Detection of paraneoplastic anti-neuronal autoantibodies on paraffin-embedded tissues

Anti-neuron-specific autoantibodies are widely recognised as useful, though non-specific, diagnostic markers of paraneoplastic neurological disorders. However, controversies on the best way to detect these autoantibodies have recently arisen, and the use of different procedures for their detection by different laboratories has made results difficult to compare. The aim of this study was to adapt the existing immunohistochemical techniques used for the detection of anit-neuron autoantibodies to improve their visualisation and to facilitate a wide application of these procedures. Sera and cerebrospinal fluid (CSF) were obtained from 15 patients known to carry paraneoplastic anti-neuronal autoantibodies; in addition, one serum with “atypical” anti-neuron autoantibody and 18 control sera were studied. Paraformaldehyde-fixed, paraffin-embedded rat nervous tissue and formalin-fixed, paraffin-embedded human nervous tissue treated in a microwave oven were used as substrate; the reactions were developed by immunoperoxidase methods. At the dilutions used for diagnostic purposes, all the sera and CSFs showed staining whose intensity and specificity was comparable to that obtained using frozen tissue; the end-point dilutions were, however, reduced. The atypical pattern of staining of one serum was confirmed and better emphasised using these procedures; all control sera and CSFs were negative. The morphology was improved by the use of paraffin-embedded tissues; moreover, the results obtained are permanent because of peroxidase staining, which makes it possible to use them as standards for further investigations and for comparison between different laboratories. The convenience of using paraffin-embedded material could facilitate a wide application of these procedures in clinical neurology. Received: 22 May 1996 / Revised, accepted: 2 July 1996     

Oligodendrocyte staining by multiple sclerosis serum is nonspecific.

The immunofluorescent staining properties of 65 serum samples from 54 patients with multiple sclerosis (MS), 63 samples from 55 patients with other neurological diseases (OND), and sera from 14 healthy normal individuals were examined on frozen sections of bovine and human brain. When tested on bovine brain sections, positive oligodendrocyte staining was present in 63% of MS sera, 43% of OND sera, and 29% of normal sera. The percentages were lower with human brain tissue. Astrocyte and myelin staining was common. F(ab')2 fragments purified from selected positive and negative MS and control sera gave no staining, though IgG fractions from the same sera prior to pepsin digestion gave positive staining. When tested against antihuman IgM and IgA conjugates, the same positive sera and their IgG-depleted globulin fractions gave minimal or no staining. These results indicate that oligodendrocyte staining is not specific for MS, is not due to specific antibody, and is probably the result of nonspecific binding to Fc receptors.     

Screening for autoantibodies in children with opsoclonus-myoclonus-ataxia

Various paraneoplastic autoantibodies have been linked to discrete neurologic syndromes and tumors in adults, but little is known about their incidence in children. We report a cross-sectional study of known paraneoplastic antibodies in 59 children with opsoclonus-myoclonus-ataxia, 86% of whom were moderately or severely symptomatic, and 68% of whom had relapsed at the time of testing. This total number of patients includes 18 children with low-stage neuroblastoma (tested after tumor resection), six of whom had never been treated with immunosuppressants. All were seronegative for anti-Hu, anti-Ri, and anti-Yo, the three paraneoplastic antibodies most associated with opsoclonus-myoclonus or ataxia in adults. These data contrast with reports of anti-Hu-positive sera in children with high-stage tumors and suggest that anti-Hu, anti-Ri, and anti-Yo do not explain relapses in pediatric opsoclonus-myoclonus-ataxia. They underscore the need to search for unique autoantibodies, as well as cellular mechanisms of pediatric paraneoplastic disease.     

Reactivity of circulating antineuronal antibodies (CANA) on peripheral nervous system structures

Summary The appearance of circulating antineuronal antibodies (CANA) in patients with malignant tumors has been correlated with the occurrence of paraneoplastic neurological syndromes. However, the effect of CANA on the peripheral nervous system is poorly understood. The reactivity of sera from CANA-positive and-negative patients were investigated on cryostat sections of peripheral nerves and skeletal muscle, and on nerve tease-fiber preparations. Only CANA-positive sera showed staining of Schwann cell nuclei on cryostat sections, whereas nerve tease-fiber preparations and sections of skeletal muscle remained negative. Positive direct immunofluorescence of small cell lung cancer (SCLS) cells was confined to CANA-positive patients only. These findings suggest the existence of a common antigen between SCLC and Schwann cells.     

