J. Lowe

Prion protein immunocytochemistry : UK five centre consensus report

Creutzfeldt‐Jakob disease (CJD) and other prion diseases are associated with the deposition of insoluble prion protein (PrPCJD) in the central nervous system (CNS). Antibodies raised against PrPCJD also react with its precursor protein, a soluble form of PrP (PrPC), which is widely distributed in the normal CNS. This cross‐reactivity has in the past raised doubts as to the specificity and diagnostic reliability of PrP immunolocalization, especially in familial cases which are atypical clinically and which lack characteristic pathology findings. Following an MRC‐funded workshop which focused on this problem, a multicentre prospective study was set up to identify a reliable protocol for PrPCJD immunocytochemistry. Five UK centres took part in this study and demonstrated consistent staining of plaques, vacuolar deposits in severe spongiform change, and perineuronal deposits using a variety of antibodies and enhancement procedures. A protocol using formic acid, guanidine thiocyanate, and hydrated autoclaving pre‐treatment in conjunction with a monoclonal PrPCJD antibody produced the clearest immunochemical results and is presented as the consensus UK recommendation for PrPCJD immunocytochemical procedures.

Ballooned neurons in several neurodegenerative diseases and stroke contain αB crystallin

αB crystallin is a protein which has homology with the small cell stress proteins. A characterized antibody to residues 1–10 of αB crystallin was used to immunostain tissues containing ballooned (chromatolytic, achromasic) neurons. The tissues included two cases of classical Picks disease, one case of dementia with swollen achromasic neurons in the cortex, two cases of Alzheimers disease with large numbers of ballooned neurons, two cases of motor neuron disease, four cases of cortico‐basal degeneration, and four cases with areas of brain showing swollen neurons adjacent to recent cerebral infarcts. The anti‐αB crystallin showed strong diffuse cytoplasmic immunoreac‐tivity of swollen cortical neurons in all the diseases. Astrocytes and oligodendroglial cells were also stained in normal tissues as previously described. Weak diffuse immunoreactivity with an antibody to ubiquitin‐conjugates was also seen in the swollen neurons from cases of neurodegenerative disease but not following infarction. Ballooned neurons have been shown to contain phosphorylated neurofilament epitopes not normally present in the perikaryonal region. The presence of αB crystallin in ballooned neurons, together with previous data which also indicate its close association with intermediate filaments, suggest that αB crystallin may be involved in aggregation and remodelling of neurofilaments in disease. The presence of αB crystallin in neurons at the edge of areas of cerebral infarction is likely to reflect cells which are regenerating following damage; its detection may therefore be a marker for such cells. On a practical level, the antibody greatly facilitates the localization of such abnormal neurons in diagnostic histology.

The cortical neuritic pathology of Huntington's disease

We have studied the brains of 10 patients with clinically and pathologically defined Huntingtons disease and graded the degree of striatal pathology according to the Vonsattel grading system. Sections from nine cerebral cortical areas (Brodmann areas 8. 10, 24, 33, 28, 38, 7, 39, 18), the cerebellum, hypothalamus, medulla and caudate nucleus were stained with antibodies to ubiquitin and ubiquitin C‐terminal hydrolase (PGP 9.5). Dystrophic neurites. immunoreactive with ubiquitin and PGP 9.5 were detected in all cortical areas. in layers 3, 5 and 6, of all brains studied. No dystrophic neurites were found in subcortical areas or cerebellum. Sections from cortical areas 8 and 24 from the two brains with the most and least ubiquitin‐immunoreactive neurites were stained with antibodies to β‐amyloid precursor protein, tau, glial fibrillary acidic protein, neurofilament protein, αB crystallin, GABA, cholecystokinin and somatostatin. The dystrophic neurites were found to also react with β‐amyloid precursor protein. Electron microscopy showed the abnormal neurites to contain granulo‐filamentous material. Granular deposits with a diameter of 40–100 nm were interspersed between randomly orientated ‘fuzzy’ or coated, straight or slightlv curved filaments measuring 10–15 nm in diameter. These structures have not been seen in control brain and differ from age‐related neuritic degeneration and neurites associated with amyloid. Immunohistochemically these structures most resemble CA 2/3 neurites seen in Lewy body disease, and, ultrastructurally, the intraneuronal filamentous inclusions in motor neuron disease. The areal density of these neurites was quantified in 20 microscopic fields in the superior frontal and anterior cingulate sections (Brodmann areas 8 and 24) and did not correlate with the Vonsattel grade, suggesting that they are an independent and possibly primary cortical pathology in Huntingtons disease.

