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Biomedical subjects

J W Neal

Publications and source records attributed to J W Neal.

At least 19 recordsLinked to original sources
PubMed2006

Complement membrane attack is required for endplate damage and clinical disease in passive experimental myasthenia gravis in Lewis rats.

Myasthenia gravis (MG) is a debilitating and potentially fatal neuromuscular disease characterized by the generation of autoantibodies reactive with nicotinic acetylcholine receptors (AChR) that cause loss of AChR from the neuromuscular endplate with resultant failure of neuromuscular transmission. A role for complement (C) in the pathology of human MG has been suggested based upon identification of C activation products in plasma and deposited at the endplate in MG. In the rat model, experimental autoimmune MG (EAMG), C depletion or inhibition restricts clinical disease, further implicating C in pathology. The mechanisms by which C activation drives pathology in MG and EAMG are unclear. Here we provide further evidence implicating C and specifically the membrane attack complex (MAC) in the Lewis rat passive EAMG model of MG. Rats deficient in C6, an essential component of the MAC, were resistant to disease induction and endplate destruction was reduced markedly compared to C6-sufficient controls. After reconstitution with C6, disease severity and endplate destruction in the C6-deficient rats was equivalent to that in controls. The data confirm the essential role of the MAC in the destruction of the endplate in EAMG and raise the prospect of specific MAC inhibition as an alternative therapy in MG patients resistant to conventional treatments.

Animals↗
PubMed2006

The membrane attack pathway of complement drives pathology in passively induced experimental autoimmune myasthenia gravis in mice.

The human neuromuscular disease myasthenia gravis (MG) is characterized by the generation of autoantibodies reactive with nicotinic acetylcholine receptors (AChR) that cause loss of AChR from the neuromuscular end-plate with resultant failure of neuromuscular transmission. A role for complement (C) in AChR loss has been suggested based upon morphological identification of C at the end-plate in MG and from the effects of C inhibition in murine models. Here we provide further evidence implicating C, and specifically the membrane attack complex (MAC), in a mouse model of MG. Mice deficient in the C regulators Daf1 and/or Cd59a were tested in the model. Wild-type mice were resistant to disease while mice deficient in Daf1 had mild disease symptoms with evidence of C activation and AChR loss at end-plates. Cd59a-deficient mice had very mild disease with some muscle inflammation and essentially undamaged end-plates. In contrast, mice deficient in both C regulators developed a severe paralytic disease with marked muscle inflammation and loss of end-plates. Inhibition of MAC assembly abrogated clinical disease in these double-deficient mice, demonstrating conclusively that MAC formation was driving pathology in the model. These findings provoke us to suggest that current anti-C therapeutics targeting MAC assembly will be beneficial in MG patients resistant to conventional therapies.

Animals↗
PubMed2006

A case of multiple cutaneous schwannomas; schwannomatosis or neurofibromatosis type 2?

A 54 year old man presented with numerous cutaneous schwannomas, cranial nerve lesions, and spinal cord lesions, but no evidence of vestibular nerve involvement. There was no family history of neurocutaneous lesions. To help discriminate between the various possible diagnoses in this patient, molecular analysis of two cutaneous schwannomas was undertaken. An identical point mutation in the NF2 gene in the two anatomically distinct tumours was found, confirming this as a case of NF2 mosaicism.

Diagnosis, Differential↗
PubMed2004

Blockade of the C5a receptor fails to protect against experimental autoimmune encephalomyelitis in rats.

Complement activation contributes to inflammation and tissue damage in human demyelinating diseases and in rodent models of demyelination. Inhibitors of complement activation ameliorate disease in the rat model antibody-dependent experimental autoimmune encephalomyelitis and rats unable to generate the membrane attack complex of complement develop inflammation without demyelination. The role of the highly active chemotactic and anaphylactic complement-derived peptide C5a in driving inflammation and pathology in rodent models of demyelination has been little explored. Here we have used a small molecule C5a receptor antagonist, AcF-[OPdChaWR], to examine the effects of C5a receptor blockade in rat models of brain inflammation and demyelination. C5a receptor antagonist therapy completely blocked neutrophil response to C5a in vivo but had no effect on clinical disease or resultant pathology in either inflammatory or demyelinating rat models. We conclude that C5a is not required for disease induction or perpetuation in these strongly complement-dependent disease models.