Localization of antibody in the central nervous system of a patient with paraneoplastic encephalomyeloneuritis.

We report a patient with severe paraneoplastic encephalomyeloneuritis, occult small-cell carcinoma of the lung, and high titers of circulating antineuronal antibody who died shortly after developing limbic encephalitis. The antibody was of IgG class and reacted specifically with nuclei and cytoplasm of all neurons in the pattern typical for encephalomyelitis and subacute sensory neuropathy associated with small-cell carcinoma (type II, anti-Hu). At autopsy, perivascular inflammatory infiltrates were prominent. All samples of serum, CSF, and postmortem peritoneal and pleural fluid contained high titers of antibody. Direct immunofluorescence of frozen tissue revealed IgG bound to most remaining neurons in multiple brain regions in a pattern similar to indirect immunofluorescence of normal brain tissue. IgG was also bound to tumor. Attempts to elute antibody from tissue decreased background staining but did not remove neuronal immunofluorescence. These results indicate that antibody can access and bind specifically to neuronal antigens in the brain during the course of paraneoplastic disease.     

Immunccytochemical Localization of Angiotensinogen in Rat Brain: Dependence of Neuronal Immunoreactivity on Method of Tissue Processing

There is disagreement between laboratories on the presence and location of angiotensinogen immunostaining in neuronal cells. We examined this issue by using different antisera and histological procedures to stain for angiotensinogen in brains from normal, colchicine-treated and nephrectomized rats. Five different antisera from three laboratories were used to stain sections of paraffin-embedded tissue, frozen sections and Vibratome sections of cerebral cortex, thalamus, hypothalamus, brainstem and cerebellum. All five antisera and all three tissue treatments were effective in showing angiotensinogen staining in glial cells, with the most intense staining being achieved in Vibratome sections. All five antisera gave identical results. Neuronal staining was also found with all antisera but mostly in paraffin-embedded sections, with occasional light staining in frozen sections. No neuronal staining was observed in Vibratome sections. Neuronal staining was frequently perivascular, tended to have a more variable anatomical localization, and to occasionally lack bilateral symmetry in coronal sections. These results provide an explanation for the disagreement between laboratories on the presence and location of angiotensinogen immunostaining in neuronal cells. Taken together with the limited concordance between published sites of angiotensinogen and angiotensin II staining, and the recent demonstration by hybridization in situ of a specifically glial cell localization of angiotensinogen mRNA, our own results suggest a need for caution in the interpretation of neuronal staining with anti-angiotensinogen antisera.     

Antineural autoantibodies in patients with paraneoplastic cerebellar degeneration

We studied serum autoantibodies from six patients with paraneoplastic cerebellar degeneration using rat brains. Immunohistochemically, serum samples from four of six patients with uterine or ovarian tumors showed a similar staining pattern, labeling Purkinje cells and various other neurons distributed throughout the brain and spinal cord. The immunoblot study showed that these serum samples recognized the common 52-kd band. Serum from a patient with duct carcinoma of the breast reacted with the 46-kd band with a similar but weaker immunohistochemical reactivity. Serum from another patient with small-cell carcinoma of the lung in whom paraneoplastic cerebellar degeneration and Eaton-Lambert myasthenic syndrome developed recognized the 40-kd and 38-kd bands with a different immunostaining pattern. Autoantibodies in patients with paraneoplastic cerebellar degeneration are directed to a variety of neuronal antigens that may differ from patient to patient.     