Protein processing in lysosomes : the new therapeutic target in neurodegenerative disease

A little recognised feature of neurons is their large complement of lysosomes. Studies of the accumulation of the abnormal isoform of the prion protein (PrPSC) in the prion encephalopathies and the formation of beta/A4 protein from its precursor in Alzheimers disease suggest that generation of these key proteins takes place in lysosome-related organelles. The release of hydrolytic enzymes from lysosomes may be a primary cause of neuronal damage. Although molecular genetic approaches have identified protein mutations central to the main neurodegenerative disease, cell biological observations are now beginning to unravel the intracellular pathways involved in the molecular pathogenesis of neurodegeneration: as a result, it is now appropriate to consider therapeutic manipulation of the lysosomal system as an approach to treatment.

Intrapleural BCG in operable lung cancer.

In a controlled randomised clinical trial of 92 patients with surgically resected lung cancer, intrapleural BCG had no clear effect on survival. Adverse effects were insignificant.

Clinical and histopathological heterogeneity in patients with 4q35 facioscapulohumeral muscular dystrophy (FSHD)

FSHD is a relatively common muscular dystrophy most often presenting in the second decade with facial or shoulder girdle weakness. Although occasionally severe, it is usually compatible with a normal lifespan and is only slowly progressive with about 20% of patients becoming wheelchair-bound. Accepted diagnostic criteria for FSHD, established for use in research trials, are shown in Table 1 [1]. A major locus for FSHD has been demonstrated on chromosome 4 [2] and a causal relationship between FSHD and a genomic rearrangement on chromosome 4 has been shown. Most cases of FSHD appear to result from deletion of repeated 3.3-kb subunits from band 4q35 within the subtelomeric region of the long arm of chromosome 4 [3]. FSHD patients have 1–10 repeats ( < 35 kb) while healthy controls have 10–100 repeats (35–100 kb). Recent data suggest that these deletions confer a toxic gain of function secondary to the derepression of neighbouring 4q35 gene transcription. This appears to occur in a dosedependent fashion with the extent of transcriptional derepression correlating with the number of 3.2 kb repeats deleted [4]. Detection of these 4q35 deletions was previously complicated by the close similarity of the genomic region at 4q35 with that at 10q26. The use of a ‘double digest’ technique, whereby the chromosome 10 fragment is digested by the restriction enzyme Bln1, allows these regions to be distinguished, resulting in a highly reliable diagnostic gene test [5]. We report here a family in whom several members are affected by an autosomal dominant myopathy associated with heterozygous deletion at 4q35 consistent with a clinical diagnosis of FSHD (Figure 1). Muscle biopsy findings in the index case were, however, suggestive of a nemaline myopathy. We also report a second, unrelated patient who presented with a ‘bent spine syndrome’ (camptocormia) who was also shown to have a heterozygous deletion at 4q35. Patient 1, a 64-year-old man, first noticed shoulder weakness and wasting at age 10. He was a hairdresser but gave up work at age 18 because of difficulty raising his arms above his head. His lower limbs became affected in his 40s and he had been using a wheelchair from the age of 50. His parents, who died in their 80s, and his siblings were unaffected. On initial neurological assessment at age 60, he was kyphotic and had weakness of neck flexion. He had bilateral facial weakness and asymmetrical scapular winging. There was marked wasting and weakness of the shoulder girdle and proximal upper limbs. His legs were proximally weak and wasted. Deep tendon reflexes were absent. He had paradoxical respiration and abdominal muscle weakness. Sensory examination was normal. At presentation his creatine kinase (CK) levels were normal. Electromyography (EMG) sampling revealed widespread myopathic changes. A quadriceps muscle biopsy showed active muscle fibre necrosis associated with a mild lymphocytic infiltration, abnormal muscle fibre size variation and focal fatty replacement. Histochemistry identified most fibres as type 1. Dystrophin, merosin and alpha sarcoglycan expression were normal on immunostaining. The most notable finding, however, was of the presence of eosinophilic inclusion bodies in approximately 10% of fibres. Electron microscopy confirmed that these inclusion bodies were nemaline rods, present in such quantity as to strongly suggest a nemaline myopathy (Figure 2). Subsequently his three children (age range 18–22, two female, one male) sought medical attention because of concern that they had developed a similar syndrome. All three children had been poor at sport in their childhood years. They were unable to raise their arms above their heads. They had severe facial weakness (Figure 3) and scapular winging. The pattern of limb weakness was simTable 1. Main clinical diagnostic criteria for facioscapulohumeral muscular dystrophy (FSHD) of the European Neuromuscular Centre FSHD Consortium