Animals↗
PubMed2003

Familial motor neurone disease with dementia: phenotypic variation and cerebellar pathology.

OBJECTIVES: To characterise the neuropathological phenotypes of two affected individuals from a family with an unusual clinical phenotype resembling motor neurone disease and dementia. METHODS: Histological sections of cerebral cortex, basal ganglia, brain stem, cerebellum, and spinal cord were stained with haematoxylin-eosin, luxol fast blue, silver stains, anti-tau, anti-ubiquitin, anti-alpha-synuclein, and anti-neurofilament. RESULTS: Numerous ubiquitin positive, tau and alpha-synuclein negative intraneuronal inclusions were present in the cerebral cortex (particularly within the dentate gyrus), cerebellar cortex, brain stem, and spinal cord. The cerebellar ubiquitinated inclusions were located in the proximal dendrite of the Purkinje cells. Loss of Purkinje cells and occasional silver and neurofilament positive axonal swellings (torpedoes) were also seen within the cerebellar cortex. The main difference between the two cases was the severity of the spinal cord involvement: no significant pathology was present within one, but obvious motor neurone disease within the other. CONCLUSIONS: The clinical and neuropathological findings in this family are best described as an example of familial motor neurone disease with dementia. Intraneuronal ubiquitin inclusions together with agyrophilic, neurofilament positive torpedoes were present within the cerebellar cortex, both previously unrecognised findings in this group of diseases.

Adult↗
PubMed2002

Possible underascertainment of variant Creutzfeldt-Jakob disease: a systematic study.

OBJECTIVES: To predict the size of the vCJD epidemic it is important to know whether the description of cases of vCJD in 1996 represent the first cases of a new disease entity or whether detection was due to increased surveillance of CJD in humans. Detection of earlier cases would suggest a shorter incubation period and might lead to predictions of epidemic size being revised. METHODS: All certified deaths (excluding external injury and poisoning) in residents of Wales aged 15-45, between 1985 and 1995, were reviewed to detect vCJD deaths that might have been overlooked. 12 091 deaths were reviewed. "Non-specific fatal disorders compatible with vCJD" were defined. Deaths recorded to diseases other than those defined were rejected from further analysis (8769). Remaining cases (3322) were subdivided. Group A comprised deaths recorded to suicide, transport accidents, and those that could not be ascertained (ICD9 rubrics E950-959, E800-848, and 7999), a total of 2698 cases. Group B comprised deaths due to neurological disease, psychiatric disease, or substance abuse (624). RESULTS: For group A, remaining brain material was identified (n=218, 8.1%) and examined by routine histology and immunocytochemistry for prion protein. No cases of vCJD were detected. For group B, review of remaining clinical information was undertaken. Of 624 cases, information was available on 447 (72%). Brain tissue was examined by routine histology and immunocytochemistry in 47 (7.5%) cases. Sufficient clinical and pathological information was available to exclude all these as potential cases of vCJD. CONCLUSION: Variant CJD is a new disease entity and not simply the result of better case ascertainment.

Adolescent↗
PubMed2001

Accumulation of calbindin in cortical pyramidal cells with ageing; a putative protective mechanism which fails in Alzheimer's disease.

There is considerable interest in the status of calbindin immunoreactive neurones in Alzheimer's disease (AD) but previous studies have produced widely differing results. Here we describe calbindin neurones in temporal neocortex from 18 severely demented patients with neuropathologically confirmed AD and 13 age and post-mortem delay matched, neurologically normal controls. Calbindin immunoreactive neurones were small and round in layers II-IV, and pyramidal in layers IIIc and V. There were significantly more calbindin positive neurones in controls than in AD (mean+/- SD, for each comparison P < 0.01): superior temporal lobe, AD = 3.32 +/- 2.24, Control (C) = 24.83 +/- 10.8; middle temporal lobe, AD = 3.6 +/- 4.94, C = 26.09 +/- 15.7; inferior temporal lobe, AD 3.69 +/- 3.6, C = 25.25 +/- 16.9. Furthermore, there was an age-related increase in immunopositive neurones in the superior (r2 = 0.37, P = 0.046) and inferior (r2 = 0.75, P = 0.01) temporal gyri in controls. In AD the number of calbindin positive neurones did not change with age. This is the first report of such an age-related increase in controls, and it suggests that this, rather than a decrease in AD, accounts for the overall difference between AD and controls. It is possible that an increase in intraneuronal calbindin protects these cells from degeneration and that failure of such a neuroprotective mechanism is a significant contributory factor in the pathogenesis of AD.

