Biomedical subjects
Publications and source records attributed to J Dalmau.
Antibodies to neuronal antigens are found in a range of different neurological disorders, both central and peripheral, and often indicate the presence of particular underlying tumours. This conference and workshop was an opportunity to discuss the clinical associations of these antibodies, the relevance of new antibody specificities, and to establish protocols for their detection.
Antibodies against the HuD antigen expressed in small-cell lung cancer (SCLC) cross-react with proteins expressed in neurons of the central and peripheral nervous system and are associated with paraneoplastic encephalomyelitis and sensory neuropathy (PEM/SN). We isolated anti-HuD Fab fragments from an antibody phage display library that was constructed from mRNA of a metastatic lymph node from a patient with SCLC and Pem/SN. Fab GLN495 recognized HuD and other related proteins (HuC and Hel-N1, or Hu antigens) in immunoblots of these recombinant proteins and in immunohistochemical and Western blot analysis of SCLC and neurons. Fab GLN495 inhibited up to 75% of the anti-Hu antibodies of the patient from which it was derived, suggesting that recognizes a dominant epitope in the polyclonal anti-Hu antibody response. Fab GLN495 also competed with anti-Hu sera from most but not all patients with PEM/SN, indicating that the same epitope is recognized by a large subgroup of patients. Human monoclonal anti-HuD antibodies may be useful in diagnosis of HuD expressing tumors and in clarifying the autoimmune etiology of PEM/SN. This study, the first to demonstrate that tumor specific recombinant antibodies can be isolated from metastatic lymph node tissue, shows that this approach may be generally applicable to isolate human antibodies against tumor specific antigens.
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Molecular mimicry is implicated in the pathogenesis of autoimmune diseases such as diabetes mellitus, rheumatoid arthritis, and multiple sclerosis (MS). Cellular and antibody-mediated immune responses to shared viral-host antigens have been associated with the development of disease in these patients. Patients infected with human T-lymphotropic virus type I (HTLV-I) develop HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), an immune-mediated disorder of the central nervous system (CNS) that resembles some forms of MS. Damage to neuronal processes in the CNS of HAM/TSP patients is associated with an activated cellular and antibody-mediated immune response. In this study, IgG isolated from HAM/TSP patients was immunoreactive with uninfected neurons and this reactivity was HTLV-I specific. HAM/TSP IgG stained uninfected neurons in human CNS and cell lines but not nonneuronal cells. Neuronal western blots showed IgG reactivity with a single 33-kd band in all HAM/TSP patients tested. By contrast, no neuron-specific IgG reactivity could be demonstrated from HTLV-I seronegative controls and, more important, from HTLV-I seropositive, neurologically asymptomatic individuals. Both immunocytochemical staining and western blot reactivity were abolished by preincubating HAM/TSP IgG with HTLV-I protein lysate but not by control proteins. Staining of CNS tissue by a monoclonal antibody to HTLV-I tax (an immunodominant HTLV-I antigen) mimicked HAM/TSP IgG immunoreactivity. There was no staining by control antibodies. Absorption of HAM/TSP IgG with recombinant HTLV-I tax protein or preincubation of CNS tissue with the monoclonal antibody to HTLV-I tax abrogated the immunocytochemical and western blot reactivity of HAM/TSP IgG. Furthermore, in situ human IgG localized to neurons in HAM/TSP brain but not in normal brain. These data indicate that HAM/TSP patients develop an antibody response that targets uninfected neurons, yet reactivity is blocked by HTLV-I, suggesting viral-specific autoimmune reactivity to the CNS, the damaged target organ in HAM/TSP.
Patients with paraneoplastic cerebellar degeneration and Hodgkin's disease develop autoantibodies (Tr-Ab) that immunoreact with the cytoplasm of the Purkinje cells and produce a characteristic punctate pattern in the molecular layer of the cerebellum. In the present study, we analyzed the structures of the adult rat cerebellar cortex identified by Tr-Ab and the expression of the antigen recognized by Tr-Ab in the developing rat brain. By laser confocal microscopy and immunoelectron microscopy, Tr-Ab immunoreactivity was found localized in the cytosol and outer surface of the endoplasmic reticulum of the perikarya of neurons of the molecular layer and the cell body and dendrites of Purkinje cells without a particular concentration in dendritic spines. Tr-Ab reactivity was more widespread in the developing rat brain. Tr-Ab labeling of Purkinje cells was already observed at P0 (day of birth). The staining of the molecular layer followed the development of the dendritic tree. The internal and inner level of the external granule cell layer were labeled with Tr-Ab with a dotted pattern that became almost negative by the 2nd postnatal week. The staining probably corresponded to granule cells as suggested by the positive Tr-Ab labeling of cultures of embryonic granule neurons. The present findings suggest that the antigen recognized by Tr-Ab appears early and is widely expressed in the developing rat brain. In the adult cerebellum, the antigen is localized in the cell body and dendrites of the Purkinje cells but is not concentrated in the dendritic spines.
