Association of 3'-UTR polymorphisms of the oxidised LDL receptor 1 (OLR1) gene with Alzheimer's disease.

Although possession of the epsilon 4 allele of the apolipoprotein E gene appears to be an important biological marker for Alzheimer's disease (AD) susceptibility, strong evidence indicates that at least one additional risk gene exists on chromosome 12. Here, we describe an association of the 3'-UTR +1073 C/T polymorphism of the OLR1 (oxidised LDL receptor 1) on chromosome 12 with AD in French sporadic (589 cases and 663 controls) and American familial (230 affected sibs and 143 unaffected sibs) populations. The age and sex adjusted odds ratio between the CC+CT genotypes versus the TT genotypes was 1.56 (p=0.001) in the French sample and 1.92 (p=0.02) in the American sample. Furthermore, we have discovered a new T/A polymorphism two bases upstream of the +1073 C/T polymorphism. This +1071 T/A polymorphism was not associated with the disease, although it may weakly modulate the impact of the +1073 C/T polymorphism. Using 3'-UTR sequence probes, we have observed specific DNA protein binding with nuclear proteins from lymphocyte, astrocytoma, and neuroblastoma cell lines, but not from the microglia cell line. This binding was modified by both the +1071 T/A and +1073 C/T polymorphisms. In addition, a trend was observed between the presence or absence of the +1073 C allele and the level of astrocytic activation in the brain of AD cases. However, Abeta(40), Abeta(42), Abeta total, and Tau loads or the level of microglial cell activation were not modulated by the 3'-UTR OLR1 polymorphisms. Finally, we assessed the impact of these polymorphisms on the level of OLR1 expression in lymphocytes from AD cases compared with controls. The OLR1 expression was significantly lower in AD cases bearing the CC and CT genotypes compared with controls with the same genotypes. In conclusion, our data suggest that genetic variation in the OLR1 gene may modify the risk of AD.

Pathological relationships between microglial cell activity and tau and amyloid beta protein in patients with Alzheimer's disease.

The extent of microglial cell activation (microglial cell load) was estimated by image analysis of ferritin-immunostained sections of frontal cortex from 72 patients with pathologically confirmed Alzheimer's disease (AD), and correlated with the amount of pathological tau and amyloid beta protein (Abeta), as both Abeta(40) and Abeta(42) load, in adjacent sections of the same cases. Microglial cell load did not correlate with either Abeta(40) or Abeta(42) load but was significantly correlated with pathological tau load. Microglial cell load was unrelated to age at onset of disease or duration of illness. It is possible that because the presence of microglial cells predates that of pathological tau proteins within the cerebral cortex in AD, neurofibrillary damage to nerve cells may stem from the release of proinflammatory and other potentially neurotoxic molecules from microglial cells.

A polymorphism within intron 11 of the tau gene is not increased in frequency in patients with sporadic Alzheimer's disease, nor does it influence the extent of tau pathology in the brain.

There are numerous polymorphisms within the tau gene but these are in complete linkage disequilibrium and exist as two common extended haplotypes H1 and H2. We have investigated the frequency of these haplotypes in 83 cases of sporadic Alzheimer's disease (AD) using the +34 polymorphism in intron 11 of the tau gene as a marker of H1 and H2 haplotypes. The total amount of hyperphosphorylated tau protein (tau load), present as neurofibrillary tangles, neuropil threads or plaque neurites, was quantified in the frontal cortex of these patients and related to tau haplotype. We found no increase in H1H1 haplotype in this autopsy population of cases with AD compared to published control data. Stratification of cases for apolipoprotein E (APO E) genotype showed a slight, but not statistically significant, overrepresentation of epsilon 4 allele amongst bearers of H2 haplotype. There were no overall differences in tau load between haplotype groups though cases within each haplotype group bearing APO E epsilon 4 allele had a significantly higher tau load than those without epsilon 4 allele. Neither age at onset or duration of illness differed according to tau haplotype. We conclude that the frequency of tau gene H1 haplotype is not elevated in AD and possession of this has no impact upon the amount of tau pathology in AD.

The angiotensin 1-converting enzyme insertion (I)/deletion (D) polymorphism does not influence the extent of amyloid or tau pathology in patients with sporadic Alzheimer's disease.

