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Biomedical subjects

A Vincent

Publications and source records attributed to A Vincent.

At least 127 records · Page 7Linked to original sources
PubMed1999

Pathogenic autoantibodies to neuronal proteins in neurological disorders.

Autoantibodies to acetylcholine receptors and to voltage-gated calcium and potassium channels are thought to be pathogenic in three peripheral neurological disorders: myasthenia gravis, the Lambert Eaton syndrome and acquired neuromyotonia. However, evidence for the role of antibodies in conditions involving the central nervous system, is scanty or unclear. This review describes the ways in which the roles of autoantibodies have been defined in the peripheral diseases, and discusses the more controversial evidence for involvement of autoantibodies in some central disorders such as multiple sclerosis.

Autoantibodies↗
PubMed1999

Postnatal development of rat nucleus tractus solitarius neurons: morphological and electrophysiological evidence.

Postnatal development of neurons in the caudal nucleus tractus solitarii of rats was studied using the Golgi-Cox technique and whole-cell recordings. Two cell classes were defined on the basis of somatic and dendritic morphology. Elongated neurons have two thick primary dendrites originating from the long axis of the soma. The primary dendrites, tapering distally, give rise to one to four secondary dendrites. Multipolar neurons have pyramidal somas. Extending from each apex of the cell body was a long primary dendrite, which gave rise to a variable number of secondary dendrites. The relative proportion of the two classes was rather constant from birth to adulthood. During the first two postnatal weeks, dendritic length and area of influence increase, but neuronal geometry is not altered in either class. Dendritic appendages appear by postnatal day 5, reach a peak at postnatal day P12 and then almost disappear in adult neurons. Combined intracellular injection of neurobiotin and whole-cell recordings indicate that morphological alteration of caudal nucleus tractus solitarius neurons occurs in parallel with changes in passive properties and spike characteristics. However, the firing pattern of discharge is not correlated with morphology. The physiological significance of these results is discussed.

Animals↗
PubMed1999

The role of T-cells in the initiation of autoantibody responses in thymoma patients.

Thymomas are associated with several different neurological disorders. Highly specific autoantibodies directed against central nervous system and muscle antigens are found in the sera of these patients. These antibodies usually have high affinity and specificity for the intact conformation of the antigen. However, some are directed against cell surface antigens, and are directly pathogenic, while others are specific for intracellular antigens which are probably not accessible to antibodies in vivo. Moreover, the intact antigens do not appear to be present in the tumour itself. A hypothesis to explain the role of the thymoma in inducing the autoimmunity must also account for the fact that the autoimmune disorders do not necessarily remit after thymomectomy, and that in some cases they only begin several years after the operation. Thymomas often generate large numbers of T-cells that appear to be sensitised to self-epitopes in the thymoma. We hypothesise that both cytotoxic and helper T-cells are induced against specific peptides in thymoma, and then move to the periphery where they can persist. At some stage, the cytotoxic T-cells recognise epitopes presented by muscle or CNS tissue, perhaps following minor tissue damage or inflammation with upregulation of class I and/or accessory molecules. Cytotoxicity results in release of other antigens, both cytoplasmic and membranous, leading to uptake and presentation by class II positive antigen presenting cells, including antigen-specific B-cells. Only when antigen, class II-restricted helper T-cells and the specific B-cells are present together, in local lymph nodes, will the characteristic high affinity autoantibodies result. Of these, only those against cell surface antigens will be pathogenic.

Autoantibodies↗
PubMed1999

A comparison of MyoD1 and fetal acetylcholine receptor expression in childhood tumors and normal tissues: implications for the molecular diagnosis of minimal disease in rhabdomyosarcomas.

Detection of minimal residual disease or micrometastases in rhabdomyosarcoma (RMS) has been an unresolved problem in 70 to 80% of RMS patients. In patients with alveolar type RMS, which harbors chromosomal translocations and produces tumor-specific fusion products, polymerase chain reaction (PCR)-based diagnosis is clear-cut. In the more frequent embryonal RMS, however, no such PCR-based marker has been described. Recently it has been suggested that the PCR-based detection of MyoD1 may be a valuable adjunct in the diagnosis of minimal disease in embryonal RMS. We report here that MyoD1 mRNA is not specific for RMS, but can be amplified from ex vivo samples of many other childhood tumors and some normal tissues. By contrast, simultaneous amplification of alpha and gamma subunit message of the fetal type acetylcholine receptor (AChR), by a novel duplex PCR, and the quantification of both transcripts resulting in a alpha/gammaAChR ratio <1 was 100% sensitive in alveolar (n = 8) and embryonal (n = 10) RMS. Moreover, gammaAChR was not detected in other childhood (n = 27) or adult tumors (n = 12), or normal tissues, except thymus. The high sensitivity and specificity of the method were confirmed by the successful detection of five cases of cytologically or molecularly verified RMS bone marrow micrometastases among 47 bone marrow samples from childhood tumor patients. By contrast, MyoD1 showed no amplification because of its low level of transcription. We conclude that mRNA of the fetal type AChR is a more specific and (about 100 times) more sensitive marker for the molecular detection of RMS than MyoD1, and thus appears to be a promising candidate for the detection of minimal disease in RMS lacking tumor-specific translocations.

