Genetic polymorphism in exon 2 of cathepsin D is not associated with vascular dementia.

BACKGROUND: Cathepsin D, the most abundant lysosomal and endosomal aspartyl protease, shows beta and gamma secretase activity in vitro by cleaving the amyloid precursor protein (APP) into amyloid beta protein (Aß). Polymorphism at position 224, C224T, on exon 2 of cathepsin D gene (CTSD) has been associated with an increased risk for Alzheimer's disease (AD) by some investigators, but there have been contrary findings by others. However, an association between CTSD polymorphism and vascular dementia (VaD) has not been reported thus far. OBJECTIVE: To investigate whether a polymorphism at CTSD C224T is associated with VaD in the Korean population. METHODS: We compared the genotype and allele frequencies at this polymorphism site in clinically assessed 162 VaD patients with those in 197 healthy Koreans. RESULTS AND CONCLUSION: The major genotype frequency at CTSD C224T in normal controls was higher in the Asian population than in various European populations. Our study does not show a significant difference in genotype (P=0.3071) and allele (P=0.2291) frequencies of CTSD C224T between VaD and normal controls. This was the first genetic association study of CTSD in a VaD population.

Decreased percentage of polymorphonuclear neutrophils in mouse peripheral blood after inoculation with material from multiple sclerosis patients.

Mice inoculated with brain homogenates from multiple sclerosis (MS) cases showed marked changes in their leukocyte differential counts, with a decrease in per cent polymorphonuclear neutrophils (PMN) and an increase in the per cent lymphocytes. These changes were based upon an absolute decrease in the number of circulating PMN. The decrease in PMN percentages was apparent at 16 hr after infection and persisted for at least 11 months. The factor responsible for the decrease in PMN was (a) recoverable from 12 hr to 8(1/2) months after inoculation, (b) present in human brain homogenate at a concentration of 3 x 10(12), and (c) between 25 and 50 nm in diameter. Inoculation of 100 units of factor into mice and subsequent titration showed that the factor had undergone a net increase in the mouse of at least 10(9)-fold. The factor causing the PMN decrease was found in all MS material thus far tested: three brains, one spleen, three sera, and two cerebrospinal fluid (CSF) from nine cases of MS. The factor was not found in normal human material that included two brains, one spleen, two sera, and two CSF.

PRNP 1368 polymorphism is not associated with sporadic Creutzfeldt-Jakob disease in the Korean population.

BACKGROUND: Human prion protein gene (PRNP) is considered a critical and fundamental gene in determining the incidence of human prion diseases. Codons 129 and 219 play an important role in the susceptibility to sporadic Creutzfeldt-Jakob disease (CJD). An association between sporadic CJD and the polymorphism (PRNP 1368) in an upstream of PRNP exon 1 has been reported in the British and German populations, but study in the Dutch population has failed to confirm an association. PURPOSE: To investigate whether the PRNP 1368 polymorphism is associated with sporadic CJD in the Korean population. METHODS: We compared the genotype and allele frequencies of PRNP 1368 polymorphism in 171 sporadic CJD patients with those in 212 healthy Koreans. RESULT AND CONCLUSION: A significant difference of genotype and allele frequencies at PRNP 1368 was found between the normal Korean population and various European populations. In contrast to the results in the British and German populations, our study does not show a significant difference in genotype (P = 0.2763) and allele (P = 0.3750) frequencies of PRNP 1368 between sporadic CJD and normal controls.

Infected splenic dendritic cells are sufficient for prion transmission to the CNS in mouse scrapie.

Transmissible spongiform encephalopathies display long incubation periods at the beginning of which the titer of infectious agents (prions) increases in peripheral lymphoid organs. This "replication" leads to a progressive invasion of the CNS. Follicular dendritic cells appear to support prion replication in lymphoid follicles. However, the subsequent steps of neuroinvasion remain obscure. CD11c(+) dendritic cells, an unrelated cell type, are candidate vectors for prion propagation. We found a high infectivity titer in splenic dendritic cells from prion-infected mice, suggesting that dendritic cells carry infection. To test this hypothesis, we injected RAG-1(0/0) mice intravenously with live spleen cell subsets from scrapie-infected donors. Injection of infected dendritic cells induced scrapie without accumulation of prions in the spleen. These results suggest that CD11c(+) dendritic cells can propagate prions from the periphery to the CNS in the absence of any additional lymphoid element.