Neurologic complications of Hodgkin's disease (HD) include both metastatic and non-metastatic involvement of the nervous system. There are at least five paraneoplastic syndromes associated with HD but chorea has not been described. We report the first choreiform disorder as a paraneoplastic complication of HD and only the second case of paraneoplastic chorea in the literature.
Anti-Hu is a polyclonal immunoglobulin G associated with a syndrome of paraneoplastic sensory neuropathy/encephalomyelitis that principally afflicts patients with small cell lung carcinoma. Anti-Hu antibodies, which identify a family of RNA-binding proteins that are normally neuron restricted and that appear to be integral to neuronal differentiation and maintenance, selectively label the nuclei (and, less strongly, the cytoplasm) of neurons throughout the human neuraxis. Small cell carcinomas of the lung and many neuroblastomas are also labeled. We screened 112 tumors of central neuroepithelial lineage for immunohistochemical evidence of Hu expression with anti-Hu immunoglobulin G that was purified from patient sera and with a recombinant Fab fragment (Fab GLN 495) selected from a patient-derived combinatorial antibody phage display library using a recombinant Hu protein (HuD). Both antibodies uniformly labeled, in addition to native neurons, the nuclei of central neurocytomas (6 of 6) and the neuronal components of "classic" (12 of 12) and desmoplastic infantile (2 of 2) gangliogliomas. Of 33 embryonal tumors, 29 were anti-Hu reactive, including 87% of medulloblastomas (26 of 30). Glial neoplasms (n = 59) were anti-Hu negative save for one "oligodendroglioma" (of 17 oligodendroglial/oligoastrocytic tumors) that may have been an extraventricular neurocytoma. Anti-Hu immunoglobulin G/Fab GLN 495 identifies neoplasms of differentiated neuronal type and embryonal tumors with neuronogenic potential.
The sera of patients with Lambert-Eaton myasthenic syndrome (LEMS) contain autoantibodies against several extracellular and intracellular components of the voltage-gated calcium channel (VGCC)/synaptic vesicle release complex. An example of the latter are anti-beta-subunit antibodies (anti-MysB antibodies). We constructed a full-length cDNA clone of a human VGCC beta-subunit to produce purified beta-subunit fusion protein (MysB protein). Using this protein, we demonstrated that anti-beta-subunit antibodies are present in the sera of 23% of LEMS patients and only, in low titer, in 2% of small cell lung cancer patients without LEMS. The presence of anti-beta-subunit antibodies was closely associated with high titers of P/Q- and N-type VGCC antibodies. Immunization of rats with the purified MysB protein induced high antibody titers, but no signs of neurologic dysfunction were found. We conclude that anti-beta-subunit antibodies are not likely to interfere with ion channel function, but their presence could explain the cross-reactivity of LEMS sera with several subtypes of VGCCs and the lack of correlation between anti-VGCC antibody titer and clinical severity of disease.
OBJECTIVE: To determine whether patients with antineuronal antibody-associated paraneoplastic cerebellar degeneration (PCD) harbor antibodies to non-N-methyl-D-aspartate glutamate receptors (GluRs) in their serum. BACKGROUND: A recent study identified antibodies to GluRs in the serum of patients with PCD. METHODS: Sera of 35 patients with PCD (20 anti-Yo, 5 anti-Ri, 5 anti-Tr, and 5 anti-Hu) were examined by immunohistochemistry and immunoblot techniques. The expression of GluRs was obtained after transfection of 293T cells with cDNA plasmids corresponding to GluR1, GluR2, GluR3, GluR4, GluR6, and GluR7. The tumors of four patients with anti-Yo-associated PCD and nine ovarian carcinomas from patients without PCD were examined for the expression of GluRs. RESULTS: The expression of each GluR subunit was confirmed using affinity-purified antibodies against the corresponding GluRs and with whole sera from two rabbits immunized with GluR3. Thirty-two sera from patients with PCD were negative and 3 showed equivocal reactivity with 293T cells expressing different GluRs. None of the 35 sera had a pattern of reactivity characteristic of any GluR antibody on immunohistochemistry of sections of rat brain. Eleven of 14 tumors did not express GluRs; only some cells of one paraneoplastic tumor expressed GluRs. CONCLUSIONS: That patients with antibody-associated PCD (anti-Yo, -Ri, -Tr, and -Hu) harbor GluR antibodies in their sera is unlikely. GluR antibodies cannot be used as markers of paraneoplastic neurologic disorders.