Base Sequence↗
PubMed1999

Physicochemical and immunological studies of the N-terminal domain of the Torpedo acetylcholine receptor alpha-subunit expressed in Escherichia coli.

The nicotinic acetylcholine receptor (AChR) from the electric organ of Torpedo species is an oligomeric protein composed of alpha2 beta gamma delta subunits. Although much is known about its tertiary and quaternary structure, the conformation of the large extracellular domains of each of the subunits has not been analysed in detail. In order to obtain information about the spatial structure of the extracellular domain, we have expressed the N-terminal fragment 1-209 of the Torpedo californica AChR alpha-subunit in Escherichia coli. Two vectors coding for a (His)6 tag, either preceding or following the 1-209 sequence, were used and the recombinant proteins obtained (designated alpha1-209pET and alpha1-209pQE, respectively) were purified by affinity chromatography on a Ni2+-agarose column. The chemical structure of both proteins was verified by Edman degradation and mass spectrometry. The proteins were soluble in aqueous buffers but to make possible a comparison with the whole AChR or its isolated subunits, the recombinant proteins were analyzed both in aqueous solution and with the addition of detergents. The two proteins bound [125I]alpha-bungarotoxin with equal potency (KD approximately 130 nm, Bmax approximately 10 nmol.mg-1). Both were shown to interact with several monoclonal antibodies raised against purified Torpedo AChR. The circular dichroism (CD) spectra of the two proteins in aqueous solution revealed predominantly beta-structure (50-56%), the fraction of alpha-helices amounting to 32-35%. Nonionic (beta-octylglucoside) and zwitterionic (CHAPS) detergents did not appreciably change the CD spectra, while the addition of SDS or trifluoroethanol decreased the percentage of beta-structure or increased the alpha-helicity, respectively. The predominance of beta-structure is in accord with recent data on the N-terminal domain of the mouse muscle AChR alpha-subunit expressed in the mammalian cells [West et al. (1997) J. Biol. Chem. 272, 25 468]. Thus, expression in E. coli provides milligram amounts of the protein that retains several structural characteristics of the N-terminal domain of the Torpedo AChR alpha-subunit and appears to share with the latter a similar secondary structure. The expression of recombinant polypeptides representing functional domains of the AChR provides an essential first step towards a more detailed structural analysis.

Amino Acid Sequence↗
PubMed1999

Immunology of the neuromuscular junction and presynaptic nerve terminal.

The prevalence and incidence of myasthenia gravis is higher than previously thought. A potentially immunodominant T cell has been defined. The specific voltage-gated calcium channel subtype that is targeted by antibodies in the Lambert-Eaton myasthenic syndrome has been identified, and there is further evidence for the pathogenic role of autoantibodies in some cases of fetal arthrogryposis and in acquired neuromyotonia, Morvan's syndrome and Miller-Fisher syndrome.

Animals↗
PubMed1999

Comparison of mouse bioassay and immunoprecipitation assay for botulinum toxin antibodies.

OBJECTIVE: To compare a recently developed immunoprecipitation assay (IPA) to the mouse protection bioassay (MPB), currently considered the "gold standard", for detecting antibodies against botulinum toxin A (BTX-A) and to correlate these assay results with clinical responses to BTX-A injections. METHODS: MPB and IPA assays were performed on serum samples from 83 patients (38 non-responders, 45 responders) who received BTX-A injections. Six non-responders had serum tested on two separate occasions. Some patients also received a "test" injection into either the right eyebrow (n=29) or right frontalis (n=19). RESULTS: All patients antibody positive (Ab+) by MPB were also Ab+ by IPA, whereas an additional 19 patients (17 with reduced or no clinical response) who were MPB Ab- were Ab+, with low titres, by IPA. Two of these 19 patients (non-responders) were initially MPB Ab- but later became MPB Ab+. Similar to previous studies, the sensitivity for the MPB was low; 50% for clinical, 38% for eyebrow, and 30% for frontalis responses whereas the IPA sensitivity was much higher at 84% for clinical (p<0.001), 77% for eyebrow (p=0.111, NS) and 90% for frontalis responses (p<0.02). The IPA specificity was 89% for clinical, 81% for eyebrow, and 89% for frontalis responses, whereas the MPB specificity was 100% for all three response types, which were all non-significant differences. CONCLUSIONS: Both assays had high specificity although the sensitivity of the IPA was higher than the MPB. In addition, the IPA seems to display positivity earlier than the MPB, and as such, it may prognosticate future non-responsiveness. Eyebrow and frontalis "test" injections correlated well with clinical and immunological results and are useful in the assessment of BTX non-responders.

