Susan A. Clawson

Uptake of systemically administered human anticerebellar antibody by rat Purkinje cells following blood-brain barrier disruption

Paraneoplastic cerebellar degeneration accompanying gynecological or breast malignancies is frequently associated with an autoantibody response, termed “type I” or “anti-Yo” directed against cytoplasmic antigens of cerebellar Purkinje cells. The role of this antibody response in the pathogenesis of paraneoplastic cerebellar degeneration is unknown; however, it is also not known whether anti-Purkinje cell antibodies from the systemic circulation bind to target Purkinje cell antigens under the conditions of brain inflammation and blood-brain barrier disruption, which are frequently present at the onset of cerebellar symptoms. Inbred Lewis rats received intraperitoneal injections of type I or normal IgG in the setting of blood-brain barrier disruption induced by adoptive transfer of experimental allergic encephalomyelitis (EAE) and were killed after 24, 48, and 96h. Brains of these animals were studied histologically for evidence of EAE and immunohistochemically for binding of human or endogenous rat IgG to target neurons. Rat IgG was detected around vessels and in Purkinje cells of all animals studied. Human IgG was detected around vessels of all animals. In animals examined 96 h after receiving type I human IgG, human IgG was identified within processes of Purkinje cells and within occasional Purkinje cell bodies. Uptake of type I IgG by other cell types was not observed, and neuronal uptake of IgG was not seen in brains of animals receiving normal human IgG. Our data demonstrate that circulating type I IgG is internalized by cerebellar Purkinje cells in the setting of blood-brain barrier disruption and suggest a mechanism by which an antibody response directed against cytoplasmic antigens of Purkinje cells may reach target antigens at the onset of paraneoplastic cerebellar degeneration.

Association of anti-Yo (type I) antibody with paraneoplastic cerebellar degeneration in the setting of transitional cell carcinoma of the bladder: detection of Yo antigen in tumor tissue and fall in antibody titers following tumor removal.

Anti‐Yo (type I) autoantibodies reactive with Purkinje cell cytoplasmic antigens of 34 and 62 kd are found in the serum and cerebrospinal fluid of patients with paraneoplastic cerebellar degeneration associated with cancer of the ovary, uterus, adnexa, or breast. Anti‐Yo antibody response is rarely associated with other tumors. Here, we present a patient who developed paraneoplastic cerebellar degeneration and anti‐Yo antibody response in association with transitional cell carcinoma of the bladder. The presence of anti‐Yo antibodies was confirmed by immunofluorescence assay and by Western blot analysis against both Purkinje cell lysates and the CDR62 fusion protein. Yo antigen was demonstrated in sections of the patients tumor. Antibody titers fell after tumor removal. Transitional cell carcinoma should be considered in patients presenting with subacute cerebellar degeneration and anti‐Yo antibody response in whom ovarian, adnexal, uterine, or breast cancer cannot be detected. Ann Neurol 1999;45:805–809

Cerebellar Purkinje cells incorporate immunoglobulins and immunotoxins in vitro: implications for human neurological disease and immunotherapeutics

BackgroundImmunoglobulin G (IgG) antibodies reactive with intracellular neuronal proteins have been described in paraneoplastic and other autoimmune disorders. Because neurons have been thought impermeable to immunoglobulins, however, such antibodies have been considered unable to enter neurons and bind to their specific antigens during life. Cerebellar Purkinje cells - an important target in paraneoplastic and other autoimmune diseases - have been shown in experimental animals to incorporate a number of molecules from cerebrospinal fluid. IgG has also been detected in Purkinje cells studied post mortem. Despite the possible significance of these findings for human disease, immunoglobulin uptake by Purkinje cells has not been demonstrated in living tissue or studied systematically.MethodsTo assess Purkinje cell uptake of immunoglobulins, organotypic cultures of rat cerebellum incubated with rat IgGs, human IgG, fluorescein-conjugated IgG, and rat IgM were studied by confocal microscopy in real time and following fixation. An IgG-daunorubicin immunotoxin was used to determine whether conjugation of pharmacological agents to IgG could be used to achieve Purkinje cell-specific drug delivery.ResultsIgG uptake was detected in Purkinje cell processes after 4 hours of incubation and in Purkinje cell cytoplasm and nuclei by 24-48 hours. Uptake could be followed in real time using IgG-fluorochrome conjugates. Purkinje cells also incorporated IgM. Intracellular immunoglobulin did not affect Purkinje cell viability, and Purkinje cells cleared intracellular IgG or IgM within 24-48 hours after transfer to media lacking immunoglobulins. The IgG-daunomycin immunotoxin was also rapidly incorporated into Purkinje cells and caused extensive, cell-specific death within 8 hours. Purkinje cell death was not produced by unconjugated daunorubicin or control IgG.ConclusionPurkinje cells in rat organotypic cultures incorporate and clear host (rat) and non-host (human or donkey) IgG or IgM, independent of the immunoglobulins reactivity with Purkinje cell antigens. This property permits real-time study of immunoglobulin-Purkinje cell interaction using fluorochrome IgG conjugates, and can allow Purkinje cell-specific delivery of IgG-conjugated pharmacological agents. Antibodies to intracellular Purkinje cell proteins could potentially be incorporated intracellularly to produce cell injury. Antibodies used therapeutically, including immunotoxins, may also be taken up and cause Purkinje cell injury, even if they do not recognize Purkinje cell antigens.

