Apolipoprotein E-ε4 deficiency and cognitive function in hepatitis C virus-infected patients.

Hepatitis C virus (HCV) causes not only liver damage in certain patients but can also lead to neuropsychiatric symptoms. Previous studies have shown that the type 4 allele of the gene for apolipoprotein E (APOE) is strongly protective against HCV-induced damage in liver. In this study, we have investigated the possibility that APOE genotype is involved in the action of HCV in brain. One hundred HCV-infected patients with mild liver disease underwent a neurological examination and a comprehensive psychometric testing of attention and memory function. In addition, patients completed questionnaires for the assessment of fatigue, health-related quality of life and mood disturbances. Apolipoprotein E gene genotyping was carried out on saliva using buccal swabs. The APOE-ε4 allele frequency was significantly lower in patients with an impairment of working memory, compared to those with a normal working memory test result (P = 0.003). A lower APOE-ε4 allele frequency was also observed in patients with definitely altered attention ability (P = 0.008), but here, the P-value missed the level of significance after application of the Bonferroni correction. Our data suggest that the APOE-ε4 allele is protective against attention deficit and especially against poor working memory in HCV-infected subjects with mild liver disease. Considering the role of apolipoprotein E in the life cycle of the virus, the findings shed interesting new light upon possible pathomechanisms behind the development of neuropsychiatric symptoms in hepatitis C infection.

Herpes simplex virus type 1 DNA is located within Alzheimer's disease amyloid plaques.

The brains of Alzheimer's disease sufferers are characterized by amyloid plaques and neurofibrillary tangles. However, the cause(s) of these features and those of the disease are unknown, in sporadic cases. We previously showed that herpes simplex virus type 1 is a strong risk factor for Alzheimer's disease when in the brains of possessors of the type 4 allele of the apolipoprotein E gene (APOE-epsilon4), and that beta-amyloid, the main component of plaques, accumulates in herpes simplex virus type 1-infected cell cultures and mouse brain. The present study aimed to elucidate the relationship of the virus to plaques by determining their proximity in human brain sections. We used in situ polymerase chain reaction to detect herpes simplex virus type 1 DNA, and immunohistochemistry or thioflavin S staining to detect amyloid plaques. We discovered a striking localization of herpes simplex virus type 1 DNA within plaques: in Alzheimer's disease brains, 90% of the plaques contained the viral DNA and 72% of the DNA was associated with plaques; in aged normal brains, which contain amyloid plaques at a lower frequency, 80% of plaques contained herpes simplex virus type 1 DNA but only 24% of the viral DNA was plaque-associated (p < 0.001). We suggest that this is because in aged normal individuals, there is a lesser production and/or greater removal of beta-amyloid (Abeta), so that less of the viral DNA is seen to be associated with Abeta in the brain. Our present data, together with our finding of Abeta accumulation in herpes simplex virus type 1-infected cells and mouse brain, suggest that this virus is a major cause of amyloid plaques and hence probably a significant aetiological factor in Alzheimer's disease. They point to the usage of antiviral agents to treat the disease and possibly of vaccination to prevent it.

Apolipoprotein E-epsilon 4 and recurrent genital herpes in individuals co-infected with herpes simplex virus type 2 and HIV.

Apolipoprotein E (APOE) alleles have been associated with the severity of, or susceptibility to, infection by various microbes. We investigated the potential association between the APOE-epsilon 4 allele and the rate of recurrence of genital herpes in patients who were HIV positive and herpes simplex virus type 2 (HSV-2) seropositive. The APOE-epsilon 4 allele was significantly associated with recurrent genital ulceration independent of ethnicity, antiretroviral therapy and CD4 count (OR 8.3; 95% CI 2.4 to 28.5). To our knowledge, this is the first published study to demonstrate this association and suggests that APOE-epsilon 4 may represent a future prognostic marker for symptomatic recurrence of genital herpes in individuals with HIV.