Aged↗
PubMed2000

Glycogen synthase kinase-3 inhibits the DNA binding activity of NFATc.

The NFAT family of transcription factors is required for the expression of numerous immunologically important genes and plays a pivotal role in both the initiation and coordination of the immune response. NFAT family members appear to be regulated primarily at the level of their subcellular localization. Here we show that NFATc is additionally regulated at the level of its DNA binding activity. Using gel mobility shift assays, we demonstrate that the intrinsic DNA binding activity of NFATc is negatively regulated by phosphorylation. We found that activation of calcineurin activity in cells and dephosphorylation of NFATc in vitro enhanced NFATc DNA binding activity, whereas phosphorylation of NFATc in vitro inhibited its ability to bind DNA. Through the analysis of NFATc mutants, we identified the conserved Ser-Pro repeat motifs as critical quantitative determinants of NFATc DNA binding activity. In addition, we provide several lines of evidence to suggest that the phosphorylation of the Ser-Pro repeats by glycogen synthase kinase-3 inhibits the ability of NFATc to bind DNA. Taken together, these studies afford new insights into the regulation of NFATc and underscore the potential role of glycogen synthase kinase-3 in the regulation of NFAT-dependent gene expression.

Amino Acid Motifs↗
PubMed2000

Spontaneous classical pathway activation and deficiency of membrane regulators render human neurons susceptible to complement lysis.

This study investigated the capacity of neurons and astrocytes to spontaneously activate the complement system and control activation by expressing complement regulators. Human fetal neurons spontaneously activated complement through the classical pathway in normal and immunoglobulin-deficient serum and C1q binding was noted on neurons but not on astrocytes. A strong staining for C4, C3b, iC3b neoepitope and C9 neoepitope was also found on neurons. More than 40% of human fetal neurons were lysed when exposed to normal human serum in the presence of a CD59-blocking antibody, whereas astrocytes were unaffected. Significant reduction in neuronal cell lysis was observed after the addition of soluble complement receptor 1 at 10 microg/ml. Fetal neurons were stained for CD59 and CD46 and were negative for CD55 and CD35. In contrast, fetal astrocytes were strongly stained for CD59, CD46, CD55, and were negative for CD35. This study demonstrates that human fetal neurons activate spontaneously the classical pathway of complement in an antibody-independent manner to assemble the cytolytic membrane attack complex on their membranes, whereas astrocytes are unaffected. One reason for the susceptibility of neurons to complement-mediated damage in vivo may reside in their poor capacity to control complement activation.

Antibodies, Blocking↗
PubMed1999

Localization of rabbit huntingtin using a new panel of monoclonal antibodies.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG repeat which is expressed as a polyglutamine tract near the N-terminus of the gene product, huntingtin. N-terminal huntingtin fragments form intranuclear aggregates in HD patients and these may be involved in the pathogenesis. Monoclonal antibodies (mAbs) against three different regions of huntingtin (amino acids 997-1276, 1844-2131 and 2703-2911) have been produced and two of the epitopes have been identified using phage displayed peptide libraries. All mAbs reacted with 350 kDa huntingtin on Western blots and one mAb from each region was selected for further study by strong immunoreactivity with neurons in different regions of rabbit brain and by ability to immunoprecipitate native huntingtin. Subcellular fractionation and sucrose density centrifugation of rabbit brain extract showed that most of the huntingtin exists as a high molecular weight complex in the cytoplasm. Two outstanding problems have been addressed; the location of huntingtin in tissues outside the central nervous system and whether huntingtin is present in the nucleus of normal cells. We conclude that huntingtin is present at low levels in most non-neuronal cells though we have identified an interstitial cell type in skin with very high immunoreactivity. Using both immunolocalization and nuclear purification methods, we were unable to exclude the possibility that a small proportion of full-length huntingtin is present in the nucleus.

Animals↗
PubMed1999

Increased complement biosynthesis by microglia and complement activation on neurons in Huntington's disease.