Adult↗
PubMed1999

Plasma from human mothers of fetuses with severe arthrogryposis multiplex congenita causes deformities in mice.

Arthrogryposis multiplex congenita (AMC) is characterized by fixed joint contractures and other deformities, sometimes resulting in fetal death. The cause is unknown in most cases, but some women with fetuses affected by severe AMC have serum antibodies that inhibit fetal acetylcholine receptor (AChR) function, and antibodies to fetal antigens might play a pathogenic role in other congenital disorders. To investigate this possibility, we have established a model by injecting pregnant mice with plasma from four anti-AChR antibody-positive women whose fetuses had severe AMC. We found that human antibodies can be transferred efficiently to the mouse fetus during the last few days of fetal life. Many of the fetuses of dams injected with AMC maternal plasmas or Ig were stillborn and showed fixed joints and other deformities. Moreover, similar changes were found in mice after injection of a serum from one anti-AChR antibody-negative mother who had had four AMC fetuses. Thus, we have confirmed the role of maternal antibodies in cases of AMC associated with maternal anti-AChR, and we have demonstrated the existence of pathogenic maternal factors in one other case. Importantly, this approach can be used to look at the effects of other maternal human antibodies on development of the fetus.

Animals↗
PubMed1999

Head versus trunk patterning in the Drosophila embryo; collier requirement for formation of the intercalary segment.

Whereas the segmental nature of the insect head is well established, relatively little is known about the genetic and molecular mechanisms governing this process. In this paper, we report the phenotypic analysis of mutations in collier (col), which encodes the Drosophila member of the COE family of HLH transcription factors and is activated at the blastoderm stage in a region overlapping a parasegment (PS0: posterior intercalary and anterior mandibular segments) and a mitotic domain, MD2. col mutant embryos specifically lack intercalary ectodermal structures. col activity is required for intercalary-segment expression both of the segment polarity genes hedgehog, engrailed, and wingless, and of the segment identity gene cap and collar. The parasegmental register of col activation is controlled by the combined activities of the head-gap genes buttonhead and empty spiracles and the pair-rule gene even skipped; it therefore integrates inputs from both the head and trunk segmentation systems, which were previously considered as being essentially independent. After gastrulation, positive autoregulation of col is limited to cells of anterior PS0. Conversely, heat-pulse induced ubiquitous expression of Col leads to disruption of the head skeleton. Together, these results indicate that col is required for establishment of the PS(-1)/PS0 parasegmental border and formation of the intercalary segment. Our data support neither a simple combinatorial model for segmental patterning of the head nor a direct activation of segment polarity gene expression by head-gap genes, but rather argue for the existence of parasegment-specific second order regulators acting in the head, at a level similar to that of pair-rule genes in the trunk.

Amino Acid Sequence↗
PubMed1999

Requirement for the Drosophila COE transcription factor Collier in formation of an embryonic muscle: transcriptional response to notch signalling.

During Drosophila embryogenesis, mesodermal cells are recruited to form a stereotyped pattern of about 30 different larval muscles per hemisegment. The formation of this pattern is initiated by the specification of a special class of myoblasts, called founder cells, that are uniquely able to fuse with neighbouring myoblasts. We report here the role of the COE transcription factor Collier in the formation of a single muscle, muscle DA3([A])(DA4([T])). Col expression is first observed in two promuscular clusters (in segments A1-A7), the two corresponding progenitors and their progeny founder cells, but its transcription is maintained in only one of these four founder cells, the founder of muscle DA3([A]). This lineage-specific restriction depends on the asymmetric segregation of Numb during the progenitor cell division and involves the repression of col transcription by Notch signalling. In col mutant embryos, the DA3([A]) founder cells form but do not maintain col transcription and are unable to fuse with neighbouring myoblasts, leading to a loss-of-muscle DA3([A]) phenotype. In wild-type embryos, each of the DA3([A])-recruited myoblasts turns on col transcription, indicating that the conversion, by the DA3([A]) founder cell, of 'naive' myoblasts to express its distinctive pattern of gene expression involves activation of col itself. We find that muscles DA3([A]) and DO5([A]) (DA4([T]) and DO5([T])) derive from a common progenitor cell. Ectopic expression of Col is not sufficient, however, to switch the DO5([A]) to a DA3([A]) fate. Together these results lead us to propose that specification of the DA3([A]) muscle lineage requires both Col and at least one other transcription factor, supporting the hypothesis of a combinatorial code of muscle-specific gene regulation controlling the formation and diversification of individual somatic muscles.

