Systemic delivery of human GlyR IgG antibody induces GlyR internalization into motor neurons of brainstem and spinal cord with motor dysfunction in mice.

AIMS: Progressive encephalomyelitis with rigidity and myoclonus (PERM) is a life-threatening condition often associated with highly raised serum antibodies to glycine receptors (GlyRs); these bind to the surface of large neurons and interneurons in rodent brain and spinal cord sections and, in vitro, inhibit function and reduce surface expression of the GlyRs. The effects in vivo have not been reported. METHODS: Purified plasma IgG from a GlyR antibody-positive patient with PERM, and a healthy control (HC), was injected daily into the peritoneal cavity of mice for 12 days; lipopolysaccharide (LPS) to open the blood-brain barrier, was injected on days 3 and 8. Based on preliminary data, behavioural tests were only performed 48 h post-LPS on days 5-7 and 10-12. RESULTS: The GlyR IgG injected mice showed impaired ability on the rotarod from days 5 to 10 but this normalized by day 12. There were no other behavioural differences but, at termination (d13), the GlyR IgG-injected mice had IgG deposits on the neurons that express GlyRs in the brainstem and spinal cord. The IgG was not only on the surface but also inside these large GlyR expressing neurons, which continued to express surface GlyR. CONCLUSIONS: Despite the partial clinical phenotype, not uncommon in passive transfer studies, the results suggest that the antibodies had accessed the GlyRs in relevant brain regions, led to antibody-mediated internalization and increased GlyR synthesis, compatible with the temporary loss of function.

Hypothermia in VGKC antibody-associated limbic encephalitis.

Voltage-gated potassium channel antibody (VGKC-Ab)-associated limbic encephalitis (LE) is a recently described syndrome that broadens the spectrum of immunotherapy-responsive central nervous system disorders. Limbic encephalitis is typically characterised by a sub-acute onset of disorientation, amnesia and seizures, but the clinical spectrum is not yet fully defined and the syndrome could be under-diagnosed. We here describe the clinical profile of four patients with VGKC-Ab-associated LE who had intermittent, episodic hypothermia. One of the patients also described a prodrome of severe neuropathic pain preceding the development of limbic symptoms. Both of these novel symptoms responded well to immunosuppressive therapy, with concurrent amelioration of amnesia/seizures.

CSF findings in patients with voltage gated potassium channel antibody associated limbic encephalitis.

UNLABELLED: Recently, a new subtype of limbic encephalitis (LE) has been described, serologically characterized by the presence of antibodies against voltage gated potassium channels (VGKC, to be called VGKC-LE). Only little is known about CSF findings in this new disorder. Here we report the results of 29 lumbar punctures in 17 patients with VGKC-LE. Slight pleocytosis, mainly consisting of lymphocytes and monocytes, and elevated total protein concentrations were present in 41 and 47%, respectively. Intrathecal immunoglobulin (Ig) synthesis as defined by the presence of CSF-specific oligoclonal IgG bands, an increased IgG index, or an elevated IgG, IgA, or IgM ratio, was not detected in any of the patients, but dysfunction of the blood-CSF barrier was found in 35%. CSF findings were normal in 23%. CONCLUSIONS: Unlike paraneoplastic LE, VGKC-LE is not frequently associated with intrathecal Ig production or markedly elevated white cell counts. Thus, normal CSF findings do not preclude the disease. VGKC-Ab should, therefore, be determined whenever LE is clinically suspected, irrespective of the presence or absence of inflammatory CSF changes.

Absence of antibodies to glutamate receptor type 3 (GluR3) in Rasmussen encephalitis.

OBJECTIVE: To determine the prevalence of serum antibodies to the ionotropic glutamate receptor 3 (GluR3) in patients with Rasmussen encephalitis (RE), a severe epileptic disorder, and to compare with serum from control subjects and patients with intractable epilepsy (IE). METHODS: The authors looked for serum immunoglobulin (Ig) G antibodies to GluR3 in 30 patients with RE, including two patients who had plasma exchange and 12 who had been treated with IV Igs with varying results, and 49 patients with IE and 23 healthy individuals, using ELISA with GluR3B peptide, Western blot analysis of recombinant full-length GluR3, immunoprecipitation of [35S]- and [125I]-labeled GluR3 extracellular domains, immunohistochemistry on rat brain sections, and electrophysiology of GluR3 expressed in Xenopus oocytes. RESULTS: Low levels of antibodies to the GluR3B peptide were detected using ELISA in only 4 of the 79 patients with epilepsy (2 with RE and 2 with IE); binding to GluR3B in other sera was shown to be nonspecific. One other patient with IE had antibodies to recombinant GluR3 on Western blot analysis. However, none of the sera tested precipitated either the [35S]- or the [125I]-labeled GluR3 domains; none bound to rat brain sections in a manner similar to rabbit antibodies to GluR3; and none of the nine sera tested affected the electrophysiologic function of GluR3. CONCLUSIONS: GluR3 antibodies were only infrequently found in Rasmussen encephalitis or intractable epilepsy.