An insertion (I)/deletion (D) polymorphism in the angiotensin 1-converting enzyme (ACE) gene has, in some studies, been associated with increased risk for Alzheimer's disease (AD), and functionally the enzyme has been implicated in the degradation of amyloid beta protein (Abeta). We have investigated the frequency of the I/D polymorphism in a clinic-based and autopsy-confirmed series of cases of AD, and investigated what impact the I/D polymorphism in ACE gene might have on the extent of Abeta and tau pathology in the frontal cortex in the autopsy-confirmed series. We found no differences in I/D allele or genotype frequencies between the clinic-based and autopsy-confirmed AD cases, or between the pooled clinic-based and autopsy-confirmed AD cases and a series of normal control subjects. Moreover, Abeta (Abeta(40) and Abeta(42)) load, tau load or extent of amyloid angiopathy did not differ between D/D, I/D and I/I genotype groups, though Abeta(42) load tended to be higher in bearers of I/I genotype (compared to D/D genotype). Neither age at onset nor duration of illness differed according to genotype. We conclude therefore that the frequency of ACE I-allele is not increased in AD and, in autopsy-confirmed AD cases, possession of the ACE I allele has no impact upon the pathology of AD, at least in terms of the amount of Abeta or tau deposited in the brain.

Causes of conjunctivitis and keratoconjunctivitis in Karachi, Pakistan.

The causes of conjunctivitis and keratoconjunctivitis in 388 patients who attended eye casualty departments in Karachi, Pakistan, during a 5 month period were investigated. Most of these infections were diagnosed as adenovirus (291, 75%) or bacterial (71, 18.3%). Of the remainder, 9 cases (2.3%) were caused by herpes simplex virus and 7 (1.8%) by Chalmydia trachomatis. There was no evidence of typical active trachoma in this urban population. Bacteria or Candida albicans were also grown from 44 of the adenovirus cases (15%). Many of the bacteria grown from eyes in this study were resistant to antibiotics, probably because of inadequate and/or inappropriate self-medication with antibiotics in this community.

A rapid and sensitive culture test for detecting herpes simplex virus from the eye.

A rapid and sensitive culture test has been developed for detecting herpes simplex virus (HSV) in ocular infections. The virus is cultured by inoculation and centrifugation of cell monolayers grown on coverslips and the inclusions detected by an indirect immunofluorescence technique. This rapid test takes only two days to complete. By comparison, in our hands the conventional culture test, which depends on the development of cytopathic effect, took between 1 and 20 days with a mean of 4.7 days. Of the 1638 ocular clinical specimens inoculated in parallel by the two methods a total of 188 were positive for HSV. The virus was detected from 184 (97.8%) specimens by the rapid test and from 144 (76.6%) by the conventional test (McNemar's test, U = 5.76, p less than 0.001).

Comparative sensitivity of a cultural test and the complement fixation test in the diagnosis of adenovirus ocular infection.

The sensitivity of human embryonic kidney (HEK) cell culture and the complement fixation test (cft) in the diagnosis of adenovirus ocular infection has been compared. The optimum time for collection of specimens to obtain the best results by each test has been examined. Sixty-one (53%) of 116 patients with moderate to severe follicular conjunctivitis, clinically indicative of adenovirus infection, had serological evidence of infection or had the virus isolated from ocular swabs. Virus was isolated from 53 patients (45%) and significant antibody rises were found in 33 (27%). When each test was carried out under optimum conditions, virus was isolated from 82% of cases and serological evidence of infection found in 72%. Serological responses as detected by the CFT appeared to be inferior in infections caused by adenovirus type 7 to those encountered in infections due to adenovirus types 3 and 8.

A comparison of the efficiency of human embryo intestine, Hep 2 cells, and human embryo kidney cells for the primary isolation of ophthalmic viruses.

A comparison has been made of the efficiency of 3 cell systems, human embryo kidney (HEK), Hep 2, and a continuous line of human embryo intestine (HEI), for the isolation of ophthalmic viruses. A total of 40 herpes simplex, 51 adenoviruses, and 2 vaccinia viruses were isolated from 323 specimens. HEK cells were found to be the optimal system, 85 out of 93 (91%) of the viruses being detected in these cells alone. However, HEK cells are difficult to obtain, and therefore the use of a combination of the continuous cell lines HEI and Hep 2 is recommended as an alternative. 89% of the viruses were detected by this combination. The use of either HEI or Hep 2 cells alone was unsatisfactory.

