Andrew R. Pachner

Localization of Borrelia Burgdorferi in the Nervous System and Other Organs in a Nonhuman Primate Model of Lyme Disease

Lyme borreliosis is caused by infection with the spirochete Borrelia burgdorferi. Nonhuman primates inoculated with the N40 strain of B. burgdorferi develop infection of multiple tissues, including the central (CNS) and peripheral nervous system. In immunocompetent nonhuman primates, spirochetes are present in low numbers in tissues. For this reason, it has been difficult to study their localization and changes in expression of surface proteins. To further investigate this, we inoculated four immunosuppressed adult Macaca mulatta with 1 million spirochetes of the N40 strain of B. burgdorferi, and compared them with three infected immunocompetent animals and two uninfected controls. The brain, spinal cord, peripheral nerves, skeletal muscle, heart, and bladder were obtained at necropsy 4 months later. The spirochetal tissue load was first studied by polymerase chain reaction (PCR)-ELISA of the outer surface protein A (ospA) gene. Immunohistochemistry was used to study the localization and numbers of spirochetes in tissues and the expression of spirochetal proteins and to characterize the inflammatory response. Hematoxylin and eosin and trichrome stains were used to study inflammation and tissue injury. The results showed that the number of spirochetes was significantly higher in immunosuppressed animals. B. burgdorferi in the CNS localized to the leptomeninges, nerve roots, and dorsal root ganglia, but not to the parenchyma. Outside of the CNS, B. burgdorferi localized to endoneurium and to connective tissues of peripheral nerves, skeletal muscle, heart, aorta, and bladder. Although ospA, ospB, ospC, and flagellin were present at the time of inoculation, only flagellin was expressed by spirochetes in tissues 4 months later. Significant inflammation occurred only in the heart, and only immunosuppressed animals had cardiac fiber degeneration and necrosis. Plasma cells were abundant in inflammatory foci of steroid-treated animals. We concluded that B. burgdorferi has a tropism for the meninges in the CNS and for connective tissues elsewhere in the body.

Neuro-ophthalmologic manifestations of lyme disease

Lyme disease is a tick-borne spirochetal infection characterized by skin rash, neurologic, cardiac, and arthritic findings. The authors report six patients with Lyme disease who had neuro-ophthalmologic manifestations. One patient had meningitis with papilledema, two had optic neuritis, and one had neuroretinitis. Three patients had sixth nerve paresis, two of whom cleared quickly, whereas multiple cranial nerve palsies and subsequent optic neuropathy developed in another. Early recognition of neuro-ophthalmologic findings can help in the diagnosis and treatment of Lyme disease.

Borrelia burgdorferi in the nervous system: the new "great imitator".

There are many obvious similarities between Lyme disease and syphilis. The major ones are their spirochetal etiology, the ability of the spirochetes to stay alive in human tissue for years, occurrence of clinical manifestations in stages, early disease in the skin and later disease in the brain, and susceptibility to antibiotic treatment. Thus, one can assume that many of the same lessons learned from the centuries of experience with syphilis will apply to Lyme disease. One of these lessons that should be constantly borne in mind is that spirochetal disease of the brain can mimic many other neurological diseases. Thus, the effective clinician must take special care to consider Lyme disease primarily because of the excellent response to antibiotics early in its course in relationship to some of the diseases it mimics. Lyme meningitis, occurring in the second stage of the disease, usually is fairly easily recognized because it occurs in the summer or early fall, often is associated with ECM or a recent history of it, and has a characteristic clinical picture of lymphocytic meningoradiculoneuritis. Many patients with Lyme meningitis or ECM have very mild symptoms, and it is likely that a large percentage of patients go undiagnosed and untreated. The frequency of progression of these patients to third-stage disease is unknown but may be quite high. This can be inferred from a similar situation in the other major late manifestation of Lyme disease: Lyme arthritis. A large number of patients present with joint involvement as their only manifestation of Lyme disease. Similarly, patients may present with symptoms of third-stage Lyme disease affecting the CNS, but they may not be recognized because of the lack of earlier stages usually associated with the disease. Thus, serology has become a very important tool for identifying patients exposed to B. burgdorferi. At the present time, serologic tests are the key to diagnosis of Lyme disease in its later stages, since, as in neurosyphilis, cultures and tests for antigen have not proven useful. Lyme arthritis and acrodermatitis atrophicans (ACA) both are associated with quite high antibody titers to the organism, while the test is understandably unreliable for identification of patients with ECM. Antibody titers in Lyme meningoradiculoneuritis are generally positive but often are not as high as those in ACA or arthritis. The antibody response in serum in CNS Lyme disease seems to be related to the presence of other manifestations; patients who have had both arthritis and CNS disease have quite high titers, while those with only CNS disease sometimes do not.(ABSTRACT TRUNCATED AT 400 WORDS)

