Philip J. Luthert

Tissue distribution of protease resistant prion protein in variant Creutzfeldt-Jakob disease using a highly sensitive immunoblotting assay

BACKGROUND Variant Creutzfeldt-Jakob disease (vCJD) has a pathogenesis distinct from other forms of human prion disease: disease-related prion protein (PrP(Sc)) is readily detectable in lymphoreticular tissues. Quantitation of risk of secondary transmission, and targeting of risk reduction strategies, is limited by lack of knowledge about relative prion titres in these and other peripheral tissues, the unknown prevalence of preclinical vCJD, and a transmission barrier which limits the sensitivity of bioassay. We aimed to improve immunoblotting methods for high sensitivity detection of PrP(Sc) to investigate the distribution of PrP(Sc) in a range of vCJD tissues. METHODS We obtained tissues at necropsy from four patients with neuropathologically confirmed vCJD and from individuals without neurological disease. Tissues were analysed by sodium phosphotungstic acid precipitation of PrP(Sc) and western blotting using high sensitivity enhanced chemiluminescence. FINDINGS We could reliably detect PrP(Sc) in the equivalent of 50 nL 10% vCJD brain homogenate, with a maximum limit of detection equivalent to 5 nl. PrP(Sc) could be detected in tissue homogenates when present at concentrations 10(4)-10(5) fold lower than those reported in brain. Tonsil, spleen, and lymph node were uniformly positive for PrP(Sc) at concentrations in the range of 0.1-15% of those found in brain: the highest concentrations were consistently seen in tonsil. PrP(Sc) was readily detected in the retina and proximal optic nerve of vCJD eye at levels of 2.5 and 25%, respectively of those found in brain. Other peripheral tissues studied were negative for PrP(Sc) with the exception of low concentrations in rectum, adrenal gland, and thymus from a single patient with vCJD. vCJD appendix and blood (Buffy coat fraction) were negative for PrP(Sc) at this level of assay sensitivity. INTERPRETATION We have developed a highly sensitive immunoblot method for detection of PrP(Sc) in vCJD tissues that can be used to provide an upper limit on PrP(Sc) concentrations in peripheral tissues, including blood, to inform risk assessment models. Rectal and other gastrointestinal tissues should be further investigated to assess risk of iatrogenic transmission via biopsy instruments. Ophthalmic surgical instruments used in procedures involving optic nerve and the posterior segment of the eye, in particular the retina, might represent a potential risk for iatrogenic transmission of vCJD. Tonsil is the tissue of choice for diagnostic biopsy and for population screening of surgical tissues to assess prevalence of preclinical vCJD infection within the UK and other populations.

Neuronal loss in the frontal cortex in HIV infection.

In an attempt to elucidate the cause and mechanism of the dementia and other neurological disorders that can occur in HIV-1 infection, we have quantitatively assessed neuronal populations, by means of a stereological technique (the disector), in the frontal cortex of patients with HIV infection. Eleven of sixty-five brains in the Medical Research Council Central AIDS Brain Bank were selected for study. The selected patients died without opportunistic infection or neoplasm affecting the brain; they had HIV encephalitis or minimal changes. We compared their neuronal counts with those of eight control subjects (seven died of systemic illness, one of pontine haemorrhage which did not affect the cerebral hemispheres). The neuronal numerical density was significantly lower in the HIV group than in the control group (mean [SD] 307 [46] vs 499 [113] x 10(2) per mm3; p less than 0.001). This difference represents a loss of about 38%. There was no significant difference between the HIV subgroups, which suggests that neuronal loss occurs in cases of minor pathology as well as in HIV encephalitis. This finding contributes to the understanding of dementia in AIDS patients and has important implications for their future treatment.

Clinical and neuropathological correlates of depression in Alzheimer's disease.

Depressive symptoms have been reported in patients with mild to moderate Alzheimers disease (AD). Recent evidence suggests that a noradrenergic deficit originating from neuronal degeneration in brainstem nuclei may represent an organic correlate of these disturbances. We examined the neuropathological changes in the locus coeruleus (LC), substantia nigra (SN), basal nucleus of Meynert and cortex of 52 patients (12 male, 40 female, mean age 83.2 +/- 6.4 years) with pathologically verified AD. Fourteen patients (1 male, 13 female) showed signs of depression. The majority of these patients suffered from severe physical disability or sensory impairment and developed persistent delusions, but had less cognitive impairment. Neuronal counts in the LC were significantly lower than in the 38 patients without depression (36.9 +/- 14.0; 51.4 +/- 28.0 neuromelanin-pigmented cells per section per nucleus; F = 3.4, df = 1, 50, P = 0.04). Neuron counts were higher in the basal nucleus of Meynert in depressed AD patients and there were no differences of the neuron numbers in the SN. Depression (main effect; F = 4.5, P = 0.04) contributed significantly to the variance of neuronal counts in the LC, even when covarying for gender, age of onset, cognitive impairment and cortical Alzheimer pathology. The observed disproportionate loss of noradrenergic and cholinergic neurons in the LC and basal nucleus of Meynert may represent an important organic substrate of depression in AD.