Evidence of underdiagnosis of myasthenia gravis in older people.

BACKGROUND: Myasthenia gravis is a potentially serious but treatable muscle disease caused by autoantibodies directed at the acetylcholine receptor (AChR) on the postsynaptic membrane of the neuromuscular junction. There is anecdotal evidence that the diagnosis is sometimes missed in older patients. OBJECTIVE: To examine the incidence and age distribution of positive AChR antibodies in samples referred to diagnostic laboratories in the UK, and the prevalence of positive AChR antibodies in samples from a cohort of older individuals. METHODS: Positive AChR antibody tests were identified from all UK centres registered for the assay with the European quality assurance scheme (EQAS) during 1997-99, and the age and sex specific incidence was calculated, based on the UK population. The prevalence of AChR antibodies in sera from a sample of 2000 individuals aged > or =60 years was determined. RESULTS: 3183 individuals had positive AChR antibody tests on routine screening during the years 1997 to 1999 in the UK, giving an annual incidence of 1.8/100 000. In both sexes, the age specific incidence rose steeply between the ages of 45 and 74, reaching 9.9/100 000 in men, and then fell, with a sharp decline above the age of 80. In the prevalence study, whereas only one serum from individuals aged 60-74 years was positive for AChR antibodies (0.12%), sera from eight individuals aged > or =75 years were positive (0.7%). Only one had a previous clinical diagnosis of myasthenia gravis but four others had histories of stroke or transient ischaemic attacks. CONCLUSIONS: The sharp fall in the incidence of clinically recognised myasthenia gravis in people over 80 years of age in our national AChR antibody incidence study, and the high prevalence of previously unrecognised positive AChR antibodies in those > or =75 years old, suggest that myasthenia gravis may be substantially underdiagnosed in older people.

A role for autoantibodies in some cases of acquired non-paraneoplastic gut dysmotility.

BACKGROUND: Antibody-mediated autoimmunity underlies a diverse range of disorders, particularly in the nervous system where domains of ion channels and receptors are potential targets. The aetiology of many adult-onset conditions of severe gut dysmotility is not known. We looked for antibodies to neuronal antigens in patients with severe (slow-transit-type) constipation (STC). METHODS: Eleven sera from adult-onset STC patients and 18 from childhood onset cases were tested by routine immunoprecipitation assays for antibodies against neuronal antigens including voltage-gated potassium channels (VGKCs), calcium channels (VGCCs), both muscle and neuronal acetylcholine receptor and glutamic acid decarboxylase (GAD). Results were compared with positive and negative control populations. RESULTS: Two of the 11 sera from patients with adult-onset STC showed highly positive anti-VGKC antibodies. Both had onset of symptoms de novo in adulthood without evidence of autoimmune, neoplastic or neurological disease. One of these patients, and one other, had anti-GAD antibodies. None of the childhood-onset STC had evidence of anti-neuronal antibodies. CONCLUSIONS: Anti-neuronal antibodies are found in some patients with a condition of severe acquired gut dysmotility of previously unknown aetiology. Future studies may demonstrate an autoimmune role for such antibodies.

Morvan's syndrome: peripheral and central nervous system and cardiac involvement with antibodies to voltage-gated potassium channels.