Clinical and epidemiological features of adenovirus keratoconjunctivitis in London.

Ninety-eight patients were studied. Ninety were consecutive patients who were isolation-positive for adenovirus, and 8, who were associated with a hospital outbreak of adenovirus serotype 8 infection, developed characteristic features of infection but were isolation-negative. The ratio of males to females was 2 to 1, and most patients were aged 20 to 39. Adenovirus serotypes 3, 7, and 8 were isolated from 86% of patients, and serotypes 2, 4, 5, 11, 15, and 15/29 from the remaining 14%. Adenovirus serotype 7 was more commonly isolated from patients under the age of 19 and was Not isolated during winter. Sources of infection could be identified in 36% of patients and included contact with upper respiratory tract of ocular infections, a hospital outbreak, and a recent visit to a swimming pool. Associated systemic disease was detected in 47% of patients, most of whom had upper respiratory tract infection. The most severe and prolonged conjunctivitis was caused by serotypes 5 and 8. Most patients developed epithelial punctate keratitis. Subepithelial punctate keratitis, which was once-considered to be a characteristic feature of adenovirus serotype 8, developed in cases of serotype 3, 4, 5, 7, and 8 infection.

Rapid culture test for adenovirus isolation.

A rapid culture test has been developed and evaluated for the detection of adenovirus in ocular infections. The test requires only two days' incubation of the inoculated cell monolayers on coverslips in flat bottomed tubes followed by detection of adenovirus inclusions using immunofluorescence staining method. The sensitivity of the rapid test is found to be comparable with that of the conventional tissue culture test which depends on the development of a cytopathic effect requiring 2-21 days' incubation (mean 9 X 4, SD 4 X 9).

An outbreak of acute haemorrhagic conjunctivitis in Kaduna, Nigeria.

Clinical studies were carried out on two groups of patients with acute haemorrhagic conjunctivitis (AHC) during an epidemic in 1985 in Northern Nigeria. Group 1 consisted of 99 students attending a girls' boarding school, group 2 of 200 patients selected randomly from 1000 examined at the local clinic. Moderate to severe hyperaemia and papillary responses were present in the palpebral conjunctiva of all patients, and 234 (66%) had subconjunctival haemorrhages. Transient superficial punctate keratitis was noted in over 60% of patients. A transient flare suggestive of a low grade iritis was seen in five patients. No neurological disorders were noted. Serological studies were carried out on patients from group 2. Fifteen paired and 20 single serum samples were titrated against adenovirus type 4 (Ad-4) and enterovirus type 70 (EV-70). Two pairs of sera showed a 4-fold rise in antibody levels to EV-70, whereas the antibody titres to EV-70 in the rest of the sera ranged from 1:20 (no antibody) to 1:160. None of the paired serum samples showed a 4-fold rise in antibody levels to adenovirus. The results of clinical studies and serological findings support EV-70 as a probable cause of AHC in Nigeria.

Adenovirus serotypes isolated from ocular infections in London.

During the period 1973-8 700 adenoviruses were isolated from the eyes of patients presenting at Moorfields Eye Hospital. Of these, 678 were serotyped by a neutralisation test. Twenty-one different serotypes were identified. Serotype 3, 7, and 10 accounted for 68% of the isolates, 4, 8, 15 (15/29), and 19 for 25%, and the other 14 serotypes for 7%. Community outbreaks of ocular infections by adenovirus 3, 4, 7, 10, and 15 (15/29) were observed. Outbreaks with adenovirus 3, 7, and 10 appeared to continue for 2 years or more, whereas outbreaks with 4 and 15 (15/29) were restricted to one year or less. Hospital outbreaks by adenovirus 8 and 19 were also recorded. During the same period 18 of these 21 adenovirus serotypes were isolated from the nonocular sites (mainly respiratory tract) in 7804 cases. There was a close association in the distribution of adenovirus 1, 2, 3, 5, and 6 in the ocular and nonocular sites. No such association was observed for adenovirus serotypes 4, 8, 10, 15 (15/29), and 19.

Genetic associations between cathepsin D exon 2 C-->T polymorphism and Alzheimer's disease, and pathological correlations with genotype.

