Clinical trials of helminth therapy in autoimmune diseases: rationale and findings.

Some helminths are major human pathogens. Recently, however, increased understanding of the immunoregulatory responses induced by this class of parasites, in combination with epidemiologic and animal studies, suggests that helminths may have therapeutic potential in autoimmune diseases (AD) and other conditions. This article reviews the rationale for and results of clinical trials to test the safety and efficacy of helminth therapy in AD. Also discussed are future prospects for investigation and the possibility that helminth treatment may serve as a probe to help reveal the pathogenesis of AD.

Helminth therapy and multiple sclerosis.

Multiple sclerosis is a common and frequently disabling neurological disease of young adults. It is characterised by recurrent areas of focal inflammation (plaques) in the CNS which give rise to episodic neurological signs and symptoms. According to the hygiene (microbial deprivation) hypothesis, evolutionarily abnormal high levels of sanitation in the environment of the developed world may contribute to disordered immunoregulation in this and other putative autoimmune disorders. Helminths have been shown to augment immunoregulation. On this basis, the possibility of treating multiple sclerosis with live helminths or helminth products has been explored in animal models, natural human infections and phase 1 clinical trials. To date helminth therapy appears safe and preliminary clinical, magnetic resonance imaging and immunological outcomes have generally been favourable. Nevertheless, serious adverse effects are always possible, particularly with live parasitic administration. Follow up studies with safety monitoring, regulatory oversight and objective outcome measures will be required to definitively assess safety and efficacy for this novel class of potential immunological therapies in multiple sclerosis.

Probiotic helminth administration in relapsing-remitting multiple sclerosis: a phase 1 study.

BACKGROUND: Probiotic treatment strategy based on the hygiene hypothesis, such as administration of ova from the non-pathogenic helminth, Trichuris suis, (TSO) has proven safe and effective in autoimmune inflammatory bowel disease. OBJECTIVE: To study the safety and effects of TSO in a second autoimmune disease, multiple sclerosis (MS), we conducted the phase 1 Helminth-induced Immunomodulatory Therapy (HINT 1) study. METHODS: Five subjects with newly diagnosed, treatment-naive relapsing-remitting multiple sclerosis (RRMS) were given 2500 TSO orally every 2 weeks for 3 months in a baseline versus treatment control exploratory trial. RESULTS: The mean number of new gadolinium-enhancing magnetic resonance imaging (MRI) lesions (n-Gd+) fell from 6.6 at baseline to 2.0 at the end of TSO administration, and 2 months after TSO was discontinued, the mean number of n-Gd+ rose to 5.8. No significant adverse effects were observed. In preliminary immunological investigations, increases in the serum level of the cytokines IL-4 and IL-10 were noted in four of the five subjects. CONCLUSION: TSO was well tolerated in the first human study of this novel probiotic in RRMS, and favorable trends were observed in exploratory MRI and immunological assessments. Further investigations will be required to fully explore the safety, effects, and mechanism of action of this immunomodulatory treatment.

A consensus protocol for the standardization of cerebrospinal fluid collection and biobanking.

There is a long history of research into body fluid biomarkers in neurodegenerative and neuroinflammatory diseases. However, only a few biomarkers in CSF are being used in clinical practice. One of the most critical factors in CSF biomarker research is the inadequate powering of studies because of the lack of sufficient samples that can be obtained in single-center studies. Therefore, collaboration between investigators is needed to establish large biobanks of well-defined samples. Standardized protocols for biobanking are a prerequisite to ensure that the statistical power gained by increasing the numbers of CSF samples is not compromised by preanalytical factors. Here, a consensus report on recommendations for CSF collection and biobanking is presented, formed by the BioMS-eu network for CSF biomarker research in multiple sclerosis. We focus on CSF collection procedures, preanalytical factors, and high-quality clinical and paraclinical information. The biobanking protocols are applicable for CSF biobanks for research targeting any neurologic disease.

Contributions of CD8+ T cells and viral spread to demyelinating disease.