Morvan's 'fibrillary chorea' or Morvan's syndrome is characterized by neuromyotonia (NMT), pain, hyperhydrosis, weight loss, severe insomnia and hallucinations. We describe a man aged 76 years with NMT, dysautonomia, cardiac arrhythmia, lack of slow-wave sleep and abnormal rapid eye movement sleep. He had raised serum antibodies to voltage-gated K(+) channels (VGKC), oligoclonal bands in his CSF, markedly increased serum norepinephrine, increased serum cortisol and reduced levels and absent circadian rhythms of prolactin and melatonin. The neurohormonal findings and many of the clinical features were very similar to those in fatal familial insomnia, a hereditary prion disease that is associated with thalamic degenerative changes. Strikingly, however, all symptoms in our MFC patient improved with plasma exchange. The patient died unexpectedly 11 months later. At autopsy, there was a pulmonary adenocarcinoma, but brain pathology showed only a microinfarct in the hippocampus and no thalamic changes. The NMT and some of the autonomic features are likely to be directly related to the VGKC antibodies acting in the periphery. The central symptoms might also be due to the direct effects of VGKC antibodies, or perhaps of other autoantibodies still to be defined, on the limbic system with secondary effects on neurohormone levels. Alternatively, changes in secretion of neurohormones in the periphery might contribute to the central disturbance. The relationship between VGKC antibodies, neurohormonal levels, autonomic, limbic and sleep disorders requires further study.

Flow cytometric measurement of intracellular Th1 and Th2 cytokine production by human villous and extravillous cytotrophoblast.

A wide variety of cytokines are present at the maternal-fetal interface, but the extreme cellular complexity of the placenta has made it difficult to determine which cytokines are produced by which cells. Hence novel flow cytometric methods have been applied to determine intracellular cytokine production by specific cell-types in placental cell suspensions. Cell suspensions were prepared from first and third trimester chorionic villi and third trimester amniochorion by enzymatic digestion and Percoll density gradient centrifugation. After overnight incubation in the presence of monensin, cells were fixed, permeabilized and labelled with antibodies for villous cytotrophoblast (cytokeratin+, MHC class I-), extravillous cytotrophoblast (cytokeratin+, MHC class 1+) and leucocytes (CD45+). These cell types were further characterized by their expression of EGFR (proliferative cytotrophoblast) and c-erbB2 (invasive cytotrophoblast). Production of IL-4, IL-10, TNF-alpha, IFN-gamma and IL-12 was determined by simultaneous labelling with the appropriate monoclonal antibodies. Only IL-4 was detected consistently in all samples of cytotrophoblast. IL-10 was not detected but IL-10 mRNA was demonstrated in third trimester chorionic villus digests by RT-PCR. Although IL-4 secretion has not been demonstrated, these data suggest that, in vivo there may be a "Th2 type cytokine bias" orchestrated by the trophoblast. It is proposed that other cytokines (including IL-10 and TNF-alpha) are produced by decidual leukocytes, and not cytotrophoblast, at the maternal-fetal interface.

Potassium channel antibodies in two patients with reversible limbic encephalitis.

Limbic encephalitis (LE) is often associated with lung, thymic, or testicular tumours and antibodies to Hu, CV2, or Ma2 (Ta) antigens. In these cases, it generally has a poor prognosis. Here we describe two patients with symptoms of LE, negative for typical paraneoplastic antibodies, in whom antibodies to voltage-gated potassium channels (VGKC) were detected retrospectively in serial serum samples. Patient 1 had a thymoma recurrence, but in patient 2 no tumour has been detected in the years following presentation. Plasma exchange was effective in reducing VGKC antibody levels, with substantial improvement in mental symptoms in patient 1. In patient 2, the VGKC antibodies fell spontaneously over two years, with almost complete recovery of mental function. Although neither patient had obvious neuromyotonia at presentation, both showed excessive secretions. We suggest that patients with limbic symptoms and excessive secretions should be tested for VGKC antibodies, and, if they are present, prompt and effective immunosuppressive treatment should be considered.

A three-colour flow cytometry technique for measuring trophoblast intracellular antigens: the relative expression of TAP1 in human cytotrophoblast and decidual cells.

Flow cytometry is conventionally used to measure cell-surface antigen expression. However, many antigens are found within the cytoplasm, and it is necessary to fix and permeabilize cells to enable antibodies to gain access to them. In this study we have established the conditions for studying intracellular antigens in human trophoblast cells by flow cytometry using an antibody to TAP1 (a key molecule in the process of Class I MHC assembly). We have previously shown by immunocytochemistry that TAP1 expression is apparently greater on Class 1 positive extravillous cytotrophoblast than on any other fetal or maternal tissue. However, as immunohistochemistry is not quantitative we have used three-colour flow cytometry to measure the expression of TAP1 in different trophoblast populations. Villous and extravillous cytotrophoblast were identified in first trimester and term placental and decidual digests on the basis of their expression of cytokeratin and Class I MHC antigens. The level of expression of TAP1 for each population was investigated using a commercial kit that determines the number of antibody-binding sites per cell. TAP expression was found to be three- to fivefold higher in extravillous cytotrophoblast, confirming our previous findings. The techniques developed here are directly applicable to the measurement of other intracellular molecules in trophoblast, in particular cytokines.