Antibodies to cerebellar Purkinje cells in patients with paraneoplastic cerebellar degeneration and ovarian carcinoma

Sera from 2 patients with ovarian carcinoma and paraneoplastic cerebellar degeneration confirmed postmortem were reacted with frozen sections of human cerebellum and stained using indirect immunofluorescence methods. Both sera produced bright cytoplasmic staining of Purkinje cells and of neurons within deep cerebellar nuclei. Titration of these sera to end point revealed staining at final dilutions of 1:640 and 1:2, 560, respectively. Neither of these sera reacted with sections of human cerebrum, basal ganglia, spinal cord, peripheral nerve, lung, liver, kidney, or ovary. Staining of Purkinje cells was not obtained with sera from 34 normal, healthy controls, 5 patients with oat cell carcinoma of the lung, 6 patients with inflammatory central nervous system disorders, or 12 of 14 neurologically normal patients with ovarian carcinoma. Sera from 2 neurologically normal patients with ovarian carcinoma, however, produced staining of Purkinje cells and deep nuclei similar to that obtained with sera from patients with paraneoplastic cerebellar degeneration. The present study documents the presence of antibodies to Purkinje cells in patients with ovarian carcinoma and cerebellar degeneration and demonstrates that development of these antibodies may antedate the onset of clinically evident cerebellar degeneration.     

Use of frozen sections to determine neuronal number in the murine hippocampus and neocortex using the optical disector and optical fractionator

Stereology is an important technique for the quantification of neurons in subregions of the central nervous system. A commonly used method of stereology relies upon embedment of tissue in glycol methacrylates to allow production of sections that are resistant to shrinkage in thickness. However, the use of glycol methacrylates for stereology has several disadvantages, including severe constraints on the size of tissue that can be processed and the long duration of time often required for infiltration. We describe a novel method of stereology utilizing tissue sections cut in the frozen state. This new methodology relies upon the staining of sections as free-floating sections and upon the mounting of these sections onto slides with a water-based mounting media. Sections cut in the frozen state and processed by these methods undergo little or no shrinkage in thickness and are ideal for stereological cell counts utilizing either the optical disector or optical fractionator methods of stereology. We demonstrate that frozen sections can be utilized to estimate neuronal number with high degrees of precision and with low coefficients of error. Because large tissue blocks can be cut as frozen sections, this method expands the range of tissues that can be processed efficiently for stereology and readily allows quantification of neurons from multiple brain regions from the same tissue sections. We applied this new methodology to estimate neuronal numbers in the neocortex and hippocampus of 10-day-old mice. The method was useful for estimation of both large, sparsely packed cell populations, such as the neocortex, and small, densely packed cells, such as the dentate gyrus granule cells. Thus, frozen section methodology offers many potential advantages over the use of glycol methacrylate embedment for stereology. These advantages include expansion of the size of tissue blocks that can be processed, reduction in expended time and costs, and ability to quantify multiple brain regions from a single set of sections.     

Autoimmune response of patients with paraneoplastic cerebellar degeneration to a Purkinje cell cytoplasmic protein antigen

Sera from 6 of 12 patients with paraneoplastic cerebellar degeneration (PCD) contained anti-Purkinje cell antibodies, as determined by indirect immunofluorescence on frozen sections of normal human cerebellum. Samples of cerebrospinal fluid from 2 of the patients with serum antibodies were tested, and both specimens contained anti-Purkinje cell antibody. The anti-Purkinje cell antibodies were polyclonal, fixed complement, and were present in all patients at serum dilutions of 1:1,000 or greater. Antibody activity could not be suppressed by preabsorption of sera with human or animal brain and tissue powders or with fresh crude human cerebellar extracts. No anti-Purkinje cell antibodies were detected in control sera from 167 neurologically normal cancer patients, 32 normal volunteers, 10 patients with other causes of cerebellar degeneration, or 8 patients with other paraneoplastic neurological diseases. Preliminary evidence suggests that the Purkinje antigen is a protein that is often concentrated in the periphery of the cytoplasm in disc-shaped structures. Patients with antibodies often developed signs of PCD near the time of detection of the tumor and had relentless progression of neurological disease. Patients without antibodies frequently had cancer for months to years before PCD developed, and often had spontaneous stabilization of neurological disease with time. Four patients without and 3 patients with antibodies underwent plasmapheresis without response.     