Polymorphisms at codons 129 and 219 of the prion protein gene (PRNP) are not associated with sporadic Alzheimer's disease in the Korean population.

Polymorphisms of prion protein gene (PRNP) at codons 129 and 219 play an important role in the susceptibility to Creutzfeldt-Jakob disease (CJD). Alzheimer's disease (AD) and prion diseases, such as CJD, are both characterized by the accumulation of abnormally folded proteins in the brain. An association between sporadic AD and the PRNP polymorphism at codon 129 has been reported in several studies, but other studies have failed to confirm an association. To investigate whether PRNP polymorphisms are associated with an increased risk for developing sporadic AD in the Korean population, we compared the genotype, allele, and haplotype frequencies of PRNP polymorphisms in 271 sporadic AD patients with those in 236 healthy Koreans. Our study does not show a significant difference in PRNP genotype, allele, and haplotype frequency at codons 129 and 219 between sporadic AD and normal controls. Analyses stratifying by age at disease onset, and gender also failed to reveal any association between these polymorphisms and sporadic AD. These results indicate that these PRNP polymorphisms have no direct influence on the susceptibility to sporadic AD in the Korean population.

An endogenous retrovirus and exogenous scrapie in a mouse model of aging.

As we enter the post-genomic era, there is an increasing need for accurate methods of identifying host and pathogen factors that contribute to bacterial, viral and fungal disease. In addition, there is a requirement for fast and precise techniques to evaluate potential therapies for the prevention of infectious diseases. The development of useful and cost-effective model systems will be crucial in advancing our knowledge of all aspects of microbial pathogenesis. In this series, we will learn of animal models used to investigate diseases caused by a wide variety of pathogens, including HIV, Vibrio cholerae and Pseudomonas aeruginosa. A description of a model system specifically designed to study intracellular pathogens will be presented, as will a variety of the techniques currently used to exploit other useful models of infection. Additionally, a description of the mathematical models used to analyse the population biology of human onchocerciasis will be discussed. The series begins with an intriguing look at the possible connections between an endogenous retrovirus, the infectious agent of scrapie and accelerated senescence in a mouse model of early aging.

Vacuolization, incubation period and survival time analyses in three mouse genotypes injected stereotactically in three brain regions with the 22L scrapie strain.

In previous studies we showed that C57BL mice injected stereotactically in the cerebellum with the 22L scrapie strain had a significantly shorter incubation period than those injected with the same agent in other brain regions. In mice injected in the cerebellum, vacuolization was limited to the cerebellum, medulla and mesencephalon, whereas injection into forebrain regions resulted in vacuolization in all brain regions. The studies suggested that the cerebellum had a selective vulnerability for 22L. In this study we examined the interaction between host genotype and selective vulnerability of specific brain regions. The mouse gene that has the most profound effect on pathogenesis, particularly incubation period, is termed Sinc (scrapie incubation). Groups of mice with three genotypes of Sinc (s7s7, p7p7 and their F1 cross, s7p7) were injected with 22L into the cerebral cortex, thalamus or cerebellum. Analysis of incubation periods showed that, regardless of the host genotype, the cerebellum injection group had a significantly shorter incubation period than groups injected in other regions. After cerebellum injection vacuolization was limited to the cerebellum, medulla and mesencephalon in all three host genotypes. The location of vacuoles within the cerebellum differed depending upon the host strain. Vacuolization developed almost exclusively in grey matter in s7s7 mice, mainly in white matter in p7p7 mice, and in both grey and white matter in F1 mice. These results demonstrate that the selective vulnerability of the cerebellum to induction of clinical disease by 22L does not depend on host genotype, but host genotype does affect lesion distribution within the cerebellum.

Pathogenesis and pathology of scrapie after stereotactic injection of strain 22L in intact and bisected cerebella.

The mechanisms involved in the spread of scrapie within the brain remain unclear. To examine this issue the 22L scrapie strain was injected in one side of the cerebellum of mice in which the cerebellum had been bisected prior to injection. Another group of animals received the same injection into intact cerebella, i.e. without prior bisection. We found that bisection of the cerebella delayed the spread of scrapie agent from the injection site to the contralateral side of the cerebellum and that the occurrence of vacuolization was not as extensive and was markedly delayed in the uninjected side compared to its occurrence after injection in the intact cerebellum. Replication of agent in an area preceded the development of vacuolization in that area by several weeks. There was marked loss of Purkinje cells on the injected side of bisected cerebella, with no loss seen on the uninjected side. The incubation period of scrapie disease in mice injected after cerebellar bisection was significantly longer than after the injection of intact cerebella. The results in this study suggest that the scrapie agent spreads along intact nerve cell tracts, probably by axonal transport.

