N. A. Gregson

GALACTOCEREBROSIDE IS A SPECIFIC CELL-SURFACE ANTIGENIC MARKER FOR OLIGODENDROCYTES IN CULTURE

A TEMPERATURE-SENSITIVE (ts) mutant of the highly oncogenic group A (ref. 1) human adenovirus type 12 (H12), H12ts401 (ref. 2) is unable to establish stable transformation of cells in restrictive conditions3. Cells transformed by ts401 at the permissive temperature and shifted to nonpermissive temperatures show a reversion to a normal phenotype; wild-type H12-transformed cells, in contrast, exhibit a transformed phenotype when grown in either restrictive or permissive conditions4. The temperature sensitivity of the transformed phenotype of the ts401 mutant-transformed cells suggests that the continued expression of gene 401 is required for maintenance of the transformed cell phenotype. This study was initiated to detect the H12 transformation-specific protein(s) in H12-transformed cell lines. We identify here a 60,000 molecular weight transformation-specific antigen in H12-transformed rat 3Y1 cells5,6 by immunoprecipitation of 35S-methionine labelled polypeptides with serum from H12 tumour-bearing hamsters. Furthermore, the expression of this antigen is temperature dependent in 3Y1 cells transformed by ts401. Further characterisation of the 60,000 MW antigen may lead to an understanding of the molecular mechanism of adenovirus cell transformation.

Campylobacter jejuni Infection and Guillain–Barré Syndrome

BACKGROUND Although infection with Campylobacter jejuni is recognized as a common antecedent of the Guillain-Barré syndrome, the clinical and epidemiologic features of this association are not well understood. METHODS We performed a prospective case-control study in a cohort of patients with Guillain-Barré syndrome (96 patients) or Miller Fisher syndrome (7 patients) who were admitted to hospitals throughout England and Wales between November 1992 and April 1994. Bacteriologic and serologic techniques were used to diagnose preceding C. jejuni infection. RESULTS There was evidence of recent C. jejuni infection in 26 percent of the patients with Guillain-Barré or Miller Fisher syndrome, as compared with 2 percent of household controls and 1 percent of age-matched hospital controls (P < 0.001). Of the 27 patients with C. jejuni infection, 19 (70 percent) reported having had a diarrheal illness within 12 weeks before the onset of the neurologic illness. No specific serotypes were associated with Guillain-Barré syndrome. C. jejuni infection was slightly more common in men (P = 0.14) and was more likely to be associated with a pure motor syndrome and a slower recovery (P = 0.03). The patients with preceding C. jejuni infection were more likely to have acute axonal neuropathy or axonal degeneration in association with acute inflammatory demyelinating polyradiculoneuropathy, and they had greater disability after one year (P = 0.02). C. jejuni infection was significantly associated with a poor outcome even after correction for other factors associated with a poor prognosis. CONCLUSIONS Infection with C. jejuni often precedes the Guillain-Barré syndrome and is associated with axonal degeneration, slow recovery, and severe residual disability.

Pathogenesis of Guillain-Barré syndrome.