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OBJECTIVE: To analyze the T-cell receptor (TCR) repertoire in the inflammatory infiltrates of the nervous system and tumor of patients with anti-Hu associated paraneoplastic encephalomyelitis and sensory neuronopathy (PEM/SN). BACKGROUND: In PEM/SN, the pathogenic role of the infiltrating T cells is speculative. TCR analysis may establish whether these lymphocytes are attracted nonspecifically by a proinflammatory environment or are driven by a specific antigen or superantigen. METHODS: We examined frozen tissues of seven patients with PEM/SN using immunohistochemical and PCR analysis. Of 62 tissue blocks from seven patients, 19 blocks from five patients had >100 CD3+ cells per section infiltrating the nervous system or tumor. These infiltrates allowed screening of the TCR Vbeta family repertoire using a panel of 18 antibodies that recognize family-specific regions of most TCR Vbeta families against which antibodies have been generated. To distinguish between antigen-driven clonal and superantigen-driven family expansion, we extracted RNA from frozen tissue and performed reverse transcriptase (RT)-PCR analysis followed by subcloning and sequencing of the antigen-specific CDR3 region of the TCR Vbeta chain. RESULTS: All five patients showed a limited Vbeta repertoire. An overrepresentation (>10% of total CD3+) of certain Vbeta families was identified in three patients (as high as 45% of total CD3+), which consisted mainly of CD8 + cells. CDR3 sequences obtained from one patient revealed an in situ expansion of two clones in the amygdala (one at a frequency of 57%) and four clones in the tumor. CONCLUSION: These findings suggest that an antigen-driven oligoclonal cytotoxic T-cell response plays a role in the pathogenesis of anti-Hu associated PEM/SN.
Increased levels of lipoprotein (a) [Lp(a)] have been considered an independent risk factor for cardiovascular disease, but the mechanism behind this relationship is not completely understood. A high concentration of Lp(a) may interfere with fibrinolysis because of the structural similarity between apo(a) and plasminogen. The aim of the present study was to examine the influence of apo(a) levels and isoforms on fibrinolytic activity in 82 subjects from 24 families in which the Lp(a) levels were > or =30 mg/dl in at least one child and one parent. Several fibrinolytic parameters, including plasmin generation by fibrin-bound tissue plasminogen activator, the lipid profile and apo(a) isoforms were studied. Subjects with high circulating Lp(a) levels (n = 44) had significantly reduced plasmin generation compared with their relatives with normal Lp(a) levels (n = 38). A significant inverse correlation between Lp(a) levels and plasmin generation was observed. The individuals with a combination of high levels of plasma Lp(a) and a major apo(a) isoform < or =580 kD molecular weight show the lowest fibrinolytic activity. A high correlation was found between the levels of apo(a) isoforms in children and the levels of the corresponding parental apo(a) isoforms. We conclude that the antifibrinolytic effect of Lp(a) in subjects with two apo(a) isoforms may depend not only on the total plasma level of Lp(a) but also on the relative concentration of the small apo(a) isoform.
The cholesterol/phospholipid ratio (C/PL) of the red blood cell membrane, plasma lipids and erythrocyte aggregability were evaluated in 20 children with familial hypercholesterolemia (age: 10.4+/-4.6 years) but without detectable vascular injury. The results indicate that hypercholesterolemic children have a higher erythrocyte membrane C/PL ratio than the control group (0.81+/-0.23 vs. 0.65+/-0.08, p < 0.01). This membrane lipid alteration correlates inversely with the plasma concentration of HDL-cholesterol (r = -0.558, p < 0.010). The patients also showed greater erythrocyte aggregability than the control group (8.21+/-1.11 vs. 6.25+/-1.24, p < 0.001), but this does not seem to correlate with the changes observed in the lipid composition of the cell membrane. These results suggest that from childhood, people with familial hyper-cholesterolemia show alterations in the lipid composition of the red blood cell membrane that are related to the changes observed in plasma lipids and appear prior to atherosclerotic vascular symptoms.