In this study complement activation and biosynthesis have been analysed in the brains of Huntington's disease (HD) (n = 9) and normal (n = 3) individuals. In HD striatum, neurons, myelin and astrocytes were strongly stained with antibodies to C1q, C4, C3, iC3b-neoepitope and C9-neoepitope. In contrast, no staining for complement components was found in the normal striatum. Marked astrogliosis and microgliosis were observed in all HD caudate and the internal capsule samples but not in normal brain. RT-PCR analysis and in-situ hybridisation were carried out to determine whether complement was synthesised locally by activated glial cells. By RT-PCR, we found that complement activators of the classical pathway C1q C chain, C1r, C4, C3, as well as the complement regulators, C1 inhibitor, clusterin, MCP, DAF, CD59, were all expressed constitutively and at much higher level in HD brains compared to normal brain. Complement anaphylatoxin receptor mRNAs (C5a receptor and C3a receptor) were strongly expressed in HD caudate. In general, we found that the level of complement mRNA in normal control brains was from 2 to 5 fold lower compared to HD striatum. Using in-situ hybridisation, we confirmed that C3 mRNA and C9 mRNA were expressed by reactive microglia in HD internal capsule. We propose that complement produced locally by reactive microglia is activated on the membranes of neurons, contributing to neuronal necrosis but also to proinflammatory activities. Complement opsonins (iC3b) and anaphylatoxins (C3a, C5a) may be involved in the recruitment and stimulation of glial cells and phagocytes bearing specific complement receptors.

Adult↗
PubMed1999

Aberrant interactions of transcriptional repressor proteins with the Huntington's disease gene product, huntingtin.

We detected an interaction of the N-terminus of huntingtin (htt171) with the C-terminal region of the nuclear receptor co-repressor (N-CoR) using the yeast two-hybrid system. This interaction was repeat length dependent and specific to htt171; the co-repressor did not interact with the repeat carrying a section of atrophin 1 nor with the androgen receptor or polyglutamine alone. The interaction was confirmed using His-tagged Escherichia coli -expressed C-terminal human and rat co-repressor protein which pulled full-length huntingtin out of homogenized rat brain and in pull-down assays. The N-CoR represses transcription from sequence-specific ligand-activated receptors such as the retinoid X-thyroid hormone receptor dimers and other nuclear receptors including Mad-Max receptor dimers. The mechanism of this repression appears to be through the formation of a complex of repressor proteins including the N-CoR, mSin3 and histone deacetylases. We have used N-CoR and mSin3A antibodies in immunohistochemical studies and find that in Huntington's disease (HD) cortex and caudate, the cellular localization of these proteins is exclusively cytoplasmic whilst in control brain they are localized in the nucleus as well as the cytoplasm; mSin3A immunoreactivity also occurred in a subset of huntingtin positive intranuclear inclusions. The relocalization of repressor proteins in HD brain may alter transcription and be involved in the pathology of the disease.

Acetyltransferases↗
PubMed1999

Differential expression of individual complement regulators in the brain and choroid plexus.

Membrane bound regulators of complement (C) control the system at key points during activation. To determine whether C regulators were expressed in the central nervous system, temporal cortex, and choroid plexus, tissues from eight adult humans were obtained at postmortem and surgery. Tissue was taken fresh for total RNA isolation, snap freezing, or processing in paraffin wax for immunocytochemistry and in situ hybridization. Immunocytochemistry of temporal cortex using anti-CD59 stained microglia intensely; astrocytes and neurons weakly. Microglia were unequivocally stained with anti-membrane cofactor protein (MCP) whereas staining on astrocytes and neurons was weak. Decay accelerating factor (DAF) was strongly expressed by microglia but weakly by astrocytes. Neurons expressed neither DAF nor complement receptor 1 (CR1). CR1 was also absent on astrocytes and microglia. The choroid plexus epithelium revealed intense apical staining with antibodies to CD59, less strongly with anti-MCP and weakly with anti-DAF. CR1 was detected only on phagocytic Kolmer cells in the choroid plexus. Reverse transcriptase-polymerase chain reaction revealed CD59, MCP, and to a lesser degree, DAF mRNA both in the choroid plexus and temporal cortex. CR1 mRNA was detected in choroid plexus samples only. Digoxigenin-UTP-labeled riboprobes to all four membrane regulators were used for in situ hybridization. DAF, MCP, and CD59 mRNA were expressed by epithelial cells of the choroid plexus and CR1 mRNA was found only in Kolmer cells. In the temporal cortex, MCP and CD59 mRNA were expressed by glia and at low level by neurons, but DAF was not detected. Previous studies have suggested that C produced in inflamed brains in conditions such as Alzheimer's and Huntington's diseases can be specifically toxic to neurons. The demonstration herein that neurons express only very low levels of CD59 and MCP and lack both CR1 and DAF might explain their susceptibility to C damage.