A review of neuronal damage in human immunodeficiency virus infection: its assessment, possible mechanism and relationship to dementia.

Over the past decade it has been realized that HIV affects the central nervous system, and various investigations have illuminated the spectrum of neuropathology in AIDS. One major advance has been the demonstration that there is substantial neuronal loss, which appears independent of the HIV-associated inflammatory lesions. Quantitative studies on neuronal populations, while fraught with methodological difficulties, are essential to the understanding of the mechanism of this neurotoxic damage. This article will review, firstly, the modern stereological procedures available for quantitative investigations; secondly, the pattern, degree and time scale of HIV-associated neuronal loss; thirdly, other morphological evidence of neuronal damage; and finally, the pathological and clinical implications of these findings.

Complement factor H deficiency in aged mice causes retinal abnormalities and visual dysfunction.

Age-related macular degeneration is the most common form of legal blindness in westernized societies, and polymorphisms in the gene encoding complement factor H (CFH) are associated with susceptibility to age-related macular degeneration in more than half of affected individuals. To investigate the relationship between complement factor H (CFH) and retinal disease, we performed functional and anatomical analysis in 2-year-old CFH-deficient (cfh−/−) mice. cfh−/− animals exhibited significantly reduced visual acuity and rod response amplitudes on electroretinography compared with age-matched controls. Retinal imaging by confocal scanning laser ophthalmoscopy revealed an increase in autofluorescent subretinal deposits in the cfh−/− mice, whereas the fundus and vasculature appeared normal. Examination of tissue sections showed an accumulation of complement C3 in the neural retina of the cfh−/− mice, together with a decrease in electron-dense material, thinning of Bruchs membrane, changes in the cellular distribution of retinal pigment epithelial cell organelles, and disorganization of rod photoreceptor outer segments. Collectively, these data show that, in the absence of any specific exogenous challenge to the innate immune system, CFH is critically required for the long-term functional health of the retina.

Cortical Lewy body dementia: clinical features and classification.

Seven patients, aged 65-72 years, are described with dementia and cortical Lewy bodies. In one patient a Parkinsonian syndrome was followed by dementia and motor neuron disease. In the remaining six patients dementia was accompanied by dysphasia, dyspraxia and agnosia. One developed a Parkinsonian syndrome before the dementia, in three cases a Parkinsonian syndrome occurred later, and in two cases not at all. All patients showed Lewy bodies and cell loss in the substantia nigra, locus coeruleus and dorsal vagal nucleus, as in Parkinsons disease. The severity of cell loss in the nucleus basalis varied from mild to severe. Lewy bodies were also present in the parahippocampus and cerebral cortex, but Alzheimer-type pathology was mild or absent, and insufficient for a diagnosis of Alzheimers disease. Patients with moderate or severe dementia, some with temporal or parietal features, may have cortical Lewy body disease, Alzheimers disease, or a combination of the two. Cortical Lewy body disease may be associated with dementia in Parkinsons disease more often than realised, but is not necessarily associated with extensive Alzheimer pathology.

Accumulation of tissue inhibitor of metalloproteinases-3 in human eyes with Sorsby’s fundus dystrophy or retinitis pigmentosa