Increased blood-brain barrier permeability in scrapie-infected mice.

The present investigation was designed to study the ultrastructural integrity of the blood-brain barrier (BBB) in the cerebral microvasculature of scrapie-infected mice showing clinical illness. Cerebral microvessels from either IM, VM, or C57BL/6J mice, terminally affected with various strains of scrapie agent showed a focal leakage of horseradish peroxidase (HRP) in all agent-strain and mouse-strain combinations. This leakage was most pronounced in and near the primary site of agent inoculation, but was also observed in microvessels scattered throughout the brain. Cytochemical studies also revealed a redistribution of plasmalemma-bound alkaline phosphatase in the endothelial cells. In control mice, the enzymatic activity was mainly concentrated in the luminal plasmalemma, while in the scrapie-infected mice the activity also appeared in the abluminal side in the majority of microvessels. Our observations are evidence that the BBB of the mouse is altered in some way by the scrapie agent. Such an alteration may have important implications for human disease, since the scrapie agent is related to the group of "slow" viral infections, including kuru and Creutzfeldt-Jakob disease. Scrapie may also serve as an important model for the study of senile dementia of the Alzheimer type (SDAT).

Senile dementia of the Alzheimer type: possibility of infectious etiology in genetically susceptible individuals.

The concept that SDAT is caused by an infectious agent acting in a genetically susceptible host was approached from a number of standpoints. The similarities between SDAT and the transmissible encephalopathies are discussed. One of the areas of similarity is the influence of genetic background on the development and expression of both conditions. Evidence is presented showing that genetics plays a role in many cases of SDAT and that there are known genetically controlled phenomena, the incidence of which is positively correlated with SDAT. For human encephalopathies the genetics of CJD and GSS are detailed. In experimental systems with scrapie, the influence of genetic control, operating through both host and agent, on the outcome of infection with scrapie is described. The events controlled include length of incubation period, type of lesions and their distribution and intensity. In the context of the human diseases, scrapie provides a model for the known human encephalopathies and for SDAT.

Titers of murine leukemia virus are higher in brains of SAMP8 than SAMR1 mice.

To elucidate possible causes of premature aging seen in a strain of senescence-accelerated prone (SAMP8) mice, levels of murine leukemia virus (MuLV) were quantitated in various tissues of SAMP8 by an SC-1/UV plaque assay. MuLV levels in SAMP8 tissues were compared to those seen in the closely related SAMR1 strain, which is resistant to premature aging. MuLV titers were found to be higher in blood and spleen and much higher in brain of SAMP8 than in the same tissues of SAMR1. MuLV levels were seen to increase in SAMP8 brain with increasing age. Virus typing experiments indicated that the MuLV from SAMP8 brain is N-tropic, as is the MuLV seen in the AKR strain, one of the SAM progenitor strains. MuLV from SAMP8 brain was able to grow well in SAMR1 mouse embryo cells, indicating that it may be possible to infect SAMR1 mice with the SAMP8 MuLV to determine the effects of the virus on aging of SAMR1 mice.

Murine leukemia virus in organs of senescence-prone and -resistant mouse strains.

A series of inbred strains of mice have been developed that are either prone (SAMP) or resistant (SAMR) to accelerated senescence. All of these strains originated from an inadvertent cross or crosses between the AKR/J mouse strain and an unknown strain(s). The characteristics of the nine senescence-prone lines differ, with all strains showing generalized aspects of accelerated aging but with each line having a specific aging-related change that is emphasized, e.g. learning and memory deficits, osteoporosis and senile amyloidosis. The senescence-resistant strains have normal patterns of aging and do not show the specific aging-related changes seen in SAMP strains. The fact that AKR mice have high levels of endogenous, ecotropic murine leukemia virus (MuLV) prompted an examination of the expression levels of MuLV in SAM strains. Analysis of brain, spleen and thymus samples revealed that seven of nine SAMP strains had high levels of MuLV and contained the Emv11 provirus (previously termed Akv1) that encodes the predominant MuLV found in AKR mice. In contrast, none of the SAMR strains had Emv11 or significant amounts of virus. The current findings represent an initial step in determining the role of MuLV in the accelerated senescence seen in SAMP strains.

