Efficacy of treatment of MS with IFNbeta-1b or glatiramer acetate by monthly brain MRI in the BECOME study.

BACKGROUND: There are no published MRI studies comparing interferon beta 1b (IFNbeta-1b) and glatiramer acetate (GA) for treatment of relapsing multiple sclerosis (MS). OBJECTIVE: To compare the efficacy of IFNbeta-1b and GA for suppression of MS disease activity as evidenced on frequent brain MRI. METHODS: A total of 75 patients with relapsing-remitting MS or clinically isolated syndromes were randomized to standard doses of IFNbeta-1b or GA and followed by monthly brain MRI for up to 2 years with a protocol optimized to detect enhancement. The primary outcome was the number of combined active lesions (CAL) per patient per scan during the first year, which included all enhancing lesions and nonenhancing new T2/fluid-attenuated inversion recovery (FLAIR) lesions. Secondary outcomes were the number of new lesions and clinical exacerbations over 2 years. RESULTS: Baseline characteristics were similar between the groups. The primary outcome showed similar median (75th percentile) CAL per patient per scan for months 1-12, 0.63 (2.76) for IFNbeta-1b, and 0.58 (2.45) for GA (p = 0.58). There were no differences in new lesion or clinical relapses for 2 years. Only 4.4% of CAL on monthly MRI scans were nonenhancing new T2/FLAIR lesions. CONCLUSION: Patients with relapsing multiple sclerosis randomized to interferon beta 1b or glatiramer acetate showed similar MRI and clinical activity.

Humoral immune response associated with lyme borreliosis in nonhuman primates: analysis by immunoblotting and enzyme-linked immunosorbent assay with sonicates or recombinant proteins.

The immune response to Borrelia burgdorferi, the causative agent of Lyme disease, is complex. We studied the immunoglobulin M (IgM) and IgG antibody response to N40Br, a sensu stricto strain, in the rhesus macaque(nonhuman primate [NHP]) model of infection to identify the spirochetal protein targets of specific antibody. Antigens used in enzyme-linked immunosorbent assays were whole-cell sonicates of the spirochete and recombinant proteins of B. burgdorferi. Immunoblotting with a commercially available strip and subsequent quantitative densitometry of the bands were also used. Sera from four different groups of NHPs were used: immunocompetent, transiently immunosuppressed, extended immunosuppressed, and uninfected. In immunocompetent and transiently immunosuppressed NHPs, there was a strong IgM and IgG response. Major proteins for the early IgM response were P39 and P41 and recombinant BmpA and OspC. Major proteins for the later IgG response were P39, P41, P18, P60, P66, and recombinant BmpA and DbpA. There was no significant response in the NHPs to recombinant OspA or to Arp, a 37-kDa protein that elicits an antibody response during infection in mice. Most antibody responses, except for that to DbpA, were markedly diminished by prolonged dexamethasone treatment. This study supports the hypothesis that recombinant proteins may provide a useful adjunct to current diagnostic testing for Lyme borreliosis.

Central and peripheral nervous system infection, immunity, and inflammation in the NHP model of Lyme borreliosis.

The relationship between chronic infection, antispirochetal immunity, and inflammation is unknown in Lyme neuroborreliosis. In the nonhuman primate model of Lyme neuroborreliosis, we measured spirochetal density in the nervous system and other tissues by polymerase chain reaction and correlated these values to anti-Borrelia burgdorferi antibody in the serum and cerebrospinal fluid, and to inflammation in tissues. Despite substantial presence of Borrelia burgdorferi, the causative agent of Lyme borreliosis, in the central nervous system, only minor inflammation was present there, though skeletal and cardiac muscle, which contained similar levels of spirochete, were highly inflamed. Anti-Borrelia burgdoferi antibody was present in the cerebrospinal fluid but was not selectively concentrated. All infected animals developed anti-Borrelia burgdorferi antibody in the serum, but increased amplitude of antibody was not predictive of higher levels of infection. These data demonstrate that Lyme neuroborreliosis is a persistent infection, that spirochetal presence is a necessary but not sufficient condition for inflammation, and that antibody measured in serum may not predict the severity of infection.