Studies on liver chromatin from rats treated with dimethylnitrosamine.

Shortly after injecting a single low dose of N,N-dimethylnitrosamine into rats, the DNA, RNA and histones are methylated, the level in the DNA greatly exceeding that in the histones. The composition of the chromatin and the electrophoretic profiles of the histone and non-histone proteins are not detectably different from those obtained from control animals. Electric birefringence studies suggest that methylation may result in both interparticle cross-linking and some localised loosening of the DNA-protein complex complex.

Methylation of DNAase-digestible DNA and of RNA in chromatin from rats treated with dimethylnitrosamine.

After injecting rats with di[14C]methylnitrosamine we have prepared liver chromatin and have examined firstly, the methylation level of the DNAase I-degradable fraction of the DNA and secondly, the level of methylation and the stability of methylated sites in chromatin RNA. Our results show that the level of 7-methylguanine in the degradable DNA is about 1.3 times that of whole DNA; therefore in the 20% or so of the DNA which is undegradable by DNAase I, the level must be very low or zero. Experiments using chromatin from rats injected with unlabelled dimethylnitrosamine plus [3H]thymidine show that the specific activity is similar in the DNAase I degradable and undegradable fractions, suggesting that there is no preferential repair in the latter region. In chromatin RNA, the level of 7-methylguanine is higher than that of whole DNA and decreases fairly rapidly within 30 h after dimethylnitrosamine treatment. Our results indicate that this decrease is due to some type of excision or repair process rather than to normal turnover.

Studies on liver chromatin RNA from rats treated with alkylating agents.

We have examined firstly some properties of rat liver chromatin RNA and nuclear sap RNA and secondly the incorporation of [3H]orotic acid into the RNA in vivo in control rats and in rats treated with the alkylating agents, N,N-dimethylnitrosamine or methyl methane sulphonate. Half or more of the nuclear RNA is associated with the chromatin and consists mainly of two species: one is labelled and probably comprises "nascent" RNA, and the other is unlabelled and of lower molecular weight. Neither species is attributable to cytoplasmic contamination. Studies with added polylysine with RNAase A and with DNAase I suggest that both species are ironically bound to protein and that the labelled species is not associated with the part of the chromatin DNA most readily degraded by DNAase I. After dimethylnitrosamine treatment, the amount of unlabelled RNA remains constant but the amount of labelled RNA increases after a low dose, and decreases after a high dose. After methyl methane sulphonate treatment, no change occurs in either species. These results can be explained by changes in extent of association of the DNA and protein within the chromatin complex.

Does apolipoprotein E polymorphism influence susceptibility to malaria?

Outcome of infection varies greatly among people, and in the case of three very different viruses, it is determined by apolipoprotein E (APOE) genotype. APOE might affect outcome of malaria infection also, since apoE protein and the protozoon (like the viruses) share cell entry mediators (heparan sulphate proteoglycans and/or specific apoE receptors). APOE polymorphisms give rise to protein variants that differ in binding strength to these mediators; thus, the extent of competition between apoE and protozoon for cell entry, and hence magnitude of protozoan damage, might depend on apoE isoform. Genotypes of infants infected with malaria were examined. It was found that APOE epsilon 2 homozygotes became infected at an earlier age than those carrying the other genotypes, the difference being statistically significant. Parasite densities, all of which were low, did not differ significantly. This effect, although based on small numbers, suggests that APOE epsilon 2 may be a risk factor for early infection.

Apolipoprotein E and herpes virus diseases: herpes simplex keratitis.