Acute and chronic demyelination are hallmarks of CNS infection by the neurotropic JHM strain of mouse hepatitis virus. Although infectious virus is cleared by CD8+ T cells, both viral RNA and activated CD8+ T cells remain in the CNS during persistence potentially contributing to pathology. To dissociate immune from virus-mediated determinants initiating and maintaining demyelinating disease, mice were infected with two attenuated viral variants differing in a hypervariable region of the spike protein. Despite similar viral replication and tropism, one infection was marked by extensive demyelination and paralysis, whereas the other resulted in no clinical symptoms and minimal neuropathology. Mononuclear cells from either infected brain exhibited virus specific ex vivo cytolytic activity, which was rapidly lost during viral clearance. As revealed by class I tetramer technology the paralytic variant was superior in inducing specific CD8+ T cells during the acute disease. However, after infectious virus was cleared, twice as many virus-specific IFN-gamma-secreting CD8+ T cells were recovered from the brains of asymptomatic mice compared with mice undergoing demyelination, suggesting that IFN-gamma ameliorates rather than perpetuates JHM strain of mouse hepatitis virus-induced demyelination. The present data thus indicate that in immunocompetent mice, effector CD8+ T cells control infection without mediating either clinical disease or demyelination. In contrast, demyelination correlated with early and sustained infection of the spinal cord. Rapid viral spread, attributed to determinants within the spike protein and possibly perpetuated by suboptimal CD8+ T cell effector function, thus ultimately leads to the process of immune-mediated demyelination.

Quantitative sense-specific determination of murine coronavirus RNA by reverse transcription polymerase chain reaction.

In many applications, it is useful to know the sense and amount of viral RNAs present in a sample. In theory, sense-specific measurement of viral RNAs may be achieved by reverse transcription polymerase chain reaction (RT-PCR) assays which utilize primers of defined polarity during the RT step. However, in practice, it has been shown that such assays are prone to artifacts, such as non-specific priming, which drastically diminish their reliability. Using murine coronavirus MHV-4 as a model, we describe and validate several modifications of the RT-PCR procedure which eliminate these artifacts. Key RT-PCR parameters which were optimized include the design of tagged primers, DNase treatment of in vitro transcribed RNA standards, specification of temperature differences between RT and PCR annealing steps, and use of competitive RNA templates for quantitative assays. The assays described may be used to determine the sense and abundance of any viral or host RNA of interest in complex biological specimens.

Quasispecies development by high frequency RNA recombination during MHV persistence.

Recent studies suggest that infectious viruses and particularly persisting viral RNAs often exist as diverse populations or "quasispecies". We have developed an approach to characterize populations of the murine coronavirus mouse hepatitis virus (MHV) generated during persistent infection which has allowed us to begin to address the role of the viral quasispecies in MHV pathogenesis. We analyzed the population of persisting viral RNAs using reverse-transcription polymerase chain reaction amplification (RT-PCR) of the S1 "hypervariable" region of the spike gene followed by differential colony hybridization to identify spike deletion variants (SDVs) from acute and persistently infected mice. Sequence analysis revealed that mice with the most severe chronic paralysis harbored the most complex quasispecies. Mapping of the SDVs to the predicted RNA secondary structure of the spike RNA revealed that an isolated stem loop structure is frequently deleted. Overall, these results are consistent with high frequency recombination at sites of RNA secondary structure contributing to expansion of the viral quasispecies and persisting viral pathogenesis.

Generation of coronavirus spike deletion variants by high-frequency recombination at regions of predicted RNA secondary structure.

Coronavirus RNA evolves in the central nervous systems (CNS) of mice during persistent infection. This evolution can be monitored by detection of a viral quasispecies of spike deletion variants (SDVs) (C. L. Rowe, S. C. Baker, M. J. Nathan, and J. O. Fleming, J. Virol. 71:2959-2969, 1997). We and others have found that the deletions cluster in the region from 1,200 to 1,800 nucleotides from the 5' end of the spike gene sequence, termed the "hypervariable" region. To address how SDVs might arise, we generated the predicted folding structures of the positive- and negative-strand senses of the entire 4,139-nt spike RNA sequence. We found that a prominent, isolated stem-loop structure is coincident with the hypervariable region in each structure. To determine if this predicted stem-loop is a "hot spot" for RNA recombination, we assessed whether this region of the spike is more frequently deleted than three other selected regions of the spike sequence in a population of viral sequences isolated from the CNS of acutely and persistently infected mice. Using differential colony hybridization of cloned spike reverse transcription-PCR products, we detected SDVs in which the hot spot was deleted but did not detect SDVs in which other regions of the spike sequence were exclusively deleted. Furthermore, sequence analysis and mapping of the crossover sites of 25 distinct patterns of SDVs showed that the majority of crossover sites clustered to two regions at the base of the isolated stem-loop, which we designated as high-frequency recombination sites 1 and 2. Interestingly, the majority of the left and right crossover sites of the SDVs were directly across from or proximal to one another, suggesting that these SDVs are likely generated by intramolecular recombination. Overall, our results are consistent with there being an important role for the spike RNA secondary structure as a contributing factor in the generation of SDVs during persistent infection.