Anti-Hu antibodies in Merkel cell carcinoma

Anti‐Hu antibody is an antineuronal autoantibody found in a subset of patients with paraneoplastic neurological disease. The antibody was first associated with small cell carcinoma of the lung and is most often used as a marker for this neoplasm in patients presenting with suspected paraneoplastic syndromes. Here we report a patient with a multifaceted neurological disorder in the setting of Merkel cell carcinoma. The patients serum contained antibodies against the Hu antigen, and the expression of the Hu antigen was demonstrated in the patients tumor.

Anti-Yo Antibody Uptake and Interaction with Its Intracellular Target Antigen Causes Purkinje Cell Death in Rat Cerebellar Slice Cultures: A Possible Mechanism for Paraneoplastic Cerebellar Degeneration in Humans with Gynecological or Breast Cancers

Anti-Yo antibodies are immunoglobulin G (IgG) autoantibodies reactive with a 62 kDa Purkinje cell cytoplasmic protein. These antibodies are closely associated with paraneoplastic cerebellar degeneration in the setting of gynecological and breast malignancies. We have previously demonstrated that incubation of rat cerebellar slice cultures with patient sera and cerebrospinal fluid containing anti-Yo antibodies resulted in Purkinje cell death. The present study addressed three fundamental questions regarding the role of anti-Yo antibodies in disease pathogenesis: 1) Whether the Purkinje cell cytotoxicity required binding of anti-Yo antibody to its intraneuronal 62 kDa target antigen; 2) whether Purkinje cell death might be initiated by antibody-dependent cellular cytotoxicity rather than intracellular antibody binding; and 3) whether Purkinje cell death might simply be a more general result of intracellular antibody accumulation, rather than of specific antibody-antigen interaction. In our study, incubation of rat cerebellar slice cultures with anti-Yo IgG resulted in intracellular antibody binding, and cell death. Infiltration of the Purkinje cell layer by cells of macrophage/microglia lineage was not observed until extensive cell death was already present. Adsorption of anti-Yo IgG with its 62 kDa target antigen abolished both antibody accumulation and cytotoxicity. Antibodies to other intracellular Purkinje cell proteins were also taken up by Purkinje cells and accumulated intracellularly; these included calbindin, calmodulin, PCP-2, and patient anti-Purkinje cell antibodies not reactive with the 62 kDa Yo antigen. However, intracellular accumulation of these antibodies did not affect Purkinje cell viability. The present study is the first to demonstrate that anti-Yo antibodies cause Purkinje cell death by binding to the intracellular 62 kDa Yo antigen. Anti-Yo antibody cytotoxicity did not involve other antibodies or factors present in patient serum and was not initiated by brain mononuclear cells. Purkinje cell death was not simply due to intraneuronal antibody accumulation.