Genetic variations represent major risk factors for Alzheimer's disease (AD). While familial early onset AD is associated with mutations in the amyloid precursor protein and presenilin genes, only the e4 allele of the apolipoprotein E (APOE) gene has so far been established as a genetic risk factor for late onset familial and sporadic AD. It has been suggested that the C-->T (224Ala-->Val) transition within exon 2 of the cathepsin D gene (CTSD) might represent a risk factor for late onset AD. The objective of this study was to investigate whether possession of the CTSD exon 2 T allele increases the risk of developing AD, and to determine whether this modulates the amyloid pathology of the disease in conjunction with, or independent of, the APOE e4 allele. Blood samples were obtained from 412 patients with possible or probable AD and brain tissues from a further 148 patients with AD confirmed by postmortem examination. CTSD and APOE genotyping were performed by PCR on DNA extracted from blood, or from frontal cortex or cerebellum in the postmortem cases. Pathological measures of amyloid beta protein (Abeta), as plaque Abeta40 and Abeta42(3) load and degree of cerebral amyloid angiopathy were made by image analysis or semiquantitative rating, respectively. CTSD genotype frequencies in AD were not significantly different from those in control subjects, nor did these differ between cases of early or late onset AD or between younger and older controls. There was no gene interaction between the CTSD T and APOE e4 alleles. The amount of plaque Abeta40 was greater in patients carrying the CTSD T allele than in non-carriers, and in patients bearing APOE e4 allele compared with non-carriers. Possession of both these alleles acted synergistically to increase levels of plaque Abeta40, especially in those individuals who were homozygous for the APOE e4 allele. Possession of the CTSD T allele had no effect on plaque Abeta42(3) load or degree of CAA. Possession of the CTSD T allele does not increase the risk of developing AD per se, but has a modulating effect on the pathogenesis of the disorder by increasing, in concert with the APOE e4 allele, the amount of Abeta deposited as senile plaques in the brain in the form of Abeta40.

Is there a relation between APOE expression and brain amyloid load in Alzheimer's disease?

BACKGROUND: It has been proposed that, independent of the epsilon4 allele, APOE promoter polymorphisms (-491 A/T and -219 G/T) may be risks factor for Alzheimer's disease by modulating APOE expression. OBJECTIVE: To measure the level of APOE expression in Alzheimer's disease. METHODS: Brains were obtained at necropsy from 114 patients with early and late onset sporadic Alzheimer's disease in Greater Manchester (UK) during years 1986 to 2001. Total RNA was extracted from 84 brains. Purified lymphocytes were obtained from fresh blood from 16 probable Alzheimer cases from Lille (France). APOE and beta-actin gene expression was determined by reverse transcriptase polymerase chain reaction in brain and lymphocytes. RESULTS: An inverse correlation between APOE expression level and A beta loads was observed. As previously described and extended to 114 cases here, an association between the -219 TT genotype and a higher level of parenchymal A beta deposition was found, irrespective of APOE epsilon4 allele status. This effect was more pronounced in older individuals, whereas higher A beta load appeared more closely related to epsilon4 in the younger age group (cut off point at the median age at death (72.5 years)). The -219 TT genotype was associated with a decrease in APOE expression. There was a 60% decrease in APOE expression in lymphocytes from probable Alzheimer cases v controls (p = 0.01). CONCLUSIONS: In the oldest individuals, reduced APOE expression, modulated in part by -219 G/T polymorphism, may influence risk and constitute a determinant A beta load in Alzheimer's disease.

Effect of centrifugation on herpes simplex virus isolation.

The effects of high-speed centrifugation on the isolation of herpes simplex virus (HSV) were studied. Aliquots of laboratory or clinical specimens were inoculated into test tubes and flat-bottomed tubes containing HEp2 monolayers. Test tubes were incubated at 35 degrees C on roller drums (standard method), and flat-bottomed tubes were centrifuged at 15,000g at 35 degrees C for 1 hr, before being incubated at 35 degrees C without rolling (centrifuged method). Centrifugation of clinical and laboratory specimens of HSV type 1 and HSV type 2 produced significantly increased isolation rates compared with the standard method. When clinical and laboratory specimens were diluted, the centrifuged method was more sensitive at all dilutions. When 20 specimens were used for end-point titrations, the centrifuged method was 10 times more sensitive for 15 specimens and 100 times more sensitive for five specimens. There was no difference in the time taken for the appearance of cytopathic effect (CPE) between the standard and centrifuged methods.

Retrospective screening for hepatitis C in a tertiary paediatric referral centre.

