Reinhard Prior

Epidermal growth factor receptor expression and growth fraction in human tumours of the nervous system.

100 tumours of the human nervous system were investigated by means of immunohistochemistry in order to determine the expression of epidermal growth factor receptor (EGFr) and the proliferative activity as evaluated by demonstration of the proliferation-associated Ki-67 antigen. Epidermal growth factor receptor immunoreactivity was present in 79% (23/29) of the high-grade malignant gliomas examined but in only 9% (2/22) of the low-grade gliomas. Besides the gliomas, EGFr-expression was detectable in smaller amounts in most (13/15) meningiomas, in one anaplastic neurinoma and in individual tumour cells of one medulloblastoma. In addition, EGFr-expression was found in 50% (6/12) of metastatic carcinomas. Seven of eight medulloblastomas, two cerebral primitive neuroectodermal tumours (PNETs), three benign neurinomas, one ganglioneuroma, one metastatic intracerebral malignant melanoma, three spinal plasmocytomas and one immunocytoma showed no detectable EGFr-expression. Our results indicate that (1) the expression of EGFr in human tumours of the nervous system depends on the histological tumour type and (2) in the glioma group is related to the grade of malignancy. A close correlation between EGFrexpression and proliferative activity as evaluated by Ki-67 staining could not, however, be established.

Transferrin receptor expression in tumours of the human nervous system: relation to tumour type, grading and tumour growth fraction

The expression of transferrin receptor (Tr) was investigated by means of immunohistochemistry in 101 tumours of the human central and peripheral nervous system. The results were compared with the proliferative activity of the tumours, determined by immunostaining for the proliferation-associated antigen Ki-67. In addition to immunostaining of normal and proliferated blood vessel endothelium and of a fraction of tumour infiltrating lymphocytes, we observed staining for Tr in a variable fraction of neoplastic cells of all histological tumour types. Immunoreactivity in the majority of tumour cells was found only in anaplastic tumours such as glioblastomas. Furthermore, a positive correlation between Tr expression and the Ki-67 growth fraction was established for gliomas. Non-glial tumours strongly expressing Tr included one metastatic rhabdomyosarcoma, one intracerebral malignant lymphoma, two of four plasmocytomas and seven of nine metastatic carcinomas. Our results indicate that immunohistochemistry for Tr and Ki-67 can provide additional information about the biological behaviour of nervous system tumours, thus complementing conventional histopathological criteria for anaplasia.

Studies on the effects of altered PMP22 expression during myelination in vitro

Severe inherited dysmyelinating diseases of the peripheral nervous system, the Charcot‐Marie‐Tooth type1A disease (CMT1A) and the hereditary neuropathy with liability to pressure palsies (HNPP) are associated with a large DNA duplication or deletion of a chromosomal region containing the peripheral myelin protein 22 (PMP22) gene. It has been suggested that a gene dosage effect involving PMP22 is responsible for the pathological phenotype. We investigated if altered PMP22 expression affects the onset of myelin formation and the ultrastructure of myelin. Rat Schwann cell cultures were stably infected with recombinant retrovirus vectors harboring the rat PMP22 cDNA in sense or antisense orientation. Schwann cells over‐ or underexpressing PMP22 were cocultured with purified DRG neurons under conditions that promote myelination. We examined PMP22 expression and localization in the myelin forming cultures by RT‐PCR, immunohistochemistry and confocal microscopy, and we analyzed myelin ultrastructure by electron microscopy. Our results demonstrate that abnormal levels of PMP22 expression do not impair the early stages of myelination and membrane compaction and do not interfere with the expression of other myelin genes. Our observations further indicate that PMP22 is involved more in controlling myelin thickness and stability than in the events determining the initial steps of myelin formation. J. Neurosci. Res. 48:31–42, 1997.

Loss of vessel wall viability in cerebral amyloid angiopathy

Cerebral amyloid angiopathy (CAA) is a neuropathological feature of Alzeheimers disease and an important cause of cerebral haemorrhage in the elderly. CAA is characterized by the deposition of Alzheimer amyloid β protein (Aβ) in cerebral and leptomeningeal vessel walls. In order to study the effect of cerebrovascular Aβ deposits in vivo, living canine leptomeninges obtained from old dogs affected by CAA were analysed by confocal laser scanning microscopy after immunofluorescence staining for Aβ and viability staining with fluorescein diacetate (FDA). Simultaneous detection of the two signals showed a segmental loss of leptomeningeal vessel wall viability at some site of Aβ deposition. Many of the non-viable vessels segments were also dilated, suggesting that Aβ-induced vascular cell death creates the loci minores resistentiae for the development of cerebral haemorrhage in CAA.