Neuronal uptake of anti-Hu antibody, but not anti-Ri antibody, leads to cell death in brain slice cultures

BackgroundAnti-Hu and anti-Ri antibodies are paraneoplastic immunoglobulin (Ig)G autoantibodies which recognize cytoplasmic and nuclear antigens present in all neurons. Although both antibodies produce similar immunohistological labeling, they recognize different neuronal proteins. Both antibodies are associated with syndromes of central nervous system dysfunction. However, the neurological deficits associated with anti-Hu antibody are associated with neuronal death and are usually irreversible, whereas neurological deficits in patients with anti-Ri antibody may diminish following tumor removal or immunosuppression.MethodsTo study the effect of anti-Hu and anti-Ri antibodies on neurons, we incubated rat hippocampal and cerebellar slice cultures with anti-Hu or anti-Ri sera from multiple patients. Cultures were evaluated in real time for neuronal antibody uptake and during prolonged incubation for neuronal death. To test the specificity of anti-Hu antibody cytotoxic effect, anti-Hu serum IgG was incubated with rat brain slice cultures prior to and after adsorption with its target Hu antigen, HuD.ResultsWe demonstrated that: 1) both anti-Hu and anti-Ri antibodies were rapidly taken up by neurons throughout both cerebellum and hippocampus; 2) antibody uptake occurred in living neurons and was not an artifact of antibody diffusion into dead cells; 3) intracellular binding of anti-Hu antibody produced neuronal cell death, whereas uptake of anti-Ri antibody did not affect cell viability during the period of study; and 4) adsorption of anti-Hu antisera against HuD greatly reduced intraneuronal IgG accumulation and abolished cytotoxicity, confirming specificity of antibody-mediated neuronal death.ConclusionsBoth anti-Hu and anti-Ri antibodies were readily taken up by viable neurons in slice cultures, but the two antibodies differed markedly in terms of their effects on neuronal viability. The ability of anti-Hu antibodies to cause neuronal death could account for the irreversible nature of paraneoplastic neurological deficits in patients with this antibody response. Our results raise questions as to whether anti-Ri antibody might initially induce reversible neuronal dysfunction, rather than causing cell death. The ability of IgG antibodies to access and react with intracellular neuronal proteins could have implications for other autoimmune diseases involving the central nervous system.

Antineuronal autoantibodies in paraneoplastic cerebellar degeneration associated with adenocarcinoma of the prostate.

Paraneoplastic neurological syndromes are unusual in prostatic cancer, and paraneoplastic cerebellar degeneration associated with adenocarcinoma of the prostate is rare. Here we report a 68year old man who developed progressive ataxia in the setting of stage D2 adenocarcinoma of the prostate and whose MRI showed cerebellar atrophy. The patients serum produced a previously undescribed pattern of immunoreactivity, binding to nuclei and cytoplasm of Purkinje cells, deep cerebellar neurons, scattered cells in the molecular and granule cell layers, and neuronal populations in thalamus, cerebral cortex, and hippocampus but not with liver or kidney. The patients IgG also labeled a 65kDa protein, discrete from Yo antigen, in Western blots of Purkinje cell lysates and did not react with blotted recombinant HuD, Ri, Yo, or amphiphysin proteins. Sera from neurologically normal patients with adenocarcinoma of the prostate did not contain this antibody, and the patients serum did not react with normal prostate or with prostatic adenocarcinomas from other individuals. Prostatic adenocarcinoma may occasionally be accompanied by development of anticerebellar antibodies. Adenocarcinoma of the prostate should be considered as a possible underlying malignancy in older males with unexplained progressive cerebellar degeneration.

Propagation of archetype and nonarchetype JC virus variants in human fetal brain cultures: demonstration of interference activity by archetype JC virus.