Isogenic serotypes of Borrelia turicatae show different localization in the brain and skin of mice.

Mice with severe combined immunodeficiency (scid mice) and infected with the relapsing fever agent Borrelia turicatae develop manifestations that resemble those of disseminated Lyme disease. We have characterized two isogenic serotypes, A and B, which differ in their variable small proteins (Vsps) and disease manifestations. Serotype A but not serotype B was cultured from the brain during early infection, and serotype B caused more severe arthritis, myocarditis, and vestibular dysfunction than serotype A. Here we compared the localization and number of spirochetes and the severity of inflammation in scid mice, using immunostained and hematoxylin-and-eosin-stained coronal sections of decalcified heads. Spirochetes in the brain localized predominantly to the leptomeninges, and those in peripheral tissues localized mainly to the extracellular matrix. There were significantly more serotype A than B spirochetes in the leptomeninges and more serotype B than A spirochetes in the skin. The first tissue where spirochetes were observed outside the vasculature was the dura mater. Inflammation was more severe in the skin than in the brain. VspA, VspB, and the periplasmic flagellin protein were expressed in all tissues examined. These findings indicate that isogenic but antigenically distinct Borrelia serotypes can have marked differences in their localization in tissues.

Lyme borreliosis in rhesus macaques: effects of corticosteroids on spirochetal load and isotype switching of anti-borrelia burgdorferi antibody.

Experimental Borrelia burgdorferi infection of rhesus monkeys is an excellent model of Lyme disease and closely parallels the infection in humans. Little is known about the interaction of host immunity with the spirochete in patients with chronic infection. We hypothesized that rapid development of anti-B. burgdorferi antibody in immunocompetent nonhuman primates (NHPs) is the major determinant of the reduction of the spirochetal load in Lyme borreliosis. This hypothesis was tested by measurement of the spirochetal load by PCR in association with characterization of the anti-B. burgdorferi humoral immune response in immunocompetent NHPs versus that in corticosteroid-treated NHPs. Although anti-B. burgdorferi immunoglobulin G (IgG) antibody was effectively inhibited in dexamethasone (Dex)-treated NHPs, anti-B. burgdorferi IgM antibody levels continued to rise after the first month and reached levels in excess of IgM levels in immunocompetent NHPs. This vigorous production of anti-B. burgdorferi IgM antibodies was also studied in vitro by measurement of antibody produced by B. burgdorferi-stimulated peripheral blood mononuclear cells. Despite these high IgM antispirochetal antibodies in Dex-treated NHPs, spirochetal loads were much higher in these animals. These data indicate that Dex treatment results in interference with isotype switching in this model and provide evidence that anti-B. burgdorferi IgG antibody is much more effective than IgM antibody in decreasing the spirochetal load in infected animals.

The rhesus model of Lyme neuroborreliosis.

Similarity of pathology and disease progression make the non-human primate (NHP) model of Lyme neuroborreliosis appropriate and valuable. In the NHP model of Lyme neuroborreliosis, spirochetal density in the nervous system and other tissues has been measured by polymerase chain reaction and correlated to anti-Borrelia burgdorferi antibody in the serum and cerebrospinal fluid and to inflammation in tissues. Despite the demonstrable presence of Borrelia burgdorferi, the causative agent of Lyme borreliosis, only minor inflammation of the central nervous system occurs, though inflammation can be demonstrated in other tissues. Infected animals also develop anti-Borrelia burgdorferi antibody in the serum, although increased amplitude of antibody is not predictive of higher levels of infection. The NHP model continues to provide important insight into the disease process in humans.

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.

Spirochetal infection of the nervous system.