Brain-reactive autoantibodies prevalent in human sera increase intraneuronal amyloid-β(1-42) deposition

Previous studies have reported immunoglobulin-positive neurons in Alzheimer's disease (AD) brains, an observation indicative of blood-brain barrier (BBB) breakdown. Recently, we demonstrated the nearly ubiquitous presence of brain-reactive autoantibodies in human sera. The significance of these observations to AD pathology is unknown. Here, we show that IgG-immunopositive neurons are abundant in brain regions exhibiting AD pathology, including intraneuronal amyloid-β(42) (Aβ(42)) and amyloid plaques, and confirm by western analysis that brain-reactive autoantibodies are nearly ubiquitous in human serum. To investigate a possible interrelationship between neuronal antibody binding and Aβ pathology, we tested the effects of human serum autoantibodies on the intraneuronal deposition of soluble Aβ(42) peptide in adult mouse neurons in vitro (organotypic brain slice cultures). Binding of human autoantibodies to mouse neurons dramatically increased the rate and extent of intraneuronal Aβ(42) accumulation in the mouse cerebral cortex and hippocampus. Additionally, individual sera exhibited variable potency related to their capacity to enhance intraneuronal Aβ(42) peptide accumulation and immunolabel neurons in AD brain sections. Replacement of human sera with antibodies targeting abundant neuronal surface proteins resulted in a comparable enhancement of Aβ(42) accumulation in mouse neurons. Overall, results suggest that brain-reactive autoantibodies are ubiquitous in the blood and that a defective BBB allows these antibodies to access the brain interstitium, bind to neuronal surfaces and enhance intraneuronal deposition of Aβ(42) in AD brains. Thus, in the context of BBB compromise, brain-reactive autoantibodies may be an important risk factor for the initiation and/or progression of AD as well as other neurodegenerative diseases.     

免疫印迹技术测定血清中抗小脑浦肯野细胞Yo抗体

本文报道4例副肿瘤性小脑变性患者血清中存在特异性抗小脑浦肯野细胞抗体，免疫组化示小脑浦肯野细胞呈特征性粗颗粒状染色，患者血清中抗体滴度为1∶8000～1∶32000。免疫印迹技术发现抗体识别小脑浦肯野细胞提取物中分子量为34000与62000两种蛋白抗原，认为这种抗体是特异性抗-Yo抗体。本文就抗-Yo在临床检查中的意义进行了讨论。     

Paraneoplastic antibodies coexist and predict cancer, not neurological syndrome

We investigated coexisting autoantibodies in sera of 553 patients with a neurological presentation and one or more paraneoplastic neuronal nuclear or cytoplasmic autoantibodies: antineuronal nuclear autoantibody type 1 (ANNA-1), ANNA-2, ANNA-3; Purkinje cell cytoplasmic autoantibody type 1 (PCA-1), PCA-2; and CRMP-5-immunoglobulin G or amphiphysin-immunoglobulin G. Except for PCA-1, which occurred alone, 31% of sera had more than one of these autoantibodies. In addition, 25% of sera had neuronal calcium channel (P/Q-type or N-type), potassium channel, ganglionic acetylcholine receptor, muscle acetylcholine receptor, or striational antibodies. The autoantibody profiles observed in patients with paraneoplastic disorders imply the targeting of multiple onconeural antigens and predict the patient's neoplasm, but not a specific neurological syndrome.     

Problems in fast intraoperative diagnosis in Hirschsprung's disease

Histopathological evaluation of the proximal segment in colonic resections/biopsies for Hirschsprung's disease (HD) is very important because of the strong link between proximal segment histology and postoperative outcome. Therefore, we investigated whether a recently described rapid immunohistochemical staining technique using EnVision-antibody-complex is suitable for intraoperative diagnosis during surgery in HD. Various antibodies were applied on frozen sections from 20 colonic tissue samples including aganglionic segments, segments from the transitional zone, and normally innervated bowel segments to show ganglion cells (GCs) and cholinergic fibres. Several antibodies revealed positive staining of GCs (neurofilament, synaptophysin, peripherin, neural cell adhesion molecule/NCAM/CD56). However, none of these antibodies selectively identifies GCs. Microtubuli-associated protein 2/MAP2 stained few GCs only when incubation times were extensively prolonged. The antibodies applied to determine cholinergic innervation by immunohistochemistry (Acetylcholinesterase/AChE, cholinergic Acetyltransferase/chAT) failed. Rapid immunohistochemical technique using EnVision-antibody-complex on frozen sections of the large bowel is a) suitable for detection of many diverse antigens, including several neuronal antigens, b) not helpful to prove the presence or the absence of ganglion cells with any of the antibodies applied, c) not suitable to display the cholinergic innervation and iv) therefore is not helpful to shorten the consumption time during surgery in HD.     