Animals↗
PubMed1999

Mutation of the acetylcholine receptor epsilon-subunit promoter in congenital myasthenic syndrome.

Congenital myasthenic syndrome comprises a heterogeneous group of inherited disorders of neuromuscular transmission. Acetylcholine receptor (AChR) deficiency is the most common form of congenital myasthenic syndrome and in most cases results from mutations within the coding region of the AChR epsilon subunit. However, studies in mice have established that synapse-specific expression of AChR is dependent on a sequence contained within the AChR-subunit promoter regions, termed an N-box. We describe a consanguineous family in which 2 of 7 siblings had clinical and electromyographic features consistent with AChR deficiency. Muscle biopsy demonstrated low AChR numbers, establishing the disorder as postsynaptic. Single-strand conformational polymorphism analysis identified an abnormal conformer in the AChR epsilon-subunit gene promoter of the patients. DNA sequence and restriction endonuclease analysis shows that the disorder cosegregates with recessive inheritance of a single point mutation, a transition (C-->T) in the N-box of the epsilon-subunit promoter. Analysis of an intercostal biopsy from 1 of the patients showed a dramatic reduction in epsilon-subunit mRNA levels compared with disease and normal controls. This is the first evidence in humans that an N-box mutation can lead to disruption of epsilon-subunit transcription, resulting in the loss of adult AChR synthesis and the clinical phenotype of AChR-deficiency congenital myasthenic syndrome.

Adult↗
PubMed1998

Induction of primary immune responses by allogeneic human myoblasts: dissection of the cell types required for proliferation, IFNgamma secretion and cytotoxicity.

Non-professional antigen-presenting cells (APC) have a limited ability to activate T lymphocytes during normal and auto-immune responses. Myoblasts could play an important role as APC in the etiology of autoimmune myasthenia gravis and polymyositis, as well as during muscle graft rejection. We examined the role of different component cell subsets in the response of human peripheral blood mononuclear cells (PBMC) to allogeneic myoblasts. Treatment of myoblasts with TNFalpha or IFNgamma led to the expression of a range of immunostimulatory molecules including MHC class I and II, and CD95 (Fas), but not B7 family molecules. Whole PBMC, cultured with allogeneic myoblasts, proliferated, secreted IFNgamma, and were cytotoxic. Proliferation and IFNgamma secretion were largely dependent on the presence of CD4+ lymphocytes, but neither CD4+ nor CD8+ T cells were responsible for cytotoxicity, which was mediated by MHC class II+ non-T mononuclear cells. However, purified CD4+ lymphocytes co-cultured with allogeneic myoblasts required co-stimulation with anti-CD28 antibodies for proliferation and IFNgamma secretion, which only induced a low level of IFNgamma secretion by CD8+ lymphocytes and did not induce cytotoxic function. These results suggest that human myoblasts can act as antigen-presenting cells for naive T lymphocytes, but only with additional co-stimulation.

Antigen Presentation↗
PubMed1998

A pathogenetic role for the thymoma in myasthenia gravis. Autosensitization of IL-4- producing T cell clones recognizing extracellular acetylcholine receptor epitopes presented by minority class II isotypes.

Myasthenia gravis (MG) is caused by helper T cell-dependent autoantibodies against the muscle acetylcholine receptor (AChR). Thymic epithelial tumors (thymomas) occur in 10% of MG patients, but their autoimmunizing potential is unclear. They express mRNAs encoding AChR alpha and epsilon subunits, and might aberrantly select or sensitize developing thymocytes or recirculating peripheral T cells against AChR epitopes. Alternatively, there could be defective self-tolerance induction in the abundant maturing thymocytes that they usually generate. For the first time, we have isolated and characterized AChR-specific T cell clones from two MG thymomas. They recognize extracellular epitopes (alpha75-90 and alpha149-158) which are processed very efficiently from muscle AChR. Both clones express CD4 and CD8alpha, and have a Th-0 cytokine profile, producing IL-4 as well as IFN-gamma. They are restricted to HLA-DP14 and DR52a; expression of these minority isotypes was strong on professional antigen-presenting cells in the donors' tumors, although it is generally weak in the periphery. The two clones' T cell receptor beta chains are different, but their alpha chain sequences are very similar. These resemblances, and the striking contrasts with T cells previously cloned from non-thymoma patients, show that thymomas generate and actively induce specific T cells rather than merely failing to tolerize them against self antigens.

Amino Acid Sequence↗