The true prevalence of hepatitis C virus in children in the UK is not known and targeted screening is not standard practice despite an anticipated rise in new cases due to vertical transmission. An extension of the Department of Health's 'look-back' exercise was undertaken in order to determine the prevalence of hepatitis C virus in high-risk patient groups who were transfused with blood and/or blood products before 1991. Five hundred and ninety-five patients transfused between 1971-91 were traced and offered counselling and testing. Blood samples from 405 were analysed for the presence of HCV antibodies and/or HCV RNA by RT-PCR and eight patients were found to be positive. The HCV seroprevalence rate in this cohort was 1.97% and the HCV genome detection rate was 1.72%. In view of the long-term complications from this infection and the availability of potentially effective anti-viral agents, we feel that targeted screening is of value in this setting.

A rapid and sensitive culture test for the laboratory diagnosis of genital herpes in women.

A rapid and sensitive cell culture test has been developed to detect herpes simplex virus (HSV) in women with genital herpes. The virus is cultured by inoculation and centrifugation of cell monolayers, and the virus inclusions are detected using an indirect immunofluorescence test. The test takes only 48 hours to complete compared with the conventional cell culture test, which may take up to eight days. Of a total of 2100 cervical specimens collected from unselected women attending a sexually transmitted diseases (STD) clinic and inoculated in parallel, HSV was isolated from 55 specimens by either or both tests. Of these 55 positive specimens, 54(98%) were positive by the rapid test but only 24(44%) by the conventional test (McNemars test; p less than 0.001).

Identification of adenoviruses in faeces from patients with diarrhoea at the Hospitals for Sick Children, London, 1989-1992.

Faecal samples from 137 patients that had been shown to contain adenoviruses by electron microscopy were identified in a series of enzyme immunoassays (EIA) using a single monoclonal antibody (Mab) to adenovirus 40 and four different Mabs to adenovirus 41. Adenoviruses were partially characterised by restriction enzyme analysis (REA) of DNA extracts using SmaI. Samples were also run in a commercial EIA (Adenovirus IDEIA; Dako, Ltd.) which detects group antigen. The majority (84%) of adenoviruses were subgenus F: adenovirus type 41, 87 (64%) and adenovirus type 40, 28 (20.4%). Subgenus A viruses were identified in ten, (7%) patients, eight were type 31, and two type 12. The adeno IDEIA test was sensitive and specific, detecting 127 of 131 positives and giving no false-positive results with other enteric viruses. Use of monoclonal-based EIAs showed significant differences depending on which adeno 41 Mab was used, although the restriction patterns obtained using SmaI appeared to be identical for 66 of 69 samples that produced recognisable bands. The Mab that performed best, M 4.3.1, was raised against strains obtained from children in England and detected 83 of 84 (99%) of the adenovirus 41 samples tested. In contrast Mab JH/41 raised against the prototype strain of adenovirus 41 (Tak) detected only 69 of 87 (79%).

Epstein-Barr virus monitoring in paediatric renal transplant recipients.

Prospective Epstein-Barr virus (EBV) surveillance post transplant was undertaken by qualitative polymerase chain reaction testing for EBV DNA in plasma so as to detect EBV viremia as early as possible and thereby attempt to pre-empt post-transplant lymphoproliferative disease by reduction of immunosuppression. Forty-three children (46 transplants) were followed for a median (range) of 15.5 (3-25) months. Thirty-one children (67%) were EBV seropositive pre transplant. Twenty children (44%) developed EBV viremia; of these 9 (60%) were seronegative and 11 (36%) seropositive recipients. Primary infection developed later (median difference 14.2 weeks, P=0.009), was more likely to be symptomatic (odds ratio 2.91, 95% confidence interval 0.95-4.88) and associated with a rise in serum creatinine (odds ratio 6.13, 95% confidence interval 4.13-8.13) than reactivation disease. There was a higher incidence of EBV disease in children receiving quadruple therapy and tacrolimus (odds ratio 13.2, 95% confidence interval 11.5-14.9) compared with those given cyclosporin-based immunosuppression. Immunosuppression was reduced when EBV infection was detected. All children became asymptomatic and renal function returned to normal by a median (range) of 17 (6-52) days, although mild relapses occurred in 3 children. Regular EBV surveillance allowed prompt reduction of immunosuppression and was associated with a good outcome in this group of children.