In order to ascertain whether young familial hypercholesterolemic subjects without atherosclerotic lesions demonstrable on a carotid echo-Doppler show decreased erythrocyte deformability, we determined the erythrocyte elongation index (EEI) by means of a shear stress diffractometer (Rheodyn SSD) in twenty-four children with heterozygous familial hypercholesterolemia (FH), aged 12 +/- 3 years, and their corresponding affected parent. The parents were twenty-three adults with heterozygous FH aged, 32 +/- 6 years. The control group was made up of a similar number of well matched healthy volunteers. The EEI at 6, 12, 30 and 60 Pa showed no statistical differences between FH children and their control group, or between FH parents and their control group. No correlations were found between EEI and plasma lipids or lipoproteins at any of the shear stresses used. These results suggest that the red blood cells of young familial hypercholesterolemic subjects who show no deterioration of the vascular tree are not less deformable than those of healthy normolipemic subjects, and that dyslipemia itself does not produce alterations that damage the rheological properties of the red blood cell.
Some patients with small cell lung cancer (SCLC) or neuroblastoma develop an immune response against HuD, a human homologue of the Drosophila protein, elav, which is expressed in the nucleus and to a lesser degree the cytoplasm of neurons and tumor cells. This immune response is characterized by antibodies (anti-Hu) that at high titers are associated with a disease called paraneoplastic encephalomyelitis/sensory neuronopathy, in which infiltrates of T cells are found in the tumor and nervous system. Although all SCLCs express HuD, anti-Hu antibodies are identified in only 17% of patients with SCLC, usually at low titers, and are associated with indolent tumor growth. To determine whether the anti-Hu immune response causes indolent tumor growth, we developed an animal model using HuD DNA immunization. We found that a plasmid coding for a secreted form of HuD induced a strong and specific anti-Hu response. Immunized animals were challenged by s.c. implantation of a neuroblastoma cell line that constitutively expresses HuD. When compared with controls, mice immunized with the secreted HuD showed significant tumor growth inhibition (51% reduction volume; P = 0.0012), and 14% of them had complete tumor rejection. Tumors from these animals showed three times more CD3+ lymphocytic infiltrates than those from control mice and had a higher CD8+:CD4+ ratio. None of the animals developed neurological deficits or neuropathological evidence of nervous system pathology. In this mouse model of neuroblastoma, DNA immunization with HuD resulted in tumor growth inhibition but did not induce neurological disease. This model closely mimics the clinical course of more indolent tumor growth seen in patients with the anti-Hu immune response.
In order to ascertain whether hemorheological alterations precede the atherosclerotic lesion in familial hypercholesterolemia (FH), we studied the lipid and hemorheological profile of 29 heterozygous FH children (12 males and 17 females), aged 12+/-3 years, and a well-matched control group (CG), and assessed their plasma thrombomodulin level as an early marker of endothelial injury. No differences were found between FH children's plasma thrombomodulin values (31.7+/-11.2 ng/ml) and those of the CG (27.8+/-15 ng/ml). However, the rheological variables of FH children and the CG were statistically different (p < 0.001) for fibrinogen (Fbg): 266+/-48 mg/dl vs. 205+/-32 mg/dl; erythrocyte aggregation at stasis (EAM0): 4.6+/-1.2 vs. 3.3+/-0.9; erythrocyte aggregation at low shear (EAM1): 7.9+/-1.7 vs. 6.1+/-0.8; and plasma viscosity (PV): 1.18+/-0.03 cP vs. 1.12+/-0.04 cP. Correlations between rheological parameters and lipids were found. The normal values obtained in FH children for plasma thrombomodulin suggest that the hemorheological alterations appear prior to the vascular injury, are in part related to dyslipemia and could contribute to the development of the atherosclerotic process by modifying blood flow conditions.
In order to ascertain whether polygenic hypercholesterolemia (PH), the most common cause of small increases in plasma lipids during childhood, is associated with rheological alterations, we determined the hemorheological and lipid profile of 21 PH children (12 males, 9 females) and a well-matched control group (CG). In addition, a carotid ultrasound was done on all the PH children, but showed no alterations. When compared with the CG, PH children showed increased erythrocyte aggregation both at stasis (EAM0) (4.39+/-1.15 vs. 3.75+/-1.02), p < 0.05, and at low shear rate (EAM1) (8.22+/-1.42 vs. 7+/-1.39), p < 0.01, and increased plasma viscosity (PV) (1.19+/-0.04 cP vs. 1.15+/-0.04 cP), p < 0.01. These results reinforce the hypothesis that lipid metabolic alterations are associated with specific rheological modifications in absence of a demonstrable atherosclerotic lesion.
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