Adult↗
PubMed1998

The receptor for complement anaphylatoxin C3a is expressed by myeloid cells and nonmyeloid cells in inflamed human central nervous system: analysis in multiple sclerosis and bacterial meningitis.

The complement anaphylatoxins C5a and C3a are released at the inflammatory site, where they contribute to the recruitment and activation of leukocytes and the activation of resident cells. The distribution of the receptor for C5a (C5aR) has been well studied; however, the receptor for C3a (C3aR) has only recently been cloned, and its distribution is uncharacterized. Using a specific affinity-purified anti-C3aR peptide Ab and oligonucleotides for reverse transcriptase-PCR analysis, C3aR expression was characterized in vitro on myeloid and nonmyeloid cells and in vivo in the brain. C3aR was expressed by adult astrocytes, astrocyte cell lines, monocyte lines THP1 and U937, neutrophils, and monocytes, but not by K562 or Ramos. C3aR staining was confirmed by flow cytometry, confocal imaging, and electron microscopy analysis. A 65-kDa protein was immunoprecipitated by the anti-C3aR from astrocyte and monocyte cell lysates. Our results at the protein level were confirmed at the mRNA level. Using reverse transcriptase-PCR, Southern blot, and sequencing we found that C3aR mRNA was expressed by fetal astrocytes, astrocyte cell lines, and THP1, but not by K562 or Ramos. The astrocyte C3aR cDNA was identical with the reported C3aR cDNA. C3aR expression was not detected in normal brain sections. However, a strong C3aR staining was evident in areas of inflammation in multiple sclerosis and bacterial meningitis. In meningitis, C3aR was abundantly expressed by reactive astrocytes, microglia, and infiltrating cells (macrophages and neutrophils). In multiple sclerosis, infiltrating lymphocytes did not express C3aR, but a strong staining was detected on smooth muscle cells (pericytes) surrounding blood vessels.

Adult↗
PubMed1998

Huntingtin protein colocalizes with lesions of neurodegenerative diseases: An investigation in Huntington's, Alzheimer's, and Pick's diseases.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease associated with a CAG trinucleotide repeat expansion in a large gene on chromosome 4. The gene encodes the protein huntingtin with a polyglutamine tract encoded by the CAG repeat at the N-terminus. The number of CAG repeats in HD are significantly increased (36 to 120+) compared with the normal population (8-39). The pathological mechanism associated with the expanded CAG repeat in HD is not clear but there is evidence that polyglutamine is directly neurotoxic. We have immunolocalized huntingtin with an in-house, well-characterised, polyclonal antibody in HD, Alzheimer's disease (AD), and Picks disease (PiD) brains. Control brain tissue sections were from head injured and cerebral ischaemia cases. In HD, huntingtin was immunopositive in the surviving but damaged neurons and reactive astrocytes of the caudate and putamen. However, in AD and PiD the immunostaining was largely restricted to the characteristic intracellular inclusion bodies associated with the disease process in each case. In AD, huntingtin was localized only in the intracellular neurofibrillary tangles and dystrophic neurites within the neuritic amyloid plaques but not with the amyloid. In PiD, strongly positive huntingtin immunostaining was present within cytoplasmic Pick bodies. Our findings suggest huntingtin selectively accumulates in association with abnormal intracytoplasmic and cytoskeletal filaments of neurons and glia in neurodegenerative diseases such as HD, AD, and PiD. Cells in the CNS appear sensitive to damage by the aggregated, toxic levels of huntingtin and evidence of its interaction with neurofilaments could provide information about its potential role in the aetiology of HD.

Adult↗