BACKGROUND/AIMS Tissue inhibitor of metalloproteinases-3 (TIMP-3) is normally synthesised by the retinal pigment epithelium (RPE) and deposited in Bruch’s membrane. Mutations in the TIMP3 gene cause Sorsby’s fundus dystrophy (SFD), which is characterised by thickening of Bruch’s membrane, choroidal neovascularisation, and photoreceptor degeneration. To elucidate the role of TIMP-3 in human retinal degenerative diseases, we immunolocalised TIMP-3 in eyes with SFD caused by the Ser-181-Cys TIMP3 gene mutation or retinitis pigmentosa (RP; not caused byTIMP3 mutations). METHODS Standard light microscopic immunocytochemistry, including antigen retrieval, was used to localise TIMP-3 in paraffin sections of human eyes: two with SFD, three with different genetic forms of RP, and two normal. RESULTS In the SFD eyes, the thickened Bruch’s membrane was strongly TIMP-3 positive except where RPE cells had degenerated. Similarly, in the RP eyes, Bruch’s membrane was TIMP-3 positive except where RPE cells were lost, consistent with ongoing RPE mediated turnover of TIMP-3 in this region. In areas of total photoreceptor loss, migrated RPE cells formed cuffs around blood vessels in the RP retinas. Thick, TIMP-3 positive extracellular matrix (ECM) deposits associated with the migrated RPE cells occluded some vascular lumina, correlating with the observed loss of inner retinal neurons in RP. CONCLUSIONS TIMP-3 is a component of the increased ECM sequestered in Bruch’s membrane in SFD. Further information is needed on normal TIMP-3/ECM interactions in Bruch’s membrane and the effect of mutant TIMP-3 on this process. The finding of TIMP-3 accumulations in retinas with RP not caused by TIMP-3 mutations emphasises the importance of ECM remodelling in normal and diseased human eyes.

How to predict proliferative vitreoretinopathy: a prospective study.

PURPOSE To determine prospectively the accuracy of a predictive risk formula for the development of postoperative proliferative vitreoretinopathy (PVR) when applied in a clinical setting. DESIGN Prospective noncomparative interventional case series. PARTICIPANTS Two hundred nineteen subjects undergoing primary vitrectomy for rhegmatogenous retinal detachment were studied. METHOD By use of a formula-based discriminant rule, subjects were classified as either high or low risk for the development of PVR. All subjects were followed prospectively. OUTCOME MEASURES Development of postoperative PVR as defined by the updated the Retina Society Classification. RESULTS Complete data were available on 212 of 219 subjects. There were 130 subjects identified as low risk and 82 subjects as high risk; 9.2% of the low-risk (12 of 130) compared with 28% (23 of 82) of the high-risk subjects had postoperative PVR develop. This difference was statistically significant (P < 0.001). CONCLUSIONS Our study has shown that using a clinical model it is possible to identify subjects at greater risk of PVR developing after primary vitrectomy.

Familial Alzheimer's disease with the amyloid precursor protein position 717 mutation and sporadic Alzheimer's disease have the same cytoskeletal pathology.

The cytoskeletal pathology of a patient with familial Alzheimers disease (AD) associated with the probably causal amyloid precursor protein (APP) codon 717 Val----Ile mutation is described. In addition to moderately extensive beta A4 protein deposition within the substance of the brain and in blood vessel walls (congophilic angiopathy), there was abundant cytoskeletal pathology in the form of neurofibrillary tangles, plaque neurites and neuropil threads. Interestingly, plentiful cortical and subcortical Lewy bodies were also seen. In order to compare the cytoskeletal pathology in this case with that seen in sporadic cases of AD we (1) studied the immunohistochemical profile of the amyloid and cytoskeletal pathology with antibodies to beta A4 protein, tau, phosphorylated neurofilament epitopes and ubiquitin and (2) performed a biochemical fractionation and Western blot analysis for the abnormally phosphorylated form of tau (A68) characteristically seen in AD. No substantial difference between the familial case and sporadic cases could be found. We conclude that it is now reasonable to hypothesise that an abnormality in APP metabolism is responsible not only for the deposition of beta A4 protein, but also for the range of cytoskeletal pathology, typical of AD.

Matrix metalloproteinase biology applied to vitreoretinal disorders

1The ECM is a complex structure that influences the behaviour of its resident cells, and those in the process of migration, by providing specific contextual information. Enzymes that modify the ECM thus have the potential to aVect basic cell biology in many physiological and pathological processes. 2 Remodelling of the ECM must occur with spatial and temporal precision for normal development, morphogenesis, homeostasis, and tissue repair. 3 As matrix degradation is a prerequisite for malignant invasion and metastasis, it is not surprising that MMP biology has attracted considerable interest, with clinical oncology trials under way in several organ systems. 4‐7 New proteolytic functions are also being defined for these enzymes. These include the degradation of non-matrix macromolecules such as myelin basic protein, inactivation of AE1 antitrypsin, release of sequestered growth factors from ECM, and cleavage of bioactive molecules from the cell surface. 8