Reduced levels of human apoE4 protein in an animal model of cognitive impairment.

The APOE4 allele is the most common genetic determinant for Alzheimer's disease (AD) in the developed world. APOE genotype specific differences in brain apolipoprotein E protein levels have been observed in numerous studies since the discovery of APOE4's link to AD. Since the human apoE4 targeted replacement mice display characteristics of cognitive impairment we sought to determine if reduced levels of apoE might provide one explanation for this impairment. We developed a novel mass spectrometry method to measure apoE protein levels in plasma. Additionally, we developed an ELISA that replicates the mass spectrometry data and enables the rapid quantitation of apoE in plasma, brain and cerebrospinal fluid. We detected a significant decrease in plasma, brain and cerebrospinal fluid apoE levels in the apoE4 mice compared to apoE2 and E3 mice. We also measured a small (∼19%) decrease in brain apoE levels from aged, non-demented APOE4 carriers. Our findings suggest that a fraction of APOE4-linked AD may be due to insufficient levels of functional apoE required to maintain neuronal health.

Progressive loss of synaptic integrity in human apolipoprotein E4 targeted replacement mice and attenuation by apolipoprotein E2.

Inheritance of the APOE4 allele is a well established genetic risk factor linked to the development of late onset Alzheimer's disease. As the major lipid transport protein in the central nervous system, apolipoprotein (apo) E plays an important role in the assembly and maintenance of synaptic connections. Our previous work showed that 7 month old human apoE4 targeted replacement (TR) mice displayed significant synaptic deficits in the principal neurons of the lateral amygdala, a region that is critical for memory formation and also one of the primary regions affected in Alzheimer's disease, compared to apoE3 TR mice. In the current study, we determined how age and varying APOE genotype affect synaptic integrity of amygdala neurons by comparing electrophysiological and morphometric properties in C57BL6, apoE knockout, and human apoE3, E4 and E2/4 TR mice at 1 month and 7 months. The apoE4 TR mice exhibited the lowest level of excitatory synaptic activity and dendritic arbor compared to other cohorts at both ages, and became progressively worse by 7 months. In contrast, the apoE3 TR mice exhibited the highest synaptic activity and dendritic arbor of all cohorts at both ages. C57BL6 mice displayed virtually identical synaptic activity to apoE3 TR mice at 1 month; however this activity decreased by 7 months. ApoE knockout mice exhibited a similar synaptic activity profile with apoE4 TR mice at 7 months. Consistent with previous reports that APOE2 confers protection, the apoE4-dependent deficits in excitatory activity were significantly attenuated in apoE2/4 TR mice at both ages. These findings demonstrate that expression of human apoE4 contributes to functional deficits in the amygdala very early in development and may be responsible for altering neuronal circuitry that eventually leads to cognitive and affective disorders later in life.

Apolipoprotein E allele-dependent pathogenesis: a model for age-related retinal degeneration.

Age-related macular degeneration (AMD) is a late-onset, multifactorial, neurodegenerative disease of the retina and the leading cause of irreversible vision loss in the elderly in the Western world. We describe here a murine model that combines three known AMD risk factors: advanced age, high fat cholesterol-rich (HF-C) diet, and apolipoprotein E (apoE) genotype. Eyes of aged, targeted replacement mice expressing human apoE2, apoE3, or apoE4 and maintained on a HF-C diet show apoE isoform-dependent pathologies of differential severity. ApoE4 mice are the most severely affected. They develop a constellation of changes that mimic the pathology associated with human AMD. These alterations include diffuse sub-retinal pigment epithelial deposits, drusenoid deposits, thickened Bruch's membrane, and atrophy, hypopigmentation, and hyperpigmentation of the retinal pigment epithelium. In extreme cases, apoE4 mice also develop marked choroidal neovascularization, a hallmark of exudative AMD. Neither age nor HF-C diet alone is sufficient to elicit these changes. We document choroidal neovascularization and other AMD-like ocular pathologies in an animal model that exploits known AMD risk factors. The model is additionally attractive because it is not complicated by invasive experimental intervention. Our findings in this model implicate the human apoE E4 allele as a susceptibility gene for AMD and support the hypothesis that common pathogenic mechanisms may underlie AMD and Alzheimer's disease.

Marked regional differences of brain human apolipoprotein E expression in targeted replacement mice.