Spinal cord involvement in the nonhuman primate model of Lyme disease

Lyme borreliosis is a multisystemic disease caused by infection with various genospecies of the spirochete Borrelia burgdorferi. The organs most often affected are the skin, joints, the heart, and the central and peripheral nervous systems. Multiple neurological complications can occur, including aseptic meningitis, encephalopathy, facial nerve palsy, radiculitis, myelitis, and peripheral neuropathy. To investigate spinal cord involvement in the nonhuman primate (NHP) model of Lyme borreliosis, we inoculated 25 adult Macaca mulatta with B. burgdorferi sensu strictu strains N40 by needle (N=9) or by tick (N=4) or 297 by needle (N=2), or with B. burgdorferi genospecies garinii strains Pbi (N=4), 793 (N=2), or Pli (N=4) by needle. Immunosuppression either transiently (TISP) or permanently (IS) was used to facilitate establishment of infection. Tissues and fluids were collected at necropsy 7–24 weeks later. Hematoxylin and eosin staining was used to study inflammation, and immunohistochemistry and digital image analysis to measure inflammation and localize spirochetes. The spirochetal load and C1q expression were measured by TaqMan RT-PCR. The results showed meningoradiculitis developed in only one of the 25 NHPs examined, TISP NHP 321 inoculated with B. garinii strain Pbi. Inflammation was localized to nerve roots, dorsal root ganglia, and leptomeninges but rarely to the spinal cord parenchyma itself. T cells and plasma cells were the predominant inflammatory cells. Significantly increased amounts of IgG, IgM, and C1q were found in inflamed spinal cord. Taqman RT-PCR found spirochetes in the spinal cord only in IS-NHPs, mostly in nerve roots and ganglia rather than in the cord parenchyma. C1q mRNA expression was significantly increased in inflamed spinal cord. This is the first comprehensive study of spinal cord involvement in Lyme borreliosis.

Profile of the regions of acetylcholine receptor α chain recognized by T-lymphocytes and by antibodies in eamg-susceptible and non-susceptible mouse strains after different periods of immunization with the receptor

C57BL/6 (B6) mice develop a neuromuscular disease, experimental autoimmune myasthenia gravis (EAMG), after two or more immunizations with Torpedo californica acetylcholine receptor (AChR). To determine whether EAMG is related to recognition of particular region(s) on the main extracellular domain of the alpha chain (residues alpha 1-210) in prolonged immunization, we have examined the differences in the antibody and T cell recognition profiles of B6 and SJL (a strain that does not develop EAMG) mice after different periods and a number of immunizations with Torpedo AChR. In a given strain, antibodies and T cells recognized immunodominant regions, which may coincide or may be uniquely B cell or T cell determinants. Both B6 and SJL exhibited similar antibody recognition profiles after the second and through the fourth immunizations with AChR. Major differences between the two strains were found in their T cell recognition of regions in the second part (residues 100-210) of the main extracellular domain of the alpha chain. T cells of SJL recognized consistently only one region (111-126) within this part of the alpha chain, whereas in B6, T cell recognition of three peptides (111-126, 146-162 and 182-198) and next neighbor regions to them persisted throughout the period. Of these three peptides, 146-162 was an immunodominant peptide unique to B6, as the other two peptides (111-126 and 182-198) were also recognized by either T cells or antibodies in SJL. To study the role of the T cells recognizing region 146-162 in EAMG, a T cell line was generated against this region and the cells transferred into B6 mice followed by one Torpedo AChR injection. Enhancement of antibody production toward alpha chain peptides was observed as an influence of T cell transfer compared to profiles at 1 week. In addition, one out of three mice examined showed signs of EAMG. These results suggest the importance of T cells recognizing residues 146-162 in EAMG. It is concluded that the presence of persistent T cell responses to the second half (residues (100-210) of the main extracellular domain of the alpha chain is associated with the development of EAMG in B6 mice, while absence of these responses in SJL mice may enable them to escape the disease. The preservation of the immunodominance of peptide 146-162 in the T cell recognition of B6 is probably most important for the pathogenesis of EAMG in this strain.

