P. van Breda Vriesman

Selection of recombinant anti-HuD Fab fragments from a phage display antibody library of a lung cancer patient with paraneoplastic encephalomyelitis.

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.

Age- and sex-related resistance to chronic experimental autoimmune myasthenia gravis (EAMG) in Brown Norway rats.

The influence of age and sex on the induction of chronic EAMG was analysed. Aged male rats, immunized with Torpedo acetylcholine receptor (tAChR), showed no clinical signs of disease or AChR loss. Immunization of young male Brown Norway (BN) rats resulted in both clinical signs of disease and 65% AChR loss. In contrast, both young and aged female BN rats showed comparable AChR loss (58% and 50%, respectively), although aged female rats did not develop clinical signs of disease. Differences in antibody titres, isotype distribution, fine specificity or complement activation could not account for the observed resistance. These results suggest that resistance against EAMG in aged rats is due to resistance of the AChR against antibody‐mediated degradation, or to mechanisms able to compensate for AChR loss.

Role of the target organ in determining susceptibility to experimental autoimmune myasthenia gravis

Injection of anti-AChR antibodies in passive transfer experimental autoimmune myasthenia gravis (EAMG) results in increased degradation of acetylcholine receptor (AChR) and increased synthesis of AChR alpha-subunit mRNA. Passive transfer of anti-Main Immunogenic Region (MIR) mAb 35 in aged rats does not induce clinical signs of disease nor AChR loss. The expression of the AChR subunit genes was analyzed in susceptible and resistant rats. In aged EAMG resistant rats, no increase in the amount of AChR alpha-subunit mRNA was measured. In vivo AChR degradation experiments did not show any increase in AChR degradation rates in aged resistant rats, in contrast to young susceptible rats. Taken together, these data demonstrate that resistance of the AChR protein to antibody-mediated degradation is the primary mechanism that accounts for the resistance to passive transfer EAMG in aged rats.

Hu antigens and anti‐Hu antibodies in a patient with myxoid chondrosarcoma

Anti-Hu antibody-associated paraneoplastic sensory neuronopathy (PSN) is a rare disorder that is associated in about 80% of cases with small-cell lung cancer (SCLC) and in 10% with other We report a patient with the unusual combination of a chondrosarcoma with anti-Hu-associated PSN, whose neurologic symptoms and signs improved after surgical treatment of the tumor in combination with immunosuppressive treatment of the paraneoplastic syndrome. Case report. A 36-year-old man was healthy until 1988, when he underwent radical quadriceps resection for extraskeletal myxoid chondrosarcoma (Tl , NO, MO, grade 1). His condition was stable until Ju ly 1991, when he developed right iliac adenopathies. Two weeks after surgical excision, he developed anorexia, nausea, vomiting, and painful paresthesias in both arms. The patient was alert. There was a moderate, diffuse weakness of the right arm. Sensory responses to touch and pain in both hands and legs were diminished. Tendon reflexes were decreased or absent. Electrodiagnostic studies showed decreased sensory nerve action potentials (SNAPS) of the right median (4 pV), right ulnar (2 pV), and left sural (5 pV) nerves, while the sensory nerve conduction velocity of the median nerve was 50 d s e c , of the ulnar nerve 43 d s e c , and of the sural nerve 39 d s e c . Two months later, no SNAPS could be obtained from these nerves, and the motor nerve conduction velocity of the median nerve was 41 m/sec. Repeated CSF studies showed a maximum of two leukocytes and seven erythr~cyteslmm~, protein content of 74 to 102 mgldl (normal range, 20 t o 451, IgG index of 0.62 to 0.71 (normal range, 0.36 to 0.56), and five oligoclonal bands. No malignant cells were detected. Glucose was normal. Immunofluorescence and Western blot analysis using a recombinant Hu protein (HuD) demonstrated the presence of the anti-Hu antibody in the patient’s serum (figure). Tumor tissue, tested as described b e f ~ r e , ~ , ~ expressed Hu antigens. No other autoantibodies (anti-DNA, antinuclear factor, rheumatoid factor) were detected. The patient’s general condition rapidly deteriorated because of severe dysphagia and vomiting. His sensory symptoms worsened and he became unable to walk. He was treated with cyclosporin A 200 mgld, prednisolone 20 mgld, plasmapheresis (4 x 2 liters, with fresh-frozen plasma reconstitution), and human immunoglobulins 12 glwk. After plasmapheresis, CSF showed one leukocyte, 10 erythrocyte~lmm~, protein 90 mgldl, and no tumor cells. Glucose was normal. IgG index was 0.40 and no oligoclonal bands were found. On a regimen of immunoglobulins, cyclosporin A, and prednisolone, the patient gradually improved. After 18 months of therapy, he was able to walk and write unassisted. Although there was a substantial (tenfold) decrease in circulating anti-Hu serum antibodies, the antibody was still present a t the last follow-up (figure). Discussion. Our patient with a metastasis of a chondrosarcoma had an anti-Hu antibody-positive PSN. He had sensory symptoms, signs of autonomic dysfunction, and weakness. Most patients have signs and symptoms suggesting multifocal lesions of the nervous system.2 History and laboratory findings excluded a Sjogren’s-syndrome-associated sensory n e ~ r o n o p a t h y . ~ CSF and EMG studies were in accord with findings previously described for PSN,2 a diagnosis confirmed by the demonstration of anti-Hu antibodies in the serum and Hu antigens in the tumor. Unlike our patient, 78% of PSN patients have SCLC,’ and there is on1 one other anti-Hu-positive chondrosarcoma in the literature. When anti-Hu antibodies a re associated with I Figure. Western blot using a recombinant Hu protein (HuD) showing a 37-kd band. Immunoblots o f recombinant protein (10 pg protein each strip) were reacted wi th the same amount of IgG (1 p g l m l ) obtained f rom serum of a normal individual (lane N: negative control), a known anti-Hu-positive serum (lane Hu: positive control), serum from our patient before treatment (lane 1, 1011 191, total serum IgG 12 .5g/ l ) , plasmapheresis filtrate (lane 2, 10l17191, total IgG9.4gl l ) , and sera obtained a t different times after treatment (lane 3, 11 121 191, total serum IgG 11.1 g l l ; lane 4, 11 19/92, total serum IgG 19.1 g l l ; and lane 5, 5 / 6 / 9 3 , total serum IgG 15.1 g l l ) .