Blood group frequencies in multiple sclerosis populations in the United States.

Multiple sclerosis (MS) patients (332) and controls (305) selected from Caucasian populations in the New York City area and in Tucson, Arizona, were tested for ABO blood group factors A and B, and Rh factors C, D, E, c, and e. There was no significant difference in the distribution frequencies of these factors in MS patients and controls.

Astrocytosis and proliferating cell nuclear antigen expression in brains of scrapie-infected hamsters.

Scrapie is a neurodegenerative disease in sheep and goats. Neuropathological examination shows astrocytosis. One issue is whether the astrocytosis seen in scrapie is a function of an increase in reactivity of individual cells, or whether there is actual replication of astrocytes. We used double-label immunohistochemistry for proliferating cell nuclear antigen (PCNA) and for glial fibrillary acidic protein (GFAP) to determine the mitotic state of cells and to confirm their identity as astrocytes. Brain sections from hamsters (strain LVG/LAK) infected with 139H or 263K scrapie isolates were examined. GFAP immunostaining was increased in astrocytes in most regions of the brains of scrapie-infected hamsters. These qualitative observations were confirmed by computerized image analysis quantification. A proportion of the hypertrophic astrocytes (0.5-10.8%, depending on specific location) were PCNA immunoreactive. The PCNA-immunopositive astrocytes were most frequently found in cerebral cortex, corpus callosum, subependymal areas, fimbria, caudate, thalamus, hypothalamus, hippocampus, and dentate gyrus. Our results suggest that the astrocytosis seen in scrapie-infected animals is, at least in part, owing to actual replication of astrocytes in these animals. We hypothesize that the astrocytes may be an important locus for the disease process.

Increased expression of prion protein is associated with changes in dopamine metabolism and MAO activity in PC12 cells.

Prion diseases of humans and animals occur following infection with infectious agents containing PrP(Sc) or in situations in which there is a mutation of the prion protein (PrP) gene. The cellular prion protein (PrP(C)) is a sialoglycoprotein that is expressed predominantly in neurons. PrP(C) is converted into a pathogenic form of PrP (PrP(Sc)), which is distinguishable from PrP(C) by its relative resistance to protease digestion. A number of postulates have been advanced for the function of normal PrP (PrP(C)), but this issue has not been resolved. To investigate the function(s) of PrP(C), we established clonal PC12 cell lines, which have elevated PrP(C) expression. The results show that there were alterations in dopamine metabolism and in monoamine oxidase (MAO) activity in transfected PC12 cells that overexpress PrP(C). There was an increase in concentration of DOPAC, a metabolite of dopamine, and in MAO activity in cells overexpressing PrP(C). MAO is involved in oxidative degradation of dopamine (DA). Our data suggest that PrP(C) plays a role in DA metabolism by regulating MAO activity.

Expression of cytokine genes and increased nuclear factor-kappa B activity in the brains of scrapie-infected mice.

A number of aspects of the pathogenesis of scrapie remain to be elucidated. The cellular and molecular aspects of the neuropathology in scrapie suggest the possibility that the proinflammatory cytokines could act as pathogenic mediators in this neurodegenerative disease. To understand this possibility, we examined the expression of proinflammatory cytokine genes in brains of IM mice-infected with 87V scrapie agent. Additionally, we also analyzed the activity of nuclear factor-kappa B (NF-kappaB), which is the major transcriptional activator for inflammatory cytokines, and formation of reactive oxygen species (ROS) as a common upstream messenger for its activation. The induction of mRNAs of the inflammatory cytokines, IL-1alpha, IL-1beta and TNF-alpha, was detected only in the brains of scrapie-infected mice. The activity of NF-kappaB was significantly increased in the nuclear extracts from brains of the scrapie-infected group and the immunoreactivity of NF-kappaB was increased in the hippocampus and thalamus in the brains of scrapie-infected mice. The NF-kappaB immunoreactivity was observed mainly in GFAP-positive astrocytes and also detected in the PrP-amyloid plaques in the brains of 87V scrapie-infected mice. Gene expression of IL-6 and iNOS, the representative target genes for NF-kappaB activation, were activated only in the infected group. The production of ROS was significantly increased in the brain mitochondrial fractions of scrapie-infected mice. These results suggest that prion accumulation in astrocytes might activate NF-kappaB through the increase of ROS generation, and thus alterations in NF-kappaB-directed gene expression may contribute to both the neurodegeneration and proinflammatory responses which occur in scrapie.

