polysulfate induced a conformational change in PrP-sen that may potentiate its PrP-res-induced conversion. These results show that certain sulfated glycosaminoglycans can directly affect the PrP conversion reaction. Therefore, depending upon the circumstances, sulfated glycans may be either cofactors or inhibitors of this apparently pathogenic process.

Major histocompatibility complex class I gene controls the generation of gamma interferon-producing CD4(+) and CD8(+) T cells important for recovery from friend retrovirus-induced leukemia.

Recovery from leukemia induced by Friend virus complex (FV) requires strong CD4(+) helper, CD8(+) cytotoxic T-lymphocyte, and B-cell responses. The development of these immune responses is dependent on the major histocompatibility complex (MHC) (H-2) genotype of the mouse. In H-2(b/b) mice, which spontaneously recover from FV-induced erythroleukemia, neutralization of gamma interferon (IFN-gamma) in vivo inhibited recovery, which indicated that IFN-gamma was a necessary component of the immune response to FV. Furthermore, in H-2(b/b) mice, high numbers of IFN-gamma-producing cells were detected after FV infection, whereas in H-2(a/b) mice, which have a low-recovery phenotype, only low numbers of IFN-gamma-producing cells were detected. Similarly, H-2(bm14/b) mice, which cannot recover from FV infection due to a point mutation in one allele of the H-2D(b) gene, also had low numbers of IFN-gamma-producing T cells. Surprisingly, this effect was observed for both CD8(+) and CD4(+) T cells. These findings reveal a novel influence of MHC class I genes on CD4(+) T-cell responses to viral infection. Furthermore, the influence of MHC class I genotype on the generation of both IFN-gamma-producing CD4(+) and CD8(+) T cells helps explain the major impact of the H-2D gene on recovery from FV disease.

A randomized controlled trial of a clinic-based support staff intervention to increase the rate of fecal occult blood test ordering.

BACKGROUND: Colorectal cancer is the second most common fatal malignancy in the United States. Early detection using fecal occult blood tests has been shown to reduce mortality, but these tests are underutilized among those eligible for this screening. Attempts to increase use of fecal occult blood tests in eligible populations have focused on the provider, patient, or system. But none have examined whether a support-staff intervention is effective in achieving this aim. We therefore conducted a randomized controlled trial to test the impact of authorizing support staff to order fecal occult blood tests in a general internal medicine clinic organized into four teams. METHODS: A total of 1,109 patients were included in the study, 545 of whom were in the two teams randomized to treatment. Univariate and multivariate regression analyses were used to evaluate the impact of the intervention. RESULTS: The intervention resulted in significantly more fecal occult blood test ordering in the treatment group than in the control group for all patients (52% vs 15%, P < 0.001). Treatment fecal occult blood test cards were returned as frequently as the control cards for all patients (44% vs 48%, P = 0.571). CONCLUSION: Delegation of selected screening tasks to support staff can enhance patient access to preventive care.

Entry versus blockade of brain infection following oral or intraperitoneal scrapie administration: role of prion protein expression in peripheral nerves and spleen.

Naturally occurring transmissible spongiform encephalopathy (TSE) diseases such as bovine spongiform encephalopathy in cattle are probably transmitted by oral or other peripheral routes of infection. While prion protein (PrP) is required for susceptibility, the mechanism of spread of infection to the brain is not clear. Two prominent possibilities include hematogenous spread by leukocytes and neural spread by axonal transport. In the present experiments, following oral or intraperitoneal infection of transgenic mice with hamster scrapie strain 263K, hamster PrP expression in peripheral nerves was sufficient for successful infection of the brain, and cells of the spleen were not required either as a site of amplification or as transporters of infectivity. The role of tissue-specific PrP expression of foreign PrP in interference with scrapie infection was also studied in these transgenic mice. Peripheral expression of heterologous PrP completely protected the majority of mice from clinical disease after oral or intraperitoneal scrapie infection. Such extensive protection has not been seen in earlier studies on interference, and these results suggested that gene therapy with mutant PrP may be effective in preventing TSE diseases.

Inhibition of formation of protease-resistant prion protein by Trypan Blue, Sirius Red and other Congo Red analogs.

Five compounds related to Congo Red were found to inhibit generation of protease-resistant prion protein in a cell-free system. In this assay Trypan Blue, Evans Blue, Sirius Red F3B, Primuline and Thioflavin-S were all more inhibitory than Congo Red itself. In scrapie-infected mouse neuroblastoma cells one compound, Sirius Red F3B, was capable of blocking the formation of protease-resistant prion protein to a similar extent as Congo Red; however, the other four compounds were less effective. Some of these compounds should be considered for testing in TSE disease models in live animals.