Apolipoprotein E, Smooth Muscle Cells and the Pathogenesis of Cerebral Amyloid Angiopathy: the Potential Role of Impaired Cerebrovascular Aβ Clearance

Abstract: Cerebral amyloid angiopathy (CAA) is caused by the deposition of β‐amyloid (Aβ) in Alzheimer disease brains. It also occurs isolated, representing a major cause for cerebral hemorrhage in the elderly. The E4 genotype of apolipoprotein E (ApoE) is a risk factor for CAA; however, the molecular mechanism underlying this genetic association is unknown. Various findings suggest that cerebrovascular Aβ is derived from the soluble Aβ contained in the cortical extracellular space or the cerebrospinal fluid (CSF) that communicates and surrounds small cortical or leptomeningeal vessels. CAA deposits are always intimately associated with smooth muscle cells (SMCs) or SMC‐derived pericytes. As we have previously reported, SMCs internalize Aβin vitro via a lipoprotein pathway involving ApoE and the low‐density lipoprotein receptor family. Internalized Aβ is subsequently located to lysosomes, suggesting its intracellular degradation. We show that Aβ is internalized via multiple pathways, because class A and class B scavenger receptors are also colocalized to Aβ‐containing endosomes in SMCs, and Aβ uptake is inhibited by various scavenger receptor antagonists. It has been recently shown for different cell types that the cellular uptake of ApoE is more efficient for the ApoE3 isoform when compared to ApoE4 and that this isoform‐specific difference depends on the presence of heparan sulfate proteoglycan (HSPG). HSPG is produced by SMCs and promotes Aβ fibrillogenesis. We propose a pathogenetic model of CAA, in which the ApoE‐ and HSPG‐mediated clearance of CSF‐derived Aβ peptides by SMCs protects the vascular extracellular matrix against critical Aβ concentrations. Impairment of this pathway or its reduced efficiency in carriers of the ApoE4 genotype may increase the risk of developing CAA.

Nerve growth factor receptor in tumours of the human nervous system: Immunohistochemical analysis of receptor expression and tumour growth fraction

The expression of nerve growth factor receptor (NGFr) was investigated by means of immunohistochemistry in 135 tumours of the human central and peripheral nervous system. The results were compared to the proliferative activity of the tumours as determined by immunostaining for the proliferation-associated antigen Ki-67. Immunoreactivity for NGFr was most consistently observed in tumours derived from the neural crest such as neurinomas, neurofibromas and ganglioneuromas. In tumours of the central nervous system, NGFr-immunostaining was particularly strong in pilocytic astrocytomas while the majority of other gliomas were either NGFr-negative or contained only a minor fraction of NGFr-positive tumour cells. Among all other investigated tumours including medulloblastomas, pituitary adenomas and meningiomas only exceptional cases demonstrated a significant number of positive tumour cells. Choroid plexus papillomas and metastatic carcinomas were always NGFr-negative. Our results indicate that NGFr-expression in tumours of the human nervous system is heterogenous with respect to tumour type and appears to be unrelated to proliferative activity.

Experimental deposition of Alzheimer amyloid β-protein in canine leptomeningeal vessels

To study the pathogenesis of cerebral amyloid angiopathy (CAA), organ cultures of canine leptomeninges were incubated with fluorescein-conjugated amyloid β- protein (FAβ, residues 1–40; 10 nM to 200 μM). Fluorescence microscopy showed focal and dose-dependent FAβ binding to blood vessels affected by CAA at FAβ- concentrations as low as 10nM. The new Aβ deposits appeared to be extracellular and were localized to the middle and outer layers of leptomeningeal arterioles. FAβ partially co-localized with apolipoprotein E (ApoE) as revealed by confocal microscopy, suggesting that Aβ in situ binds to ApoE. Young dogs or old dogs without CAA showed no deposition of FAβ. Our results indicate that after initiation of CAA pathology, physiological concentrations of soluble Aβ are sufficient to sustain its further deposition and therefore the progression of CAA.

Canine leptomeningeal organ culture: a new experimental model for cerebrovascular β-amyloidosis

Cerebral amyloid angiopathy (CAA) is a neuropathological feature of Alzheimers disease and a common cause of cerebral hemorrhage in the elderly. The pathogenetic mechanisms leading to the deposition of Alzheimer amyloid beta-protein (A beta) in cortical and leptomeningeal vessel walls are unknown. There are no experimental models which reproduce the pathological changes of CAA. In this study, leptomeninges from young and old dogs with pre-existing CAA were cultured in cell culture medium or cerebrospinal fluid and their viability, histological appearance and metabolic activity were analyzed during the culture. In addition, living leptomeninges of old and young dogs were incubated with fluorescein-conjugated A beta and the uptake of A beta was studied by fluorescence microscopy. Leptomeninges from young and old dogs were viable up to 8 weeks in culture. They contain many small- and medium-sized arterioles, the main vessel type affected by CAA. Histology and immunohistochemistry showed excellent preservation of the vessel wall microarchitecture up to 4 weeks in culture. The cultures were metabolically active as shown by the de novo production of beta-amyloid precursor protein. Exogenously added A beta was focally deposited in the vessel walls of old, but not young dogs. In conclusion, the organ culture of canine leptomeninges is easy to perform and appears suitable to investigate the pathogenesis and the progression of CAA.

Amyloid β Protein Internalization and Production by Canine Smooth Muscle Cells

Aging dogs show progressive β-amyloid (Aβ) deposition within the walls of small cortical and leptomeningeal arteries and develop cerebral amyloid angiopathy (CAA) that is morphologically identical to human CAA associated with Alzheimer’s disease and with aging. The canine and the human Aβ amino acid sequences are identical, which is an essential requirement for an animal model of β-amyloidosis, because subtle changes within the Aβ peptide sequence may impredictibly change its aggregation properties. Since cerebrovascular Aβ deposits are always closely associated with vascular smooth muscle cells (SMCs) or the SMC-related parenchymal pericytes, primary canine cerebrovascular SMC cultures have been used to investigate the molecular mechanisms underlying the development of cerebrovascular Aβ deposition. This review summarizes the results obtained with canine SMC cultures and discusses their potential implications for the pathogenesis of CAA.