Our previous studies showed that herpes simplex type 1 virus (HSV1) is present in a high proportion of the brain of elderly normal people and Alzheimer's disease (AD) patients. We subsequently discovered that the combination of HSV1 in brain and carriage of the type 4 allele of the gene for apolipoprotein E (apoE-epsilon 4) is a strong risk factor for AD, and also that apoE-epsilon 4 is a strong risk factor for herpes labialis. In this study we have examined apoE genotypes of sufferers from another disorder caused by HSV1, namely, herpes simplex keratitis (HSK), to find if an apoE allele is involved in the disorder. In 46 HSK patients the apoE-epsilon 4 allele frequency was 15%-the same as that found in 238 unaffected controls. The apoE-epsilon 2 allele frequency was 13%-higher than the value of 7% for unaffected people, but the difference is not statistically significant.

Herpes simplex virus type 1 and Alzheimer's disease.

Until recently, the only risk factors implicated in noninherited cases of Alzheimer's disease were increasing age, Down's syndrome, and probably, head injury. Having found that herpes simplex type 1 virus (HSV1) is present in the brain of many elderly people, we discovered that it is a risk factor for Alzheimer's disease when in the central nervous system of APOE-epsilon4 allele carriers. On the basis of this result and our finding that apoE-epsilon4 is a risk factor for herpes labialis, we suggested that the combination of virus and genetic factor is particularly damaging in the nervous system. The present review describes 1) the search for HSV1 in human brain; 2) HSV1 infection of the peripheral nervous system; 3) HSV1 infection of the central nervous system; 4) how APOE genotype might influence HSV1 infection; 5) possible APOE genotype effect on viral latency and its reactivation; 6) interactions of viruses with lipoproteins, their components, and lipoprotein receptors; 7) the role of APOE in repair; 8) pathological processes in AD and their relationship to prior damage; and 9) implications for the prevention or treatment of Alzheimer's disease.

Infiltration of the brain by pathogens causes Alzheimer's disease.

Despite very numerous studies on Alzheimer's disease (AD), especially on amyloid plaques and neurofibrillary tangles, little information has been obtained thus on the causes of the disease. Evidence is described here that implicates firstly herpes simplex virus type 1 (HSV1) as a strong risk factor when it is present in brain of carriers of the type 4 allele of the gene for apolipoprotein E (APOE-4). Indirect support comes from studies indicating the role of APOE in several diverse diseases of known pathogen cause. A second putative risk factor is the bacterium, Chlamydia pneumoniae. This pathogen has been identified and localized in AD brain. Current studies aimed at "proof of principle" address the entry of the organism into the CNS, the neuroinflammatory response to the organism, and the role that the organism plays in triggering AD pathology. An infection-based animal model demonstrates that following intranasal inoculation of BALB/c mice with C. pneumoniae, amyloid plaques/deposits consistent with those observed in the AD brain develop, thus implicating this infection in the etiology of AD.

Vaccination prevents latent HSV1 infection of mouse brain.

Herpes simplex encephalitis (HSE) is a rare but very serious disorder caused by herpes simplex type 1 virus (HSV-1). Treatment with acyclovir decreases mortality but many patients still suffer cognitive impairment subsequently. A vaccine against HSV1 would therefore be of great value. HSV-1 has been implicated also in Alzheimer's disease (AD): we established that HSV1 resides in the brain of about two thirds of AD patients and aged normal people, and that in carriers of the type 4 allele of the apolipoprotein E gene, it is a strong risk factor for AD. Thus a vaccine against HSV-1 might prevent development of AD in some cases. To find whether a vaccine of mixed HSV-1 glycoproteins (ISCOMs), which protects mice from latent HSV-1 infection of sensory ganglia, prevents HSV1 latency in the CNS, ISCOM-vaccinated or unvaccinated animals were infected with HSV-1. Using polymerase chain reaction (PCR) we detected HSV-1 in brain from 16 of 39 unvaccinated mice (41%), but only 3 of 41 vaccinated mice (7%) (P < 0.001). Thus, ISCOMs protect the CNS also, suggesting their possible future usage in humans.

Possible factors in the etiology of Alzheimer's disease.