Recent neurophysiological and pathological studies have led to a reclassification of the diseases that underlie Guillain-Barré syndrome (GBS) into acute inflammatory demyelinating polyradiculoneuropathy (AIDP), acute motor and sensory axonal neuropathy (AMSAN) and acute motor axonal neuropathy (AMAN). The Fisher syndrome of ophthalmoplegia, ataxia and areflexia is the most striking of several related conditions. Significant antecedent events include Campylobacter jejuni (4-66%), cytomegalovirus (5-15%), Epstein-Barr virus (2-10%), and Mycoplasma pneumoniae (1-5%) infections. These infections are not uniquely associated with any clinical subtype but severe axonal degeneration is more common following C. jejuni and severe sensory impairment following cytomegalovirus. Strong evidence supports an important role for antibodies to gangliosides in pathogenesis. In particular antibodies to ganglioside GM1 are present in 14-50% of patients with GBS, and are more common in cases with severe axonal degeneration associated with any subtype. Antibodies to ganglioside GQ1b are very closely associated with Fisher syndrome, its formes frustes and related syndromes. Ganglioside-like epitopes exist in the bacterial wall of C. jejuni. Infection by this and other organisms triggers an antibody response in patients with GBS but not in those with uncomplicated enteritis. The development of GBS is likely to be a consequence of special properties of the infecting organism, since some strains such as Penner 0:19 and 0:41 are particularly associated with GBS but not with enteritis. It is also likely to be a consequence of the immunogenetic background of the patient since few patients develop GBS after infection even with one of these strains. Attempts to match the subtypes of GBS to the fine specificity of anti-ganglioside antibodies and to functional effects in experimental models continue but have not yet fully explained the pathogenesis. T cells are also involved in the pathogenesis of most or perhaps all forms of GBS. T cell responses to any of three myelin proteins, P2, PO and PMP22, are sufficient to induce experimental autoimmune neuritis. Activated T cells are present in the circulation in the acute stage, up-regulate matrix metalloproteinases, cross the blood-nerve barrier and encounter their cognate antigens. Identification of the specificity of these T cell responses is still at a preliminary stage. The invasion of intact myelin sheaths by activated macrophages is difficult to explain according to a purely T cell mediated mechanism. The different patterns of GBS are probably due to the diverse interplay between antibodies and T cells of differing specificities.

Genetic and biochemical evidence of a Campylobacter jejuni capsular polysaccharide that accounts for Penner serotype specificity

Campylobacter jejuni, a Gram‐negative spiral bacterium, is the most common bacterial cause of acute human gastroenteritis and is increasingly recognized for its association with the serious post‐infection neurological complications of the Miller–Fisher and Guillain–Barré syndromes. C. jejuni lipopolysaccharide (LPS) is thought to be involved in the pathogenesis of both uncomplicated infection and more serious sequelae, yet the LPS remains poorly characterized. Current studies on C. jejuni suggest that all strains produce lipooligosaccharide (LOS), with about one‐third of strains also producing high‐molecular‐weight LPS (referred to as O‐antigen). In this report, we demonstrate the presence of the high‐molecular‐weight LPS in all C. jejuni strains tested. Furthermore, we show that this LPS is biochemically and genetically unrelated to LOS and is similar to group II and group III capsular polysaccharides. All tested kpsM, kpsS and kpsC mutants of C. jejuni lost the ability to produce O‐antigen. Moreover, this correlated with serotype changes. We demonstrate for the first time that the previously described O‐antigen of C. jejuni is a capsular polysaccharide and a common component of the thermostable antigen used for serotyping of C. jejuni.

Phase variation of a β-1,3 galactosyltransferase involved in generation of the ganglioside GM1-like lipo-oligosaccharide of Campylobacter jejuni

Ganglioside mimicry by Campylobacter jejuni lipo‐oligosaccharide (LOS) is thought to be a critical factor in the triggering of the Guillain–Barré and Miller–Fisher syndrome neuropathies after C. jejuni infection. The combination of a completed genome sequence and a ganglioside GM1‐like LOS structure makes C. jejuni NCTC 11168 a useful model strain for the identification and characterization of the genes involved in the biosynthesis of ganglioside‐mimicking LOS. Genome analysis identified a putative LOS biosynthetic cluster and, from this, we describe a putative gene (ORF Cj1139c), which we have termed wlaN, with a significant level of similarity to a number of bacterial glycosyltransferases. Mutation of this gene in C. jejuni NCTC 11168 resulted in a LOS molecule of increased electrophoretic mobility, which also failed to bind cholera toxin. Comparison of LOS structural data from wild type and the mutant strain indicated lack of a terminal β‐1,3‐linked galactose residue in the latter. The wlaN gene product was demonstrated unambiguously as a β‐1,3 galactosyltransferase responsible for converting GM2‐like LOS structures to GM1‐like by in vitro expression. We also show that the presence of an intragenic homopolymeric tract renders the expression of a functional wlaN gene product phase variable, resulting in distinct C. jejuni NCTC 11168 cell populations with alternate GM1 or GM2 ganglioside‐mimicking LOS structures. The distribution of wlaN among a number of C. jejuni strains with known LOS structure was determined and, for C. jejuni NCTC 12500, similar wlaN gene phase variation was shown to occur, so that this strain has the potential to synthesize a GM1‐like LOS structure as well as the ganglioside GM2‐like LOS structure proposed in the literature.