Monoclonal Antibodies to Human Thyroglobulin as Probes for Thyroglobulin Structure

A panel of monoclonal antibodies (mAbs) against human thyroglobulin (hTg) was obtained by somatic fusion of the nonsecreting myeloma cell line P3X66 Ag8/0 and spleen cells of Balb/c mice immunized with purified hTg. Antibody secreting clones were selected by solid phase enzyme immunoassay and analyzed for cross-reaction with Tg from several animal species. Twelve out of 15 mAbs cross-reacted with both rat and mouse Tg and 11 Mabs cross-reacted with bovine Tg. The cross-reaction with mouse Tg paralleled that of rat Tg, whereas discrete differences between the cross-reactivity patterns with bovine Tg were observed. Two clones secreted mAbs specific for hTg. We further characterized the mAbs and found that three mAbs recognized T4-containing determinants and one mAb reacted with both T4 and T3-containing determinants on the Tg molecule. The binding of the mAbs to hormonogenic determinants depended upon the thyroid hormone content of the molecule and the integrity of the three dimensional structure of Tg. One other mAb reacted with four peptides of CNBr-cleaved hTg, indicating the recognition of a repetitive determinant in the hTg molecule distinct from the hormonogenic regions.

Costimulatory molecules CD80 and CD86 in the rat; tissue distribution and expression by antigen‐presenting cells

The CD28‐CD80/CD86 costimulatory pathway provides a critical signal for T cell activation. Only recently rat CD80 and CD86 have been cloned and monoclonal antibodies have been generated. In this study we examined the expression of these molecules in lymphoid tissue and on purified subsets of antigen‐presenting cells (APC). The target tissue of cyclosporin A‐induced autoimmunity, i.e. the skin and tongue, were also examined for expression of CD80 and CD86. Whereas CD80 was hardly detected in the lymphoid tissues, CD86 was clearly expressed by non‐lymphoid cells in the thymus, as well as in the secondary lymphoid organs. With respect to lymphoid cells, only germinal center B cells exhibited clear CD86 expression. Phenotypic analysis by flow cytometry revealed that only dendritic cells, both of thymic and splenic origin, expressed the full array of stimulatory molecules required for the proper activation of naive T cells. On development of cyclosporin A‐induced autoimmunity, non‐professional APC, i.e. epithelial cells, started to express MHC class II, but not the costimulatory ligands CD80 and CD86. However, CD86 staining was observed in the target tissue and was associated with Langerhans cells as well as infiltrating leukocytes. Altogether, our results show that also in the rat strong stimulatory capacity for primary immune responses is associated with the expression of the costimulatory ligands CD80 and CD86. As concluded from the in situ expression CD86 may be the predominant costimulatory ligand early in immune responses. J. Leukoc. Biol. 64: 803–809; 1998.

The effects of in vivo cyclosporin A administration on rat thymic dendritic cells.

Cyclosporin A (CsA) induces a graft‐versus‐host‐like disease (GVHD) in lethally irradiated Lewis rats reconstituted with syngeneic bone marrow. The role of the thymus in the generation of disease has been unequivocally established. It has been suggested that the CsA‐induced disappearance of thymic dendritic cells (DC) is responsible for the generation of the autoaggressive cells. In this study we quantify the loss of DC upon in vivo CsA administration in normal and bone marrow‐reconstituted rats using an isolation technique. The phenotype of the DC is determined using MoAbs recognizing antigens which are expressed on thymic medullary DC. Furthermore, the functional aspects are assessed by determining the antigen presentation capacity. Short‐term CsA exposure clearly affects the number of DC isolated from the thymus in a concentration‐dependent manner. However, in all instances a substantial number of DC can be isolated from CsA‐treated animals. These isolated DC exhibit an identical phenotype and function as DC isolated from control animals. Therefore, the partial deficiency of DC can not be held as essential for loss of tolerance.