Assessing the contribution of birth asphyxia to cerebral palsy in term singletons.

In a geographically-based study, we investigated the risk of cerebral palsy following intrapartum asphyxia at term, and the contribution of intrapartum asphyxia at term to the overall rate of cerebral palsy. We used stringent criteria for identifying intrapartum asphyxia, while recognising that the initial hypoxial insult might have occurred in the antenatal period. In the first part of the investigation, a cohort of 160 term, singleton infants, with a low (< or = 3) 1-minute Apgar score, was followed to the age of 5 years. Six infants in the cohort had presumed intrapartum asphyxia, of whom two died in the neonatal period, three had spastic quadriparesis, profound developmental delay and visual impairment, and one was unimpaired. The frequency of cerebral palsy associated with birth asphyxia was estimated as one in 3700 full-term livebirths. To assess the impact of birth asphyxia on the overall rate of cerebral palsy, all cases of cerebral palsy born in the study period were identified. Of the 30 cases, the three identified in the follow-up study were the only ones whose impairment could be attributed to birth asphyxia in a full-term birth. Birth asphyxia at term therefore was associated with 10% [95% confidence interval (CI) 2.1, 26.5] of all cases of cerebral palsy and with 20% (95% CI 4.3, 48.1) of the 15 cases of cerebral palsy in children born at term.

Expression of TAP1 by human trophoblast.

Successful placentation in the human is dependent on the trophoblast evading recognition and destruction by the maternal immune system. However, invasive cytotrophoblast express HLA-G which may be able to present peptide to T cells. Transporter proteins are essential for peptide presentation and major histocompatibility complex (MHC) class I assembly. We have determined their expression by trophoblast in relation to HLA-G, using immunohistochemistry. Anti-transporter protein antibody (TAP1) labeling closely paralleled that of MHC class I, but the intensity of its expression was much greater on the HLA-G+ extravillous cytotrophoblast than any other fetal or maternal tissue in the first trimester and at term. This suggests that the extravillous cytotrophoblast are very actively assembling MHC class I antigens with peptides. However, expression of MHC class I by the cytotrophoblast was not correspondingly elevated. This pattern could result from HLA-G being shed from the surface of the trophoblast, a process which may play a central role in protecting the fetus from maternal immune attack.

Clustering of perinatal markers of birth asphyxia and outcome at age five years.

OBJECTIVES: In a cohort of term infants with cerebral depression at delivery, to investigate the association of perinatal signs of birth asphyxia, particularly abnormal fetal heart rate patterns in labour, acidaemia, and serious neonatal encephalopathy, with neurodevelopmental outcome at age five years. DESIGN: Five year follow up study of a birth cohort. SETTING: Regional maternity hospital. SUBJECTS: One hundred and eighty-four singleton infants with a 1 min Apgar score < or = 3, born at term between January 1984 and September 1985. MAIN OUTCOME MEASURES: Neonatal death, cerebral palsy, and scores on a battery of neurodevelopmental tests at age five. RESULTS: Seven infants had a cluster of perinatal signs suggestive of birth asphyxia; all included serious neonatal encephalopathy. Three of these infants died neonatally, three had spastic quadriparesis with profound developmental delay, and one was unimpaired at the age of five. Among the remaining infants, no association was found between severely abnormal fetal heart rate patterns in labour and scores on neurodevelopmental tests, or between acid-base status at delivery and test scores. CONCLUSIONS: Birth asphyxia, identified by a cluster of abnormal perinatal signs, including serious neonatal encephalopathy, has a poor prognosis. If serious encephalopathy is not present, cerebral depression at birth preceded by abnormal fetal heart rate patterns in labour, or with acid-base derangement, is not predictive of later impairment.

Expression of tumour necrosis factor alpha and its receptors in carcinoma of the breast.

The expression of tumour necrosis factor alpha (TNF-alpha) and its two distinct receptors, TNF-R p55 and TNF-R p75, was assessed by immunocytochemistry in 28 primary breast cancer and three reduction mammoplasty specimens ('normal' breast tissue). Expression of TNF-alpha or TNF-R p75 was not detectable in normal breast tissue or in non-malignant breast tissue adjacent to the tumours. By contrast, TNF-R p55 was expressed by occasional stromal cells in normal tissue. TNF-alpha was expressed focally in 50% of the tumours studied, being largely localised to macrophage-like cells in the stroma. TNF-R p55 was expressed by a population of stromal cells in all the tumours examined, and a varying proportion of neoplastic cells in 75% of these tissues. TNF-R p75 was detected in about 70% of the tumours, immunoreactivity being confined mainly to cells in the stroma. In this preliminary study there was no association between the above cytokine parameters and such measures of tumour biology as lymph node status, tumour grade, proliferative activity or degree of angiogenesis. However, there was a correlation between the expression of TNF-R p55 by blood vessels and the number of leucocytes present.