Experimental allergic neuritis in the Lewis rat: Characterization of the activity of peripheral myelin and its major basic protein,P2

Experimental allergic neuritis has been produced in the inbred Lewis rat in the absence of experimental allergic encephalomyelitis (EAE) using bovine intradural root myelin. The lack of EAE is probably because P1 is only weakly encephalitogenic in the rat. One of the basic proteins of bovine peripheral myelin, P2, was isolated and demonstrated to be pure by amino acid analysis and SDS PAGE. It was found to have a molecular weight of 15,400 and contained 4 mol 1/2-cystine/mol. This P2 was found to be highly neuritogenic and is probably the sole neuritogenic antigen in this system. The successful demonstration of its neuritogenicity must be due in large part to the use of the inbred Lewis rat and bovine P2, but an explanation could also involve the omission of denaturing organic solvents, the prevention of oxidative denaturation and presumably the fact that any changes which may occur are not sufficient to prevent recognition of the active site by the immune system of the inbred Lewis rat. P2 was neuritogenic down to 5 micrograms/animal. Its activity was enhanced by but not dependent on the presence of Mycobacterium in the adjuvant. This suggested that release of P2 could possibly break tolerance and produce an auto-immune disease such as the Guillain--Barre syndrome.

A novel paralogous gene family involved in phase-variable flagella-mediated motility in Campylobacter jejuni

Flagella-mediated motility is recognized to be one of the major factors contributing to virulence in Campylobacter jejuni. Motility of this bacterium is known to be phase variable, although the mechanism of such variation remains unknown. C. jejuni genome sequencing revealed a number of genes prone to phase variation via a slipped-strand mispairing mechanism. Many of these genes are hypothetical and are clustered in the regions involved in formation of three major cell surface structures: capsular polysaccharide, lipooligosaccharide and flagella. Among the genes of unknown function, the flagellar biosynthesis and modification region contains seven hypothetical paralogous genes designated as the motility accessory factor (maf) family. Remarkably, two of these genes (maf1 and maf4) were found to be identical and both contain homopolymeric G tracts. Using insertional mutagenesis it was demonstrated that one of the genes, maf5, is involved in formation of flagella. Phase variation of the maf1 gene via slipped-strand mispairing partially restored motility of the maf5 mutant. The maf family represents a new class of bacterial genes related to flagellar biosynthesis and phase variation. Reversible expression of flagella may be advantageous for the adaptation of C. jejunito the varied in vivo and ex vivo environments encountered during its life cycle, as well in evasion of the host immune response.

Pathogenesis of chronic inflammatory demyelinating polyradiculoneuropathy.

Abstract  The acute lesions of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) consist of endoneurial foci of chemokine and chemokine receptor expression and T cell and macrophage activation. The myelin protein antigens, P2, P0, and PMP22, each induce experimental autoimmune neuritis in rodent models and might be autoantigens in CIDP. The strongest evidence incriminates P0, to which antibodies have been found in 20% of cases. Failure of regulatory T‐cell mechanism is thought to underlie persistent or recurrent disease, differentiating CIDP from the acute inflammatory demyelinating polyradiculoneuropathy form of Guillain‐Barré syndrome. Corticosteroids, intravenous immunoglobulin and plasma exchange each provide short term benefit but the possible long‐term benefits of immunosuppressive drugs have yet to be confirmed in randomised, controlled trials.