A dominant role for non-MHC gene effects in susceptibility to cyclosporin A (CsA)-induced autoimmunity

Lethally irradiated LEW rats reconstituted with syngeneic bone marrow and given CsA for a 4‐week period develop a graft‐versus‐host‐like disease upon withdrawal of CsA. This T cell‐mediated autoimmune disease is referred to as CsA‐induced autoimmunity (CsA‐AI). CsA‐AI‐susceptible LEW rats and resistant BN rats differ greatly in the composition of their peripheral T cell compartment. To dissect the role of MHC and non‐MHC genes in the development of peripheral T cell subsets in combination with susceptibility to CsA‐AI the respective MHC congenic strains (LEW‐1N and BN‐1L) were examined for their T cell subsets and for their ability to develop CsA‐AI. In this study we show that the Th1/Th2‐like cell ratio as well as susceptibility to CsA‐AI are under control of the non‐MHC genes. This suggests that the Th1/Th2‐like cell ratio is a critical determinant for development of CsA‐AI. Alternatively, resistance can be attributed to lack of target organ susceptibility due to the absence of the target autoantigen in resistant rat strains. This interpretation is rejected, since both BN as well as BN‐1L rats consistently develop the characteristic macroscopic and microscopic signs of CsA‐AI upon adoptive transfer with autoreactive LEW‐1N and LEW T cells, respectively. Therefore, it can be concluded that the non‐MHC genes encode for immune deviation and thereby determine susceptibility or resistance to CsA‐AI.

Defective de novo thymocyte maturation in cyclosporin A (CsA)-induced autoimmunity: expression of costimulatory and activation molecules

Lethally x‐irradiated Lewis rats, reconstituted with syngeneic bone marrow and transiently treated with CsA for 4 weeks, will develop an autoimmune disease about 2–3 weeks after cessation of CsA therapy. CsA‐induced autoimmunity is a thymus‐dependent and T cell‐mediated autoimmune disease. CsA is thought to generate autoreactive T cells by interference with negative selection in the thymus; x‐irradiation is required to eliminate the peripheral autoregulatory T cell circuit. In this study we re‐evaluate the effect of CsA on thymic atrophy and thymocyte maturation. Subsequently we examine the expression of costimulatory and activation molecules (CD2, CD5, CD11a, CD11b, CD25, CD28, CD43, CD54, OX‐40, RT‐1A, RT‐1B and RT‐1D) during distinct maturational stages in order to detect possible clues to the observed effects of CsA on thymocyte maturation and selection. The results revealed that CsA blocks maturation of double‐positive TCRint to double‐positive TCRhigh thymocytes and preferentially inhibits the development of mature CD4 single‐positive thymocytes. Furthermore, CsA administration resulted in a reduced expression of the costimulatory CD2 molecule. Although it is a matter of debate whether this defective CD2 expression is involved in the aberrant maturation and selection of thymocytes, it is speculated that reduced costimulation via CD2 may influence differentiation into distinct T cell subsets.

Infection with rat cytomegalovirus (CMV) in the immunocompromised host is associated with the appearance of a T cell population with reduced CD8 and T cell receptor (TCR) expression.

Infection with human cytomegalovirus (HCMV) mostly results in a chronic subclinical infection; the immune system is unable to eliminate the virus and is apparently in equilibrium with the persistent virus. In the immunosuppressed host this equilibrium is disturbed, resulting in clinical infection. Rat cytomegalovirus (RCMV) infection in its host can be used as a model for HCMV infection. Using flow cytometry we examined the effect of acute RCMV infection on the composition of leucocyte subsets in the peripheral blood of both immunocompetent and immunosuppressed (5 Gy total body irradiation) Lewis rats. Special attention was paid to the natural killer (NK) cells and the CD8+ T cells known to be involved in the control of viral infections. Furthermore, we determined the presence of leucocyte subsets in the internal organs by immunohistochemistry. In immunocompetent rats, infection caused a small increase in NK cells and a large increase in CD8+ T cells. In contrast, infection of immunosuppressed rats caused a marked increase in NK cells and a small increase in CD8+ T cells, consisting of T cells with reduced expression of both CD8 and TCR. This phenomenon is characteristic of anergic CD8+ T cells, possibly explaining the ability of the virus to escape elimination by the immune system. The increase of NK cells in the peripheral blood of immunosuppressed, RCMV‐infected rats could also be detected in kidney, liver, lung and pancreas, but not in salivary gland. This could explain the long persistence of infectious virus in the salivary gland.