Increased neurovirulence of polytropic mouse retroviruses delivered by inoculation of brain with infected neural stem cells.

Following intraperitoneal (IP) inoculation of neonatal mice, the polytropic recombinant murine leukemia virus (MuLV), Fr98, induces a severe brain disease characterized by ataxia, seizures and death. In contrast, no apparent clinical neurological disease is seen after IP infection with Fr54, a polytropic MuLV differing from Fr98 in its envelope gene sequences. In the brain both Fr98 and Fr54 infect primarily capillary endothelial cells and microglia. However, the level of microglial infection by Fr98 is twofold higher than by Fr54, which might account for the difference in neurovirulence. In the present study, in order to test directly whether an increase in the number of microglia infected by Fr54 would be sufficient to induce clinical disease, we attempted to increase the level of Fr54 in the brain by changing the route of infection. After intraventricular inoculation with Fr54-infected neural stem cells (clone C17.2), a well-established vehicle for delivery of viruses and genes to the brain, mice became ataxic and died 4 weeks postinfection. In these mice induction of brain disease was correlated with a higher level of viral antigen in the cerebrum and an increase in the number of infected microglial cells in all brain regions examined compared with mice inoculated IP. In contrast, mice inoculated with neural stem cells infected with an ecotropic nonneurovirulent murine leukemia virus, FB29, developed no clinical disease in spite of evidence for widespread infection of microglia in brain. Since the main differences between Fr54 and FB29 are in the SU (gp70) region of the envelope gene, this region is most likely to account for the differences in induction of CNS disease seen in the current experiments.

Fine mapping of the friend retrovirus resistance gene, Rfv3, on mouse chromosome 15.

Rfv3 is a host resistance gene that operates through an unknown mechanism to control the development of the virus-neutralizing antibody response required for recovery from infection with Friend retrovirus. The Rfv3 gene was previously mapped to an approximately 20-centimorgan (cM) region of chromosome 15. More refined mapping was not possible, due to a lack of microsatellite markers and leakiness in the Rfv3 phenotype, which prevented definitive phenotyping of individual recombinant mice. In the present study, we overcame these difficulties by taking advantage of seven new microsatellite markers in the Rfv3 region and by using progeny tests to accurately determine the Rfv3 phenotype of recombinant mice. Detailed linkage analysis of relevant crossovers narrowed the location of Rfv3 to a 0.83-cM region. Mapping of closely linked genes in an interspecific backcross panel allowed us to exclude two previous candidate genes, Ly6 and Wnt7b. These studies also showed for the first time that the Hsf1 gene maps to the Rfv3-linked cluster of genes including Il2rb, Il3rb, and Pdgfb. This localization of Rfv3 to a region of less than 1 cM now makes it feasible to attempt the cloning of Rfv3 by physical methods.

Species-independent inhibition of abnormal prion protein (PrP) formation by a peptide containing a conserved PrP sequence.

Conversion of the normal protease-sensitive prion protein (PrP) to its abnormal protease-resistant isoform (PrP-res) is a major feature of the pathogenesis associated with transmissible spongiform encephalopathy (TSE) diseases. In previous experiments, PrP conversion was inhibited by a peptide composed of hamster PrP residues 109 to 141, suggesting that this region of the PrP molecule plays a crucial role in the conversion process. In this study, we used PrP-res derived from animals infected with two different mouse scrapie strains and one hamster scrapie strain to investigate the species specificity of these conversion reactions. Conversion of PrP was found to be completely species specific; however, despite having three amino acid differences, peptides corresponding to the hamster and mouse PrP sequences from residues 109 to 141 inhibited both the mouse and hamster PrP conversion systems equally. Furthermore, a peptide corresponding to hamster PrP residues 119 to 136, which was identical in both mouse and hamster PrP, was able to inhibit PrP-res formation in both the mouse and hamster cell-free systems as well as in scrapie-infected mouse neuroblastoma cell cultures. Because the PrP region from 119 to 136 is very conserved in most species, this peptide may have inhibitory effects on PrP conversion in a wide variety of TSE diseases.

Appearance of mink cell focus-inducing recombinants during in vivo infection by moloney murine leukemia virus (M-MuLV) or the Mo+PyF101 M-MuLV enhancer variant: implications for sites of generation and roles in leukemogenesis.