Neurologic infection is a characteristic feature of spirochetes. The neurologic manifestations of spirochetal infection are a source of continuing public concern: Lyme neuroborreliosis in endemic areas, neurosyphilis in HIV infected patients, and neuroborreliosis during outbreaks of relapsing fever. These are reviewed in this article. The techniques for diagnosis and recommendations in the management of these infections are also discussed.

Protection from Lyme neuroborreliosis in nonhuman primates with a multiantigenic vaccine.

In an effort to develop an effective and safe vaccine for lyme disease, rhesus macaques were injected with a multiantigenic preparation of Borrelia burgdorferi, strain N40. One month later animals were boosted before intradermal challenge with infectious B. burgdorferi. Challenges were performed at 1 and again at 5 months after the booster vaccination. Vaccinated and control nonvaccinated animals were monitored for development of systemic infection by measurement of serum anti-spirochetal antibodies by ELISA and Western blotting, and neurological involvement was monitored by testing of cerebrospinal fluid (CSF) and PCR analysis of central nervous system (CNS) tissue obtained at necropsy. Two control nonhuman primates (NHPs), given saline injections instead of vaccine and then challenged with B. burgdorferi, developed CSF pleocytosis, PCR positivity of the brain, and high levels of specific anti-B. burgdorferi antibody in the serum and CSF. Necropsy studies revealed widespread invasion of the CNS of one of these animals by the spirochete. In contrast, none of the four vaccinated animals developed evidence of invasion of the CNS after either of two challenge inoculations with infectious B. burgdorferi. In addition to resisting infection, no vaccinated animal demonstrated any untoward consequence of vaccination. These data demonstrate that a multiantigenic vaccine is effective in preventing systemic infection and lyme neuroboreliosis in NHPs and suggest that a successful vaccine could be developed in humans which would prevent lyme disease.

Isolation of DNA after extraction of RNA To detect the presence of Borrelia burgdorferi and expression of host cellular genes from the same tissue sample.

We are investigating the neuropathogenesis of Lyme disease caused by Borrelia burgdorferi in a nonhuman primate model. In the past, two separate pieces of tissue had to be used when both analyzing for the presence of the spirochete and examining the host response to infection. We have modified a procedure to purify DNA from the same sample after the extraction of RNA. The remaining material containing the DNA was precipitated, and residual organic reagent was removed prior to deproteinization and extraction of the DNA. This procedure now allows us to both assay for the presence of the Lyme microorganism and analyze the host response in the same tissue preparation.

Detection of active infection in nonhuman primates with Lyme neuroborreliosis: comparison of PCR, culture, and a bioassay.

Ideally a diagnosis of infection of the central nervous system (CNS) is made by culture of the etiologic pathogen, but Borrelia burgdorferi, the causative agent of Lyme neuroborreliosis (LNB), is rarely cultured from the cerebrospinal fluid (CSF). PCR and measurement of specific antibody in the CSF also have their limitations. The role of available assays for LNB has not been studied carefully in a comparative investigation. There is a need to assess the reliability of assays and to increase the ability to document active infection in the CNS. The recent development of the nonhuman primate (NHP) model of LNB allowed us to address this need in a faithful model of human LNB. In this study we compared the abilities of PCR and culture to detect the presence of spirochetes in the CSF and brain tissue of infected NHPs and related these measures of infection to the development of anti-B. burgdorferi antibody. We also tested a bioassay, the mouse infectivity test (MIT), in this model. Fourteen of 16 CSFs from four NHPs were positive by at least one of these techniques. Detection of spirochetes in the CSF by PCR, the MIT, and culture was inversely related to the concomitant presence of anti-B. burgdorferi antibody intrathecally. The performance of any particular test was associated with the strength of the host immune response. In early CNS infection, when anti-B. burgdorferi antibody had not yet appeared, or in immunocompromised hosts, the MIT compared favorably to culture and PCR for infected NHPs; antibody in the CSF was the most useful assay for immunocompetent NHPs.