Antigenic heterogeneity of human cytotrophoblast and evidence for the transient expression of MHC class I antigens distinct from HLA-G.

Expression of MHC class I antigens on trophoblast populations in first trimester human chorionic villous tissue was assessed by immunohistology. Antibodies used were W6/32 which recognizes a non-polymorphic framework determinant of HLA- A, -B, -C, MHM5 specific for HLA-B, C and 4E and B23.1 which are specific for HLA-B. Syncytiotrophoblast and villous cytotrophoblast were negative with all the anti (HLA class I) antibodies tested. Interstitial trophoblast cells within the maternal decidua were identified with a new antibody, NDOG5, which is specific for extravillous cytotrophoblast. Double labelling showed that they bind W6/32 but not 4E, MHM5 or B23.1; consistent with the expression of the monomorphic HLA-G. In contrast the cytotrophoblast cells of the cell islands and cytotrophoblast shell, which also express the NDOG5 antigen, were positive with W6/32, 4E, MHM5 and B23.1. Cell column cytotrophoblast cells were negative with all four MHC class I antibodies. These results suggest that differentiation of cytotrophoblast from noninvasive to invasive forms is associated with transient expression of class I antigens other than HLA-G on cytotrophoblast shell and cell island cytotrophoblast.

Spinal cord ependymomas treated with surgery and radiation therapy. A review of 11 cases.

Between 1971 and 1990, 11 patients with primary spinal cord ependymomas were treated with surgery and postoperative irradiation or surgery alone at the University of Colorado Health Sciences Center. Of the 11 patients, 6 (54%) were subclassified with myxopapillary ependymomas that were located in the lumbosacral region of the spinal cord: 2 patients underwent complete resections, 8 had subtotal resections, and 1 had a biopsy only; 8 patients received postoperative irradiation (range: 4,500-5,482 cGy) with 7 of 8 patients treated to involved spinal fields. With a mean follow-up of 7.4 years, 3 patients (27%) have developed recurrent disease, 2 in the combined treatment group, and 1 in the surgery alone group. The 5- and 10-year actuarial survival rates were 100% and 80%, respectively. Eight of nine patients (89%) demonstrated clinical improvement after postoperative irradiation which suggests that the irradiation may have contributed to the improvement. The present study supports the long-term survival of patients with spinal cord ependymomas. Results from this series and a review of the literature indicate that complete surgical resection is only possible in about one-quarter of cases. Local spinal irradiation should continue to be utilized when surgery is incomplete.

Localization of tumour necrosis factor production in cells at the materno/fetal interface in human pregnancy.

Biologically active tumour necrosis factor (TNF) was detected in medium conditioned by incubation with explants of human pregnancy decidua or fetal chorionic villous tissue, taken in the first trimester and at term. Addition of endotoxin increased TNF release in most cases. ELISA assays gave similar results for TNF-alpha and also demonstrated low levels of TNF-beta. Using cell populations purified by flow cytometry, secretion of biologically active TNF was shown to be localized to the macrophages. Cytotrophoblast purified from term amniochorion produced no TNF. Both decidual and chorionic villous tissue at term contained mRNA for TNF-alpha and TNF-beta. TNF-alpha mRNA was confined to decidual macrophages in first trimester tissue, and was not present in chorionic cytotrophoblast. TNF-beta mRNA, in contrast, was detected in both macrophage and non-macrophage populations in term decidua.

Variation during the menstrual cycle of immune cell populations in human endometrium.

Morphometric analysis and immunohistology of tissue sections have been used to assess variation, during the normal menstrual cycle, of the bone marrow-derived cell populations in human endometrium. Levels of T cells and macrophages were found to be relatively constant throughout the cycle. In contrast, numbers of large granular lymphocytes, identified as being CD56-positive, were generally low between days 10 and 19, but increased sharply in the latter part of the luteal phase, decreasing again after menstruation. This LGL population is known to be abundant in first trimester pregnancy decidua, and is presumed to play a role in early pregnancy success.