Histopathological changes in the islets of Langerhans in hamsters infected with the 139H strain of scrapie: semi-thin section study.

Using histopathological analysis of semi-thin sections stained with toluidine blue, we observed profound pathological changes in the islets of Langerhans of hamsters infected with the scrapie agent (strain 139H). These included cytoplasmic vesicles, nuclear swelling, and vacuolization in the islet cells. Two types of vacuolization were seen. "Localized vacuolization" (LV) has a distinct edge and is restricted or confined within the cell. "Diffuse vacuolization" (DV) has no distinct edge and is scattered within tissues either inside or outside of cells. DV may span intracellular and extracellular regions of the islet tissues. There were abnormal structures which we termed blood vessel cores (BVCs) in the islets of 139H-infected hamsters. BVC is a hollow space filled up with blood cells. Immunocytochemical staining for insulin antibody suggested that BVC was surrounded by the B cells of the islet. In the present study, we observed that many inflammatory cells passed through the blood-tissue barriers using pathways between cell-junction in the lumen of BVC. We also observed many necklace-like hollow spaces between islet cells. They are the pockets of extracellular space. A novel concept of "the accordion effect" was described to explain a function of the extracellular space. Under normal physiological conditions, as the synthesis of insulin increase in B cells, the volume of the B cells will increase while the volume of the extracellular space will decrease. After a synchronized secretory response from the stimulated B cells, the secretory product would move from the intracellular space into the extracellular space, the volume of the B cells would be decreased and the volume of the extracellular space would be increased. Most of the secretory product might be released into the blood stream immediately, causing an insulin releasing peak in the blood stream, whereas the rest would remain in the enlarged extracellular space. As the cycle repeat, the increasing volume of the B cells will squeeze the remaining insulin into the blood stream gradually. Thus, the expandable extracellular space would serve as buffer system and a reservoir to collect and store some secretory products for future use. We refer to this concept as "the accordion effect". The concept of "the accordion effect" may also be true in other endocrine organs such as pituitary gland and adrenal gland.

The pathological changes in peripheral organs of scrapie-infected animals.

Scrapie is an unconventional neurodegenerative disease in sheep and goats that has been known in Europe for over 260 Years. The scrapie agents affect the brain and are transmissible from animal to animal. Key features of scrapie infections are abnormal behavior and deficits in motor function. These clinical findings can be related to the damage found in the central nervous system. In some scrapie strain-host model systems there are other manifestations of disease that appear to be related to pathological changes found in the peripheral organs, especially in the endocrine organs such as pituitary, adrenal glands, the islet of Langerhans and ovary. In those model systems in which extensive histopathological changes have been seen in peripheral organs, the titers of scrapie infectivity and the levels of the scrapie specific protein, PrPSc, are relatively low in the affected organs. These data suggest but do not prove that changes in peripheral organs are secondary to the scrapie-induced neurodegeneration that is occurring in the brain. In some scrapie strain-host combinations, obesity and aberrant glucose metabolism are seen in the preclinical and clinical phases of the incubation period. There appear to be two pathways that lead to these particular clinical manifestations. In SJL mice infected by the ME7 or 22L strains of mouse-adapted scrapie and in some scrapie-infected sheep, the mechanism is related to changes induced in the hypothalamic-pituitary-adrenal axis. The other pathway is exemplified by hamsters infected with two hamster-adapted scrapie strains, 139H and 22CH; it appears that lesions found in the hypothalamic-islets of Langerhans axis are critical. A number of reviews on the pathological changes in the central nervous system have been published and therefore, in this review article, we focus on the gross and histopathological changes in peripheral organs in several scrapie strain-host combinations. The changes induced in peripheral organs in a number of scrapie strain-host combinations expand the number of diseases in which the unconventional slow infections could serve as a model. Further work in this area could help us to understand the mechanisms and pathways of the pathological changes found in the peripheral organs of the scrapie-infected animals.