Immunofluorescent labeling of nonhuman cerebellar tissue with sera from patients with systemic cancer and paraneoplastic cerebellar degeneration

Summary Sera from patients with paraneoplastic cerebellar degeneration have been shown to contain high titers of antibody to human Purkinje cells. It is not known, however, whether these sera react with cerebellar material from species other than man. The present study was conducted to determine whether cerebellar tissue of nonhuman species could be used to screen human sera for anticerebellar antibodies and whether similarities in cerebellar antigens between nonhuman and human material might permit attempts to transmit cerebellar degeneration to experimental animals by passive transfer of patient sera. Sections of human, monkey, pig, sheep, cat, rabbit, and rat cerebellar tissue were overlaid with serial dilutions of positive sera from patients with cancer and with paraneoplastic cerebellar degeneration and were stained using indirect immunofluorescence methods. All sera stained specific Purkinje cells when reacted with monkey, pig, and rabbit cerebellar sections, but not all sera stained sheep, cat, or rat tissue. Immunofluorescent labeling of animal cerebellar tissue was less bright than that obtained with human cerebellar sections, and anticerebellar antibody titers were invariably lower when assayed with nonhuman than with human material. Although human anticerebellar antibodies react with cerebellar tissue from other animal species, patient-to-patient variation in staining is sufficiently great that not all patient sera might be suitable for passive transfer experiments, and that attempts to identify anticerebellar antibodies by reacting patient sera with nonhuman cerebellar tissue could be negative where these antibodies are in fact present and could be demonstrated using human material.     

Expression of glutamic acid decarboxylase in nervous tissue structures targeted by autoantibodies in patients with diabetic autonomic neuropathy

We have previously identified an association between symptomatic diabetic autonomic neuropathy (DAN) and autoantibodies to sympathetic and parasympathetic nervous structures. The antigens identified by these autoantibodies are not known, but glutamic acid decarboxylase (GAD) has been suggested as a candidate target, since anti-GAD autoantibodies are present in patients with long-term diabetes and GAD is expressed in a variety of cell types and structures in the nervous system. The aim of this study was to examine GAD expression in sympathetic ganglia and vagus nerve and to compare the distribution of GAD within these tissues with that of anti-sympathetic ganglia and anti-vagus nerve autoantibodies from patients with DAN, using single and double indirect immunofluorescence on tissue sections. The monoclonal antibody GAD-6, specific for GAD₆₅, gave a granular, peripheral, cytoplasmic staining pattern in sympathetic ganglion cells. Dual immunofluorescence demonstrated that serum from a patient with anti-sympathetic ganglion autoantibodies stained the same cells, but homogeneously throughout the cytoplasm. In the vagus nerve, patient's serum stained the fibres only; GAD-6 stained the cytoplasm of parasympathetic ganglion cells but only occasional fibres. In addition, GAD enzymatic activity was detectable in both sympathetic ganglia and vagus nerve. Incubation of sera or GAD-6 overnight with a crude homogenate of human brain as an antigen source abolished staining of the nervous tissues by GAD-6, but not by patients' sera. The different localisation of GAD and the autoantigens targeted by patients' sera indicates that GAD is not the target of the autoantibodies characteristic of DAN. Moreover, absorption studies using human brain homogenate suggest that the targets of anti-sympathetic ganglion and anti-vagus nerve autoantibodies are absent or represented only at low levels in the central nervous system and may be confined to the periphery.     