Inherited cases of Alzheimer's disease (AD) comprise only a very small proportion of the total. The remainder are of unknown etiopathogenesis, but they are very probably multifactorial in origin. This article describes studies on four possible factors: aluminum; viruses--in particular, herpes simplex type I virus (HSV1); defective DNA repair; and head trauma. Specific problems associated with aluminum, such as inadvertent contamination and its insolubility, have led to some controversy over its usage. Nonetheless, the effects of aluminum on animals and neuronal cells in culture have been studied intensively. Changes in protein structure and location in the cell are described, including the finding in this laboratory of a change in tau resembling that in AD neurofibrillary tangles, and also the lack of appreciable binding of aluminum to DNA. As for HSV1, there has previously been uncertainty about whether HSV1 DNA is present in human brain. Work in this laboratory using polymerase chain reaction has shown that HSV1 DNA is present in many normal aged brains and AD brains, but is absent in brains from younger people. Studies on DNA damage and repair in AD and normal cells are described, and finally, the possible involvement of head trauma is discussed.

The role of viruses and of APOE in dementia.

The virus, herpes simplex virus type 1 (HSV1), when present in brain, acts together with the type 4 allele of the APOE gene, a known susceptibility factor in Alzheimer disease (AD), to confer a strong risk of AD; in carriers of the other two main alleles of the gene, the virus does not confer a risk. It also has been shown that the outcome of infection in the case of five diseases known to be caused by viruses is determined by APOE. It is hoped that the discovery of the involvement of HSV1 in AD will lead to future antiviral therapy and possibly to immunization against the virus in infancy.

An improved method for preparing DNA from human brain.

The preparation of DNA from human brain by conventional protease and RNAase digestion, phenol-chloroform extraction and ethanol precipitation results in very low yields. This is probably due to interference by lipid present at very high levels in this tissue. We have overcome this by solubilizing the initial tissue homogenate by heating at 60 degrees C for 30 min in sodium dodecyl sulphate. This results in a threefold increase in yield and a considerable reduction in preparation time. The molecular weight of the DNA is greater than 20 X 10(6) milion.

Human neuroblastoma cells treated with aluminium express an epitope associated with Alzheimer's disease neurofibrillary tangles.

A number of studies have implicated aluminium as a possible factor in the pathogenesis of Alzheimer's disease (AD). Following an examination of the uptake of aluminium by human neuroblastoma cells in culture, treated with a range of concentrations of aluminium complexed with ethylene-diaminetetra-acetic acid (EDTA), we have now carried out an immunocytochemical study. Using an antibody to phosphorylated tau protein, which reacts specifically with AD neurofibrillary tangles (NFT), we have found that after treatment periods of 16 days to 8 weeks with aluminium-EDTA, the cells show positive staining with this antibody. No such reaction was detected in cells grown in medium alone, nor in aluminium-EDTA-treated cells subjected to the same immunocytochemical procedure but without added primary antibody. Cells grown in medium plus EDTA, which contains a low level of aluminium contamination, showed a slight reaction. Our system may provide a suitable model for studying the early changes which lead to NFT formation.

DNA double-strand breaks measured by pulsed-field gel electrophoresis in irradiated lymphocytes from normal humans and those with Alzheimer's disease.

We have previously found that radiation-induced chromosome aberrations (dicentrics) are more numerous in lymphocytes from Alzheimer's disease (AD) patients than in those from age-matched normal individuals (Tobi et al. 1990). To investigate this further, we have examined double-strand breaks (dsb) produced by gamma-irradiation in the DNA of AD and normal lymphocytes by using pulsed-field gel electrophoresis. The percentage of DNA migrating into the gels is an indirect measure of the number of dsb; we have assayed the DNA content of sequential slices of the gel by direct fluorometry and have found that the percentage migrating is dose dependent. Our results show that the level of damage is similar in AD and normal lymphocytes and preliminary assays of the rate of repair suggest that the half-time is also similar, the value being > 1 h. The latter is consistent with the known rate of rejoining of chromosome fragments in interphase lymphocytes (Pantelias and Maillie 1985). The results suggest that at a gross level dsb repair is not impaired in AD cells; however, we cannot exclude the possibility that there is misrepair or non-repair of a small fraction of the dsb, which might account for the greater radiosensitivity of the AD cells.