We used three human apolipoprotein (apo) E targeted replacement mouse lines, each expressing one of the three common human apoE isoforms to study the pattern of apoE expression in the central nervous system (CNS). Immunocytochemistry on brain sections from all three lines of targeted replacement mice, wild type mice, African green monkeys, and humans show a predominantly glial pattern of apoE expression. The levels of human apoE protein in hippocampus and frontal cortex were similar between targeted replacement mice and non-demented human tissue. Within a given brain region, the levels of apoE were very similar amongst all three isoforms, which contrasts sharply with plasma, where apoE2 levels are 16-fold higher than apoE3 and E4 levels. Across brain regions, cerebellar apoE levels were significantly higher than cerebral apoE levels. In conclusion, we provide detailed analysis of a human apoE animal model system that recapitulates both the pattern and level of apoE expression in non-demented humans. The neurobiology of human apoE isoforms can now be studied in both the normal and post-injury state, since all apoE regulatory sequences are intact. Finally, the differences in apoE levels we observed may explain the regional vulnerability of neuronal degeneration in Alzheimer's disease.

Probing HLA-B7 conformational shifts induced by peptide-binding groove mutations and bound peptide with anti-HLA monoclonal antibodies.

To determine the influence of peptide-binding groove residues and MHC-bound peptide on HLA-B7 conformation, we investigated the binding sites of nine locus- or allele-specific mAbs using a panel of 82 HLA-B7 variants. The functional mAb epitopes encircle the HLA-B7 peptide-binding groove. Three mAbs are affected by mutations at solvent-accessible peptide-binding groove mutations. Mutations in peptide-binding groove residues 45, 63, and 150 affect multiple nonoverlapping mAb epitopes, probably by interaction with other MHC residues or bound peptide. However, 18 of 24 peptide-binding groove mutations do not affect mAb binding, indicating that the conformation of solvent-accessible HLA-B7 structures is largely dissociated from changes in the peptide-binding groove. To test whether bound peptides alter HLA-B7 conformation, we loaded HLA-B7 heavy chains on acid-stripped cells with beta2-microglobulin and 20 individual synthetic peptides. Two of eight mAbs are sensitive to HLA-B7-bound peptides. A likely interpretation of these data is that the conformational flexibility of HLA-B7 is due to peptide-induced conformational shifts in MHC side chains, rather than major shifts in the MHC main chain. These results suggest that HLA-B7 conformation is largely maintained in the context of different bound peptides and different peptide-binding grooves.

Bw4-reactive and Bw6-reactive antibodies recognize multiple distinct HLA structures that partially overlap in the alpha-1 helix.

Bw4 and BW6 epitopes are expressed by mutually exclusive sets of HLA-B alleles and some HLA-A and HLA-C alleles. To test whether antithetical structures are required to express Bw4 and Bw6 epitopes, we measured binding of Bw4-reactive and Bw6-reactive alloantibodies and mAbs to HLA-B7 variants. A triple substitution of HLA-B7 alpha-1 helix residues 80, 82, and 83 created Bw4 and destroyed Bw6 epitopes detected by alloantibodies and mAbs. Both Bw4-reactive and Bw6-reactive mAbs competed for binding to HLA-B7 variants with single substitutions at residues 82 and 83. Substitutions of residues H93 and D119 which form a salt bridge in HLA-A2 also permitted binding by both Bw4-reactive and Bw6-reactive mAbs, suggesting that Bw4 and Bw6 epitopes are conformationally dependent. Six Bw4-reactive mAbs showed four distinct patterns of binding to HLA-B7 variants. Detailed analysis of 74 HLA-B7 single-residue variants showed that Bw6-reactive SFR8-B6 binding was prohibited by mutations altering the distal end of the alpha-1 helix and the nearby connecting loop. In contrast, Bw6-reactive BB7.6 binding required both alpha-1 and alpha-2 helix residues. Thus, Bw4-reactive and Bw6-reactive Abs recognize multiple distinct HLA structures that partially overlap in the alpha-1 helix. As both Bw4 and Bw6 epitopes are expressed by some HLA-B7 variants, mutually exclusive expression of Bw4 and Bw6 epitopes in naturally occurring HLA class 1 molecules may reflect evolutionary pressure.

Anti-keratin antibodies in rheumatoid arthritis: frequency and correlation with other features of the disease.