In immunologically normal individuals, the polyomavirus, JC virus (JCV), produces an asymptomatic primary infection followed by lifelong persistence of the virus in renal tubular epithelial cells. In some immunocompromised patients, however, in particular acquired immunodeficiency syndrome (AIDS) patients, JCV causes an opportunistic central nervous system (CNS) disorder, progressive multifocal leukoencephalopathy (PML). JCV DNA as it persists in kidneys (archetypal JCV) and JCV DNA isolated from PML lesions show differences in their regulatory regions in which transcription and replication are controlled. Archetypal JCV DNA has a single enhancer and no rearrangements or deletions in the regulatory region. In contrast, JCV DNA from PML isolates is characterized by alterations in the regulatory region. Some PML-associated JCVs can be grown in cultures of human fetal brain (HFB) cells. Growth of archetypal JCV in cultured cells has not been reported, however. Here we demonstrate successful propagation of the archetypal JCV, strain GS/K, in HFB cells. Growth occurred more slowly and to lower titers than is seen with the prototypical PML JCV strain Mad-1, with relatively few cells containing viral T antigen (T-Ag) or viral capsid protein, Vp1. Interestingly, GS/K growth could be enhanced, with a large increase in viral DNA and cytopathic effect, by coinfection with GS/B, a nonarchetypal brain-derived JCV variant isolated from the same PML patient as GS/K. The amount of GS/KDNA was also greatly enhanced when it was cotransfected with Mad-1 JCV DNA, the prototypical PML isolate. In contrast to GS/K plus GS/B—cotransfected cells, in GS/K plus Mad-1-infected cells, cytopathic effect was not increased. On subsequent passage of culture lysates to naïve cells, however, the infection produced by either combination of viral DNAs slowed, no cytopathic effect (CPE) was present, and the amount of GS/B or Mad-1 viral DNA was greatly reduced as compared to that of GS/K DNA. These data suggest that GS/K was able to use either GS/B or Mad-1 as a helper and that GS/K was in turn able to interfere with the growth of either helper virus. Archetype JCV can be successfully propagated in HFB cells, although infection develops much more slowly than that caused by the PML JCV variant Mad-1. The ability of archetypal and variant JCVs to enhance or retard each other’s replication may have implications in vivo for the maintenance of JCV persistence and the growth of JCV variants.

Voltage-gated calcium channel autoimmune cerebellar degeneration: Case and study of cytotoxicity.

Objectives: To describe response to treatment in a patient with autoantibodies against voltage-gated calcium channels (VGCCs) who presented with autoimmune cerebellar degeneration and subsequently developed Lambert-Eaton myasthenic syndrome (LEMS), and to study the effect of the patients autoantibodies on Purkinje cells in rat cerebellar slice cultures. Methods: Case report and study of rat cerebellar slice cultures incubated with patient VGCC autoantibodies. Results: A 53-year-old man developed progressive incoordination with ataxic speech. Laboratory evaluation revealed VGCC autoantibodies without other antineuronal autoantibodies. Whole-body PET scans 6 and 12 months after presentation detected no malignancy. The patient improved significantly with IV immunoglobulin G (IgG), prednisone, and mycophenolate mofetil, but worsened after IV IgG was halted secondary to aseptic meningitis. He subsequently developed weakness with electrodiagnostic evidence of LEMS. The patients IgG bound to Purkinje cells in rat cerebellar slice cultures, followed by neuronal death. Reactivity of the patients autoantibodies with VGCCs was confirmed by blocking studies with defined VGCC antibodies. Conclusions: Autoimmune cerebellar degeneration associated with VGCC autoantibodies may precede onset of LEMS and may improve with immunosuppressive treatment. Binding of anti-VGCC antibodies to Purkinje cells in cerebellar slice cultures may be followed by cell death. Patients with anti-VGCC autoantibodies may be at risk of irreversible neurologic injury over time, and treatment should be initiated early.

Comparative neuronal uptake and cytotoxicity of anti-Hu and anti-Ri antibodies in rat cerebellar and hippocampal slice cultures