Studies on histones and non-histone proteins from rats treated with dimethylnitrosamine.

A study has been made of the histone and non-histone chromosomal proteins of rat liver after treatment in vivo with dimethylnitrosamine (DMN) (2 mg/kg). DMN was found not to affect histone turnover, as measured by 3H-labelled amino-acids incorporation. A decrease was observed in specific activity of the histones with time after injection of [14C]DMN or [14C]-formate and this was attributable to demethylation of both abnormal and normal methylation sites in these proteins. In the case of the non-histone proteins, DMN was found to increase greatly the turnover of those non-histone proteins loosely associated with chromatin DNA and RNA; turnover of those non-histone proteins tightly bound to chromatin DNA and RNA was unaffected. Demethylation of both normal and abnormal methylation sites was found to take place from both non-histone protein fractions. In the case of the loosely bound non-histone proteins a lower rate of demethylation was observed after DMN treatment.

Effect of N-methyl-N-nitrosourea on avian erythrocyte nuclei reactivation.

Avian erythrocytes are terminally differentiated cells but they can be reactivated by fusion with actively metabolising cells. We have examined the effects of treating the erythrocytes with a carcinogenic methylating agent, N-methyl-N-Nitrosourea (MNU), on the process of reactivation of adult and embryonic nuclei. We have found that the rate of nuclear enlargement is slightly lower in nuclei from MNU-treated cells than from control cells and that there is a marked delay of about 24 h in the appearance of nucleoli in both adult and embryonic cells. This is not due to an effect of MNU on ribosomal (r)DNA: the number of rDNA genes appears to be similar in treated and control cells. Also, the number of rDNA genes appears to be similar in adult and embryonic cells and in unreactivated and reactivated embryonic nuclei: thus, differences in reactivation rate between adult and embryonic cells, observed by us and others, can not be attributed to a gross difference in their ribosomal DNA contents, and reappearance of nucleoli on reactivation can not be due to an amplification of rDNA (i.e., to recovery of such genes if lost on terminal differentiation). We suggest that MNU, although a monofunctional alkylating agent, may cause increased association--possibly cross-linkage--between DNA and protein in chromatin, thereby hindering access of host cell reactivating proteins, especially to the nucleolar regions.

Distribution and repair of O6-methylguanine in different fractions of rat liver DNA after dimethylnitrosamine administration.

The removal of the promutagenic DNA alkylation product O6-methylguanine from different fractions of rat liver DNA has been examined using the technique of DNA-DNA reassociation. Male Wistar rats were given a low non-toxic dose of N,N-dimethylnitrosamine (DMN) (2 mg/kg) and killed 3 or 18 h later (a period corresponding to the removal of 50% of the O6-methylguanine from 'total' liver (DNA). DNA was extracted from liver, denatured in alkali and incubated at 60 degrees C for periods corresponding to the reassociation of highly repetitive (polycopy), middle repetitive and 'unique' sequences i.e. different 'kinetic' classes of DNA. Reassociated and single-stranded DNA were separated by hydroxyapatite chromatography and analyse for O6-methylguanine content. Three hours after administration of DMN the levels of O6-methylguanine in the reassociated and single-stranded DNA were the same after each period of reassociation indicating that O6-methylguanine was randomly distributed among the DNA classes. At 18 h the levels of O6-methylguanine were again the same in the reassociated and single-stranded DNA but approx. 50% lower than in the 3 h DNA samples. The rate of loss of O6-methylguanine from the three DNA classes was thus the same and there was therefore no indication of preferential removal of this base from any one kinetic class of DNA under the conditions used.