Pathogenesis of neuroborreliosis--lessons from a monkey model.

The diagnosis of human LNB can be difficult, because its major clinical manifestations--meningitis, facial palsy, radiculitis, and neuritis--are non-specific and the characteristic skin lesion is usually absent at the time of neurological involvement. Thus, CSF assays are often used in diagnosis. Culture of CSF is rarely performed because it has a low yield and requires special culture medium. PCR of the CSF identified spirochetal DNA in clinical specimens with greater sensitivity, but it suffers from a number of disadvantages. Measurement of specific antibody in the CSF also has its limitations. The role of available assays for LNB has not been studied carefully in a comparative investigation. The recent development of the nonhumane primate (NHP) model of LNB allows us to address this need in a faithful model of human LNB. We compared PCR and culture in their ability to detect spirochetal presence in the CSF and brain tissue of infected NHPs, and related these measures of infection to the development of anti-B. burgdorferi antibody. We also tested a bioassay, the mouse infectivity test (MIT), in this model. Using these four assays (PCR, culture, MIT, and CSF Ab) at least one assay for spirochetal presence in CSFs from NHPs was positive in 87% of CSFs tested during early infection in the CNS. Detection of spirochetal presence by PCR, MIT, and culture in the CSF was inversely related to the concomitant presence of anti-B. burgdorferi antibody intrathecally. The performance of any particular test was associated with the strength of the host immune response. In early CNS infection, when anti-B. burgdorferi antibody had not yet appeared, or in immunocompromised hosts, the MIT compared favorably to culture and PCR in infected NHPs; antibody in the CSF was the most useful assay in immunocompetent NHPs. This is the first study demonstrating that a bioassay using inoculation of mice, the mouse infectivity test (MIT), has potential as a useful adjunct in the diagnosis of LNB. The MIT for LNB was modeled after the rabbit infectivity test or RIT, which is considered the "gold standard" for the diagnosis of the related CNS infection, neurosyphilis, and felt to be very sensitive and specific. The presence of specific anti-B. burgdorferi antibody in the CSF is the most widely used assay for Lyme neuroborreliosis. In the immunocompetent NHPs in our study it was a very successful assay for detection of CNS invasion. However, it is frequently false-negative, especially early in the course of the infection, or if there is transient immunosuppression. Transient suppression of the anti-B. burgdorferi immune response in the human could occur in instances of co-infection, i.e. simultaneous transmission via the tick of another pathogen other than B. burgdorferi. Thus, mild immunosuppression as accomplished in our NHPs with corticosteroids was designed to mimic conditions in the human host which allow B. burgdorferi in the natural state to gain a firm foothold in the central nervous system in the 10-15% of B. burgdorferi-infected patients who develop clinically symptomatic nervous system disease. This study is the first to compare utility of available diagnostic techniques in LNB in which necropsy proved presence of infection in the CNS. None of the assays was ideal for all conditions, and the utility of the assay was associated with the host immune status. The differences in the responses of immunocompromised and immunocompetent NHPs in this study were striking. In immunocompetent NHPs the window of opportunity for CNS invasion prior to the development of CSF antibody was brief, and the chance of detection of spirochete low by any of the three techniques used (i.e. culture, PCR, or MIT); in this group, measurement of CSF antibody was generally diagnostic. In immunocompromised NHPs, intrathecal antibody production was delayed, and this helpful diagnostic assay was false-negative; diagnosis required more labor-intensive assays such as PCR, culture, an

Interleukin-6 is expressed at high levels in the CNS in Lyme neuroborreliosis.

In patients with Lyme neuroborreliosis, inflammation and symptoms of fatigue and malaise occur out of proportion to the relatively low number of spirochetes present. Previous studies have identified interleukin-6 (IL-6) as a candidate molecule for amplification of CNS inflammation in this disease. We pursued this possibility by measuring cytokine gene expression by reverse-transcriptase polymerase chain reaction (RT-PCR) in the brain of rhesus macaques actively infected with Borrelia burgdorferi. Samples of brain tissue were screened for IL-6 and interferon gamma using RT-PCR-ELISA, a technique that uses RT-PCR, subsequent hybridization of the PCR product with a biotinylated probe, and capture and ELISA readout of hybridization product. The number of copies in positive samples was then quantitated using qRT-PCR-ELISA, in which wild-type cytokine cDNA competes with recombinant competitor DNA in the PCR. Elevated levels of IL-6 cDNA and, to a lesser extent, interferon gamma were detected in three of three nonhuman primates with persistent infection with B burgdorferi, whereas the brains of three uninfected animals and undetectable levels of gene expression of these cytokines. These data support the hypothesis that cytokines such as IL-6 are important amplification molecules for CNS inflammation in Lyme neuroborreliosis.

The immune response to infectious diseases of the central nervous system: a tenuous balance.

The outcome of CNS infection is dependent on both the organism and host. Acute infections are usually cleared with minimal residua. However, chronic infections of the CNS, such as HIV, PML, HSV, polio, Lyme neuroborreliosis, and neurosyphilis, are becoming increasingly recognized as a cause of severe neurological morbidity, and are poorly understood. We will need to learn more about the CNS as an immune compartment to increase our knowledge of these infections.

Neuroborreliosis in the nonhuman primate: Borrelia burgdorferi persists in the central nervous system.

Neurological involvement in Lyme disease is common, and is frequently difficult to diagnose and treat. Little is known about the fate of the causative spirochete Borrelia burgdorferi in the central nervous system (CNS). To determine the frequency of parenchymal infection and to determine localization of the organism, polymerase chain reaction/hybridization assays were performed in a newly described model of Lyme neuroborreliosis in nonhuman primates infected with B. burgdorferi. Polymerase chain reaction/hybridization of CNS tissues from 5 infected nonhuman primates was performed. Substantial amounts of B. burgdorferi DNA were detected in the CNS in all infected animals, with a predilection toward subtentorial structures. These data suggest that Lyme neuroborreliosis represents persistent infection with B. burgdorferi.

Early disseminated Lyme disease: Lyme meningitis.

Lyme meningitis is the direct result of invasion of the nervous system by Borrelia burgdorferi. Occurring within the first few months of infection, it initially presents as a chronic basilar meningitis. Much about the pathogenesis of Lyme meningitis has been learned from animal models, the best being the adult Rhesus macaque. Injection of these animals with a highly infective strain of B. burgdorferi has led to a very predictable course of events: erythema migrans within the first few weeks after injection, development of anti-B. burgdorferi antibody, detection of spirochetemia in weeks 3 and 4, and central nervous system (CNS) invasion within 1 month with cerebrospinal fluid (CSF) pleocytosis. In humans, facial palsy is the earliest clinical indicator. Headache and meningismus are symptoms of inflammation of the subarachnoid space. Severe fatigue and arthralgia are common extra-CNS symptoms. Culture is not generally useful for detecting or confirming Lyme meningitis. False-positive serologic tests may occur in patients with other infections, inflammatory processes, or malignancies. Immunoblotting will differentiate true-from false-positive antibody reactivity. Lack of a consistently positive serum antibody titer should make the diagnosis of Lyme meningitis suspect. Positive CSF antibody is almost universal in patients with Lyme meningitis. Polymerase chain reaction is a direct test that is highly specific and sensitive. The antibiotic treatment of choice is intravenous (i.v.) cephalosporins or penicillin for 2-3 weeks. If the clinical picture is anything less than absolutely classic, a lumbar puncture and Western blot of serum should be obtained in a seropositive patient before initiating intravenous antibiotic therapy. There is no role at this time for long-term (> 1 month) intravenous antibiotics. Nonsteroidal antiinflammatory agents can also be of benefit.