WCN 2013 No: 1897 Topic: 36 — Other Topic Comparative neuronal uptake and cytotoxicity of anti-Hu and anti-Ri antibodies in rat cerebellar and hippocampal slice cultures J.E. Greenlee, S.A. Clawson, B. Wood, K.E. Hill, N.G. Carlson. Neurology Service, Veterans Affairs Medical Center, Salt Lake City, UT, USA; Neurology, University of Utah Health Sciences Center, Salt Lake City, UT, USA; Research Service, Veterans Affairs Medical Center, Salt Lake City, UT, USA; Neurobiology and Anatomy, University of Utah School of Medicine, Salt Lake City, UT, USA; Center on Aging, University of Utah School of Medicine, Salt Lake City, UT, USA Background: Anti-Hu and anti-Ri are paraneoplastic autoantibodies recognizing intracellular antigens present in essentially all neurons. At autopsy, brains of patients with anti-Hu antibody show neuronal destruction. In contrast, anti-Ri antibody is less clearly associated with neuronal death, and patients with anti-Ri antibodies may respond to treatment. We have demonstrated that anti-Yo antibodies, associated with paraneoplastic cerebellar degeneration, accumulate intracellularly in cerebellar Purkinje cells in slice cultures of rat cerebellum and that antibody accumulation is followed by cell death. The present study was conducted to determine whether anti-Hu and anti-Ri antibodies are also taken up by neurons and whether uptake of either antibody is cytotoxic. Objective: To evaluate neuronal uptake and cytotoxicity of anti-Hu and anti-Ri antibodies in slice cultures of rat cerebellum and hippocampus. Materials and methods: Rat cerebellar and hippocampal slice cultures were incubated with anti-Hu or anti-Ri antibodies and evaluated over time for antibody uptake and for cell death. Specificity of anti-Hu cytotoxicity was confirmed by adsorbing anti-Hu IgGwith recombinant HuD protein. Results: Anti-Hu and anti-Ri antibodies accumulated in cerebellar and hippocampal neurons. Anti-Hu antibodies produced cell death which was significantly reduced by adsorption of anti-Hu IgG with recombinant HuD protein. In contrast, neurons accumulating anti-Ri antibodies showed no evidence of cell death as compared to controls. Conclusions: Anti-Hu and anti-Ri antibodies entered and accumulated in cerebellar and hippocampal neurons. Anti-Hu antibody associated neuronal death involved reactivity with HuD protein. Anti-Ri antibody did not affect neuronal viability and may cause neuronal dysfunction rather than cell death. doi:10.1016/j.jns.2013.07.2239 Abstract — WCN 2013 No: 1902 Topic: 36 — Other Topic An evaluation of the risks involved in ischemic encephalopathy among Mongolian children T. Sosorburam, B. Batbayar. Anesthesiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Children Hospital, Ulan-Bator, Mongolia Background: Ischemic encephalopathy is one of the emerging issues among third world countries. Hypoxia seems to be the main cause of this problem, but many risk factors are associated in causation of hypoxic ischemic encephalopathy (HIE). The aim of our study was to evaluate these risk factors among Mongolian children. Methods: This study was carried out at 3 childrens hospitals, 85 neonates diagnosed with HIE over a year. Efforts were made to evaluate the questionnaire including details on parity, pre and postnatal histories, and specially the events of birth from labour till delivery of placenta. Results: There was a remarkable decreased antenatal hospital visits and almost 52% reported that they visited just once or twice during the whole pregnancy period. In 21%, there was history of increased mother age above 40 years while 62% reported that some incidents of hypoxia occurred at the time of birth. 20% used drugs or medication during the course of pregnancy without the consultation of the physician.31 % had a history of prolonged 2nd stage of labour. 49% were delivered by unskilled birth attendants. Conclusion: The data clearly showed that there are risks that can be avoided easily and a leading cause of mental retardation can be prevented. Lack of health awareness and decreased consultation from physicians on time seems to be the bulk of the problem in addition to lack of trained birth staff and health facilities. Efforts should be made to educate the mothers of child bearing ages, and counsel them to make the antenatal hospital visits more frequent as possible. doi:10.1016/j.jns.2013.07.2240 Abstract — WCN 2013 No: 1907 Topic: 36 — Other Topic Different emotions and strong stimuli: How do we make choices? WCN 2013 No: 1907 Topic: 36 — Other Topic Different emotions and strong stimuli: How do we make choices? C.V.D.S. Vilharba, A.J. Godoy. Marketing, University Sao Judas, Sao Paulo, Brazil; Neurology, University City of Sao Paulo, Sao Paulo, Brazil Our understanding of the way the brain makes a decision is still incomplete. We decided to analyze the relationships between color and different emotions and sound and different emotions and their influences to make a choice. Forty-eight undergraduate students of marketing courses were interviewed. We presented 7 objects with different colors while they were listening to a “sad” song and again while listening to a “happy” song. In each case they had to choose one of them. Then we presented six identical objects associated with different sounds while listening to those songs. Forty-six percent of the students selected the red color during the sad moment (SM) and 40% during the happy moment (HM). None of them chose white during SM (however 10% selected white during HM). During HM the “forgotten” color was orange (2% of the students). Twenty-nine percent of the volunteers chose the object associated with the sound of a bomb during SM (the same sound was selected only by 12% during HM). Only 2% selected the sound of the wind (during SM or HM). Our results suggest that strong stimuli (visual or auditory) activate many neural circuits, making someone choose a red object or one associated with the sound of a bomb. Nevertheless a so called Abstracts / Journal of the Neurological Sciences e629 (2013) e629–e678 e645