Anti-keratin antibodies (AKA) were detected in 68 out of 98 patients (69%) with classical or definite rheumatoid arthritis (RA). The intensity of the AKA reaction correlated significantly with articular index (AI), grip strength (GS), erythrocyte sedimentation rate (ESR), serum C-reactive protein (CRP) concentration, serum amyloid A (SAA) protein concentration, the level of antibodies against single stranded DNA (ssDNA) and the IgM rheumatoid factor (RF) titre. A significantly higher number of patients with nodules and Sjögren's syndrome were AKA positive compared with patients without extra-articular features (EAFs) and the AKA titre was significantly greater in the former group. The mechanisms underlying appearance of AKA are not known but may relate to an as yet unidentified structural alteration of keratin in this disease or may just reflect the rheumatoid autoimmune diathesis.

Correlation in rheumatoid arthritis of concentrations of plasma C3d, serum rheumatoid factor, immune complexes and C-reactive protein with each other and with clinical features of disease activity.

The concentrations of C3d in the plasma and of C-reactive protein (CRP), immune complexes and rheumatoid factor in the serum were measured in 99 patients with rheumatoid arthritis. Most patients had raised levels, the values of which correlated with disease activity assessed according to a newly described index based on multivariate analysis of subjective, semi-objective and objective features of the disease. There were also significant correlations between the values for plasma C3d and circulating immune complexes, immune complexes and rheumatoid factor, serum CRP and immune complexes, and serum CRP and plasma C3d. Measurement of plasma C3d provides a useful means of detecting in vivo complement activation, which may be involved in the pathogenesis of rheumatoid arthritis, but neither C3d levels nor any of the other variables correlated as closely with disease activity as did the serum CRP concentration.

Correlation of clinical parameters of disease activity in rheumatoid arthritis with serum concentration of C-reactive protein and erythrocyte sedimentation rate.

Serum C-reactive protein (CRP) levels were measured in 99 patients with definite or classical rheumatoid arthritis (RA). At the same time as the serum was obtained, the activity of the RA was assessed by assigning scores to a comprehensive set of subjective, semi-objective and objective clinical features. The results confirmed that the serum CRP concentration closely reflects activity of RA and is of value in its objective assessment. Erythrocyte sedimentation rate (ESR) followed the same pattern as CRP levels but all of the subjective and semi-objective criteria correlated more significantly with CRP levels than with ESR.

The assessment of disease activity in rheumatoid arthritis using a multivariate analysis.

A simple method of assessing 'index of disease activity' (IDA) in rheumatoid arthritis (RA) using a multivariate analysis (MVA) comprising morning stiffness (MS), pain scale (PS), grip strength (GS), articular index (AI), haemoglobin (Hb) and erythrocyte sedimentation rate (ESR) is described. The IDA of 99 patients with RA was assessed using MVA. The method could be used reliably and readily for random or longitudinal assessment, in drug trials and for comparing disease activity with other objective indices.

Expectation and patient preference -- does it matter?

Admission to clinical trials is often based on the assumption of homogeneity of the population. A group of 60 patients, all with pain in the neck or shoulder of at least 3 months duration, were studied. Expectation was graded before treatments were started by sharing out 100 points between freedom from side effects, pain relief, depression relief, improved mobility, improved sleep and speed of action. A double-blind crossover study of two established anti-inflammatory analgesics and placebo was carried out. Using analogue scales, patients were asked to grade their response. Side effects were recorded, and preference was established at the end of the study.Although all the patients were in sufficient pain to require medical attention, some rated relief of depression, improvement in sleep or lack of side effects as more important than pain relief. Differences between drug and placebo were most clearly demonstrated in those patients whose main concern was improved mobility. In our view it is important to select patients who are in need of a dominant property of a drug for a trial of this property and this may have ramifications across the medical spectrum.

Ascending lumbar venography in the diagnosis of lumbar disc lesions.

Fifty-five patients suffering from low back pain with or without sciatica were submitted to ascending lumbar venography. Six patients had anatomical variations which prevented adequate catheterization and 6 had equivocal radiographic appearances, which we could not assess. Fourteen patients had normal venograms but in 29 an abnormality was demonstrated. These 43 patients were then submitted to radiculography and, where appropriate, surgery. The 14 patients with normal venograms also had normal radiculograms; while the 29 with abnormal venograms had an abnormality confirmed on radiculography and/or surgery. However, in 10 of the patients the lesion was found to be one disc space lower than that demonstrated on venography; Side-effects were very few, and the procedure can be used on out-patients. This technique would appear to be a useful addition to the radiological investigation of the spine.