An immunodominant site of acetylcholine receptor in experimental myasthenia mapped with T lymphocyte clones and synthetic peptides.

Myasthenia gravis (MG) is an autoimmune disease of man caused by antibodies directed against the acetylcholine receptor (AChR). In the experimental model of MG in mice, murine experimental autoimmune myasthenia gravis (EAMG), an anti-AChR immune response is induced by immunization with Torpedo AChR, and anti-AChR antibodies. AChR-sensitized T cells, and neuromuscular dysfunction result. The production of antibodies to AChR is thymus-dependent. In order to define the epitopes of the AChR identified by AChR-specific T cells, we generated T cell populations and T cell hybridoma clones and tested their reactivity to synthetic uniform-sized overlapping peptides representing the entire extracellular portion of the alpha-chain of the AChR. The predominant reactivity of the T cell clones and the parent lines was to a peptide corresponding to residues 146-162 of Torpedo AChR. This data is consistent with a highly limited recognition of AChR determinants in murine EAMG by AChR-specific T cells.

Chronic murine experimental myasthenia gravis: Strength testing and serology

CB6 (Balb/c x C57Bl/6 F1) and C57Bl/6 (B6) mice were hyperimmunized with Torpedo acetylcholine receptor (AChR) for 7 months. Control groups were hyperimmunized with bovine serum albumin. Antibody titers against Torpedo AChR rose quickly, reaching plateau levels by 3-4 months, while antibody to mouse AChR lagged by a few months, reaching plateau levels in 5 months. After the last immunization the mice maintained a state of stable autoimmunity for 9 months with high levels of antibodies against Torpedo and mouse AChR. Fatigability was measured on a programmable treadmill and remained present through the 9 months after the last immunization. CB6 mice had less weakness than the B6 mice, but the latter strain when immunized with BSA had more false-positive weakness. Titers of antibodies did not correlate with the degree of weakness measured on the treadmill. Despite the weakness and the high titers of anti-AChR antibodies, sera from myasthenic mice, in contrast to sera from myasthenic humans, were not able to block bungarotoxin binding to native AChR on the surface of BC3H1 cells.

In vitro neutralization by monoclonal antibodies of α-bungarotoxin binding to acetylcholine receptor

In order to develop monoclonal antibodies that would neutralize binding of alpha-bungarotoxin to acetylcholine receptor in vitro, mice were hyperimmunized with native toxin. Frequent small doses of toxin were used. Hybridoma supernatants were screened by ELISA and six monoclonal antibodies isolated and tested. The anti-alpha-bungarotoxin monoclonal antibodies consisted of IgM, IgG1 or IgG2a antibodies. In an in vitro neutralization assay measuring the effect of the antibodies on the binding of iodinated alpha-bungarotoxin to BC3H1 and TE671 (mouse and human cell lines bearing acetylcholine receptor), three of the six monoclonal antibodies were able to neutralize toxin binding. These studies demonstrate the feasibility of using native toxin for the generation of hybridomas, and the potential of using in vitro neutralization assays to screen hybridomas for in vivo neutralization.

Anti-ligand antibodies as potential autoantigens: in vitro and in vivo characteristics of anti-bungarotoxin antibodies in the idiotype network.

Some antibodies to ligands of a receptor will have combining sites that structurally resemble the receptors binding site for that ligand. The network hypothesis predicts that anti-idiotypic antibodies to these anti-ligand antibodies will also bind to the receptor. We pursued these hypotheses in the well-defined ligand-receptor system, alpha-bungarotoxin(BTX)-acetylcholine receptor (AChR). Monoclonal antibodies (mAbs) to BTX were generated; native BTX was used as the immunogen to optimize the probability of obtaining mAbs to the AChR binding site. These mAbs were then characterized for their ability to mimic AChR in the following in vitro assays: neutralization of BTX binding to native AChR on the surface of the cell line TE671, formation of a ternary complex with 125BTX-AChR, and ability of cholinergic ligands to interfere with binding to BTX. Three aBTX mAbs which had in vitro attributes of the AChR on the basis of these assays, were injected into C3H mice and serial sera tested for antibodies to Torpedo and murine AChR. Anti-AChR antibodies directed primarily to the gamma and delta subunits of the Torpedo AChR were detected, as well as low amounts of anti-mouse AChR antibody. The generation of anti-AChR antibodies by immunization with aBTX antibodies supports the network hypothesis and provides a theoretical basis for initiation of autoimmunity to cell receptors.