One hallmark of murine leukemia virus (MuLV) leukemogenesis in mice is the appearance of env gene recombinants known as mink cell focus-inducing (MCF) viruses. The site(s) of MCF recombinant generation in the animal during Moloney MuLV (M-MuLV) infection is unknown, and the exact roles of MCF viruses in disease induction remain unclear. Previous comparative studies between M-MuLV and an enhancer variant, Mo+PyF101 MuLV, suggested that MCF generation or early propagation might take place in the bone marrow under conditions of efficient leukemogenesis. Moreover, M-MuLV induces disease efficiently following both intraperitoneal (i.p.) and subcutaneous (s.c.) inoculation but leukemogenicity by Mo+PyF101 M-MuLV is efficient following i.p. inoculation but attenuated upon s. c. inoculation. Time course studies of MCF recombinant appearance in the bone marrow, spleen, and thymus of wild-type and Mo+PyF101 M-MuLV i.p.- and s.c.-inoculated mice were carried out by performing focal immunofluorescence assays. Both the route of inoculation and the presence of the PyF101 enhancer sequences affected the patterns of MCF generation or early propagation. The bone marrow was a likely site of MCF recombinant generation and/or early propagation following i.p. inoculation of M-MuLV. On the other hand, when the same virus was inoculated s.c., the primary site of MCF generation appeared to be the thymus. Also, when Mo+PyF101 M-MuLV was inoculated i.p., MCF generation appeared to occur primarily in the thymus. The time course studies indicated that MCF recombinants are not involved in preleukemic changes such as splenic hyperplasia. On the other hand, MCFs were detected in tumors from Mo+PyF101 M-MuLV s. c.-inoculated mice even though they were largely undetectable at preleukemic times. These results support a role for MCF recombinants late in disease induction.

Effectiveness of polyene antibiotics in treatment of transmissible spongiform encephalopathy in transgenic mice expressing Syrian hamster PrP only in neurons.

To date very few drugs have favorably influenced the course of transmissible spongiform encephalopathies. In previous studies, the polyene antibiotics amphotericin B (AmB) and MS-8209 prolonged the incubation time in Syrian hamsters of the 263K strain of scrapie, but AmB had no effect against other scrapie strains in Syrian hamsters. In the present experiments using transgenic mice expressing Syrian hamster PrP in neurons only, MS-8209 extended the life spans of animals infected with the 263K strain but not the DY strain. AmB was effective against both 263K and DY and prevented death in 18% of DY-infected animals. The AmB effect against strain 263K was more prominent in mice whose endogenous PrP gene had been inactivated by homologous recombination. It was unclear whether this difference was due to a change in the duration of the disease or to possible interactive effects between the mouse PrP gene and the drugs themselves. The effectiveness of treatment after intracerebral scrapie infection in transgenic mice expressing PrP only in neurons suggested that neurons are important sites of action for these drugs.

V3 recombinants indicate a central role for CCR5 as a coreceptor in tissue infection by human immunodeficiency virus type 1.

Binding of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 to both CD4 and one of several chemokine receptors (coreceptors) permits entry of virus into target cells. Infection of tissues may establish latent viral reservoirs as well as cause direct pathologic effects that manifest as clinical disease such as HIV-associated dementia. We sought to identify the critical coreceptors recognized by HIV-1 tissue-derived strains as well as to correlate these coreceptor preferences with site of infection and dementia diagnosis. To reconstitute coreceptor use, we cloned HIV-1 envelope V3 sequences encoding the primary determinants of coreceptor specificity from 13 brain-derived and 6 colon-derived viruses into an isogenic (NL4-3) viral background. All V3 recombinants utilized the chemokine receptor CCR5 uniformly and efficiently as a coreceptor but not CXCR4, BOB/GPR15, or Bonzo/STRL33. Other receptors such as CCR3, CCR8, and US28 were inefficiently and variably used as coreceptors by various envelopes. CCR5 without CD4 present did not allow for detectable infection by any of the tested recombinants. In contrast to the pathogenic switch in coreceptor specificity frequently observed in comparisons of blood-derived viruses early after HIV-1 seroconversion and after onset of AIDS, the characteristics of these V3 recombinants suggest that CCR5 is a primary coreceptor for brain- and colon-derived viruses regardless of tissue source or diagnosis of dementia. Therefore, tissue infection may not depend significantly on viral envelope quasispeciation to broaden coreceptor range but rather selects for CCR5 use throughout disease progression.