Inflammation and primary demyelination induced by the intraspinal injection of lipopolysaccharide

Abstract Inflammation is a prominent feature of several disorders characterized by primary demyelination, but it is not clear whether a relationship exists between inflammation and myelin damage. We have found that substantial demyelination results from the focal inflammatory lesion caused by the injection of lipopolysaccharide (LPS; 200 ng) directly into the rat dorsal funiculus. Within 24 h, such injections caused a focal inflammatory response consisting of a substantial number of polymorphonuclear cells and ED1-positive and inducible nitric oxide synthase (iNOS)-positive macrophages/microglia. The number of inflammatory cells was substantially reduced by day 7. OX-52-positive T-cells were less frequently observed but were present in the meninges at 8 h, reached a maximum in the dorsal funiculus at 7 days, and were rare at 14 days. The inflammation was followed by the appearance of a large lesion of primary demyelination that encompassed up to ∼75% of the cross-sectional area of the dorsal funiculus. Treatment with dexamethasone significantly reduced the number of cells expressing iNOS, but did not prevent the demyelination. By 28 days the lesions were largely remyelinated, usually by Schwann cells. These changes were not observed in control, saline-injected animals. We conclude that the intraspinal injection of LPS results in inflammation and subsequently in prominent demyelination. The mechanisms underlying the demyelination are not clear, but it is notable that it typically begins with disruption of the adaxonal myelin. Indeed, there is an early loss of myelin-associated glycoprotein within the lesion, despite the persistence of proteolipid protein. This combination is a feature of the pattern III lesion recently described in multiple sclerosis (Lucchinetti et al., 2000), and we therefore suggest that LPS-induced demyelination may serve as the first experimental model available for the study of this type of multiple sclerosis lesion.

Multiple N-acetyl neuraminic acid synthetase (neuB) genes in Campylobacter jejuni: identification and characterization of the gene involved in sialylation of lipo-oligosaccharide.

N‐acetyl neuraminic acid (NANA) is a common constituent of Campylobacter jejuni lipo‐oligosaccharide (LOS). Such structures often mimic human gangliosides and are thought to be involved in the triggering of Guillain–Barré syndrome (GBS) and Miller–Fisher syndrome (MFS) following C. jejuni infection. Analysis of the C. jejuni NCTC 11168 genome sequence identified three putative NANA synthetase genes termed neuB1, neuB2 and neuB3. The NANA synthetase activity of all three C. jejuni neuB gene products was confirmed by complementation experiments in an Escherichia coli neuB‐deficient strain. Isogenic mutants were created in all three neuB genes, and for one such mutant (neuB1) LOS was shown to have increased mobility. C. jejuni NCTC 11168 wild‐type LOS bound cholera toxin, indicating the presence of NANA in a LOS structure mimicking the ganglioside GM1. This property was lost in the neuB1 mutant. Gas chromatography–mass spectrometry and fast atom bombardment–mass spectrometry analysis of LOS from wild‐type and the neuB1 mutant strain demonstrated the lack of NANA in the latter. Expression of the neuB1 gene in E. coli confirmed that NeuB1 was capable of in vitro NANA biosynthesis through condensation of N‐acetyl‐d‐mannosamine and phosphoenolpyruvate. Southern analysis demonstrated that the neuB1 gene was confined to strains of C. jejuni with LOS containing a single NANA residue. Mutagenesis of neuB2 and neuB3 did not affect LOS, but neuB3 mutants were aflagellate and non‐motile. No phenotype was evident for neuB2 mutants in strain NCTC 11168, but for strain G1 the flagellin protein from the neuB2 mutant showed an apparent reduction in molecular size relative to the wild type. Thus, the neuB genes of C. jejuni appear to be involved in the biosynthesis of at least two distinct surface structures: LOS and flagella.