Detection of plasma autoantibodies to brain tissue in young children with and without autism spectrum disorders

Autism spectrum disorders (ASDs) are characterized by impaired language and social skills, often with restricted interests and stereotyped behaviors. A previous investigation of blood plasma from children with ASDs (mean age = 5½ years) demonstrated that 21% of samples contained autoantibodies that reacted intensely with GABAergic Golgi neurons of the cerebellum while no samples from non-sibling, typically developing children showed similar staining (Wills et al., 2009). In order to characterize the clinical features of children positive for these autoantibodies, we analyzed plasma samples from children enrolled in the Autism Phenome Project, a multidisciplinary project aimed at identifying subtypes of ASD. Plasma from male and female children (mean age = 3.2 years) was analyzed immunohistochemically for the presence of autoantibodies using histological sections of macaque monkey brain. Immunoreactivity to cerebellar Golgi neurons and other presumed interneurons was observed for some samples but there was no difference in the rate of occurrence of these autoantibodies between children with ASD and their typically developing peers. Staining of neurons, punctate profiles in the molecular layer of the dentate gyrus, and neuronal nuclei were also observed. Taken together, 42% of controls and subjects with ASD demonstrated immunoreactivity to some neural element. Interestingly, children whose plasma reacted to brain tissue had scores on the Child Behavior Checklist (CBCL) that indicated increased behavioral and emotional problems. Children whose plasma was immunoreactive with neuronal cell bodies scored higher on multiple CBCL scales. These studies indicate that additional research into the genesis and prevalence of brain-directed autoantibodies is warranted.     

Impairment of Trk-neurotrophin receptor by the serum of a patient with subacute sensory neuropathy

BACKGROUND: Paraneoplastic peripheral neuropathy is sometimes associated with unidentified neuronal autoantibodies. OBJECTIVE: To examine the effects of serum from a patient with subacute sensory axonopathy on the function of the Trk high-affinity nerve growth factor receptor. PATIENT: An 86-year-old man with sensory neuropathy exhibiting an autoantibody to Trk. METHODS: Immunoblot analyses of the brain homogenates and immunoprecipitation were performed with human sera. We further examined the effect of sera on nerve growth factor-induced neurite outgrowth and Trk autophosphorylation. RESULTS: The patient showed sensory nerve axonopathy without well-known paraneoplastic autoantibodies. His serum inhibited nerve growth factor-induced neurite outgrowth and Trk autophosphorylation in PCtrk cells. Moreover, the patient's serum, but not control serum, immunoprecipitated Trk and recognized Trk in brain homogenates as well as in Trk immunoprecipitates. CONCLUSION: These data strongly suggest that an anti-Trk autoantibody might cause subacute sensory neuropathy.     

Regional quantitative determination of lactate in brain sections. A bioluminescent approach

Regional lactate distribution in brain was assessed quantitatively in coronal sections using a bioluminescent technique. This bioluminescence can be induced by covering freeze-dried and heat-inactivated brain sections with a frozen solution containing enzymes and coenzymes both for lactate-dependent NADH formation and NADH-dependent bioluminescence, which was recorded photographically. Quantification and density coding of bioluminescent images were carried out by utilizing the regression coefficients of the correlation between the optical density of bioluminescent pictures and the lactate content measured in tissue samples. Regional quantitative lactate images were obtained from brain tissue taken from brain tumors or after experimental cerebral ischemia.     

Detection of the anti-Hu antibody in specific regions of the nervous system and tumor from patients with paraneoplastic encephalomyelitis/sensory neuronopathy

We studied the nervous systems and tumors of five patients with anti-Hu-positive paraneoplastic encephalomyelitis/sensory neuronopathy (PEM/PSN) to determine if the autoantibody found in the serum and CSF was also present in those tissues. Immunohistochemical studies of the nervous system revealed the presence of IgG bound predominantly to the nuclei of most of the neurons and the cytoplasm of some glial cells. IgG was also present to a lesser degree in the neuropil. In brains of patients who died of cancer without the paraneoplastic syndrome, IgG was present in the immediate perivascular areas and to a very limited degree in the neuropil. There was no IgG in neurons, and in only some of the controls a few glial cells showed IgG immunoreactivity in the cytoplasm. The amount of anti-Hu IgG relative to total IgG in various brain regions and tumor was determined by quantitative Western blot analysis. The proportion of anti-Hu IgG was greater in some areas of the brain and tumor than in serum and CSF. Control brains did not contain anti-Hu IgG. There was a limited correlation among (1) the principal clinical symptoms, (2) regions of major tissue injury, and (3) the quantitative anti-Hu IgG distribution. We conclude that although the role of the antibody in the pathogenesis of the disease is still uncertain, its specific localization in the nervous system and tumor suggests an immunologic etiology of this paraneoplastic syndrome.