Wernicke's encephalopathy with ballooned neurons in the mamillary bodies: an immunohistochemical study

Two cases of Wernickes encephalopathy with the rare phenomenon of ballooned neurons in the mamillary bodies are reported. Both patients suffered from acute Wernickes symptoms starting approximately two weeks before death. The mamillary bodies contained grossly enlarged, ballooned neurons, in one case associated with focal necrosis. The affected neurons were immunoreactive for phosphorylated neurofilament (160 and 200u2003kDa), and synaptophysin. Ubiquitin and αβ crystallin expression were not detected. The mamillothalamic tract appeared normal in both cases. There was a marked associated microglial reaction, as shown by the antibody Ki‐M1P. It is concluded that the ballooning of mamillary neurons reflects an acute retrograde reaction to primarily axonal damage. Rather than being a rare manifestation of the disease, these cases may constitute a typical intermediate early stage (10–15 days) in the development of Wernickes encephalopathy.

Endosome-lysosomes and neurodegeneration

A number of the major human and animal neurodegenerative diseases, such as Alzheimers disease and sheep scrapie, are characterised by deposits of amyloid, arising through incomplete breakdown of membrane proteins. Although our knowledge concerning these diseases is increasing, they remain largely untreatable. Recently, attention has focussed on the mechanisms of production of different types of amyloid and the likely involvement within cells of acid compartments called endosome-lysosomes. These organelles may be bioreactor sites for the unfolding and partial degradation of membrane proteins to generate the amyloid materials. These subsequently become expelled from the cell, or are released from dead cells, and accumulate as pathological entities. Common features of the disease processes give new direction to therapeutic intervention.

Haemorrhagic shock encephalopathy and sudden infant death

In 2 pairs of non-identical twins, haemorrhagic-shock encephalopathy syndrome developed in 1 co-twin while the other died of sudden infant death syndrome. The twin pairs were aged 3 and 4 months, respectively, and no cause was identified. We suggest that stress protein deficiency may underlie both syndromes.

Ubiquitin and the Lysosomal System: Molecular Pathological and Experimental Findings

Considerably advances in our understanding of the molecular pathology of the major human neurodegenerative diseases have been made by the use of ubiquitin immunocytochemistry. The technique demonstrates that filamentous inclusions and vacuoles contain ubiquitin-protein conjugates. The molecular structure of the filaments and the morphological type of vacuoles is not completely understood but there is evidence that some of the filamentous inclusions contain intermediate filaments and the perinuclear distribution of the vacuoles resembles the distribution of intraneuronal lysosomes. Intermediate filaments and lysosomes are involved in the sequestration and degradation of viral membrane proteins in tissue culture cells. Immunogold electron microscopical and biochemical evidence indicates that ubiquitin-protein conjugates are normally considerably enriched in the lysosomes of fibroblasts relative to all other organelles. Immunogold electron microscopy showsasimilar enrichment ofubiquitin-protein conjugates in the dense lysosomes of granulocytic precursor cells in the bone marrow. Filamentous inclusions showing several of the features seen in inclusions in the neurodegenerative diseases are seen in Epstein-Barr virus transformed lymphoblastoid cells. Immunohistochemistiy shows that the inclusions contain vimentin intermediate filaments, the latent membrane transforming protein of the virus, ubiquitin-protein conjugates, and heat-shock protein 70 (HSP70). Immunogold electron microscopy demonstrates that the latent membrane protein, ubiquitin-protein conjugates and HSP70 are in lysosomes entwined in a intermediate filament cage centred on the microtubule organising centre. The implications of the combined observations for our understanding of the cell stress response in degenerating neurones and in virally infected cells are discussed.