Evolution of mouse hepatitis virus: detection and characterization of spike deletion variants during persistent infection.

High-frequency RNA recombination has been proposed as an important mechanism for generating viral deletion variants of murine coronavirus. Indeed, a number of variants with deletions in the spike glycoprotein have been isolated from persistently infected animals. However, the significance of generating and potentially accumulating deletion variants in the persisting viral RNA population is unclear. To study this issue, we evaluated the evolution of spike variants by examining the population of spike RNA sequences detected in the brains and spinal cords of mice inoculated with coronavirus and sacrificed at 4, 42, or 100 days postinoculation. We focused on the S1 hypervariable region since previous investigators had shown that this region is subject to recombination and deletion. RNA isolated from the brains or spinal cords of infected mice was rescued by reverse transcription-PCR, and the amplified products were cloned and used in differential colony hybridizations to identify individual isolates with deletions. We found that 11 of 20 persistently infected mice harbored spike deletion variants (SDVs), indicating that deletions are common but not required for persistent infection. To determine if a specific type of SDV accumulated during persistence, we sequenced 106 of the deletion isolates. We identified 23 distinct patterns of SDVs, including 5 double-deletion variants. Furthermore, we found that each mouse harbored distinct variants in its central nervous system (CNS), suggesting that SDVs are generated during viral replication in the CNS. Interestingly, mice with the most severe and persisting neurological disease harbored the most prevalent and diverse quasispecies of SDVs. Overall, these findings illustrate the complexity of the population of persisting viral RNAs which may contribute to chronic disease.

Pathogenesis of mouse hepatitis virus-induced demyelination.

Infection of rodents with neurotropic mouse hepatitis virus (MHV) may result in lethal encephalitis or paralytic demyelinating disease resembling the human disease multiple sclerosis. The outcome of MHV infection is dependent on a number of variables, including the passage history of the viral isolate, dose and route of inoculation, and the age and immune status of the host. Alterations in surface glycoproteins, especially the spike protein, can profoundly influence pathogenesis. Innate resistance to MHV infection may be related to the expression of cellular receptors or to immunological factors. The immune system plays a major role in MHV pathogenesis, affecting encephalitis, viral clearance, and demyelination. Antiviral antibodies, CD4+ T lymphocytes, or CD8+ T lymphocytes may protect infected animals from lethal encephalitis, but both CD4+ and CD8+ T lymphocytes are required for effective viral clearance. Demyelination in MHV-infected animals has been attributed to the cytolytic effects of viral infection on myelin-producing oligodendrocytes, but more recent evidence supports an immunopathological mechanism for demyelination. Immunopathological models for demyelination include autoimmunity, direct immune cytotoxicity, and indirect 'bystander' damage. Although evidence exists supporting all of these models, the authors favor the bystander demyelination model. Much remains to be revealed about the processes leading to demyelination in MHV-infected mice, and information gained from these investigations may aid in the study of demyelinating disease in humans.

Dissociation of demyelination and viral clearance in congenitally immunodeficient mice infected with murine coronavirus JHM.

Infection of rodents with murine coronavirus JHM results in a subacute or chronic demyelinating disease which serves as a model for the human disease multiple sclerosis. Previous studies with JHMV have established a role for the immune system in both viral clearance and demyelination. To further clarify the role of the immune system in JHMV pathogenesis, several strains of congenitally immunodeficient mice were studied. Infection of immunocompetent C57BL/6 mice with JHMV resulted in severe paralysis and demyelination and complete clearance of infectious virus from the brain (C+D+ phenotype). In contrast, infected SCID mice showed little or no paralysis or demyelination and were unable to clear infectious virus (C-D- phenotype). Athymic nude mice and a proportion of mice lacking MHC Class I or II expression exhibited robust demyelination but did not completely clear infectious virus from the brain (C-D+ phenotype). These results are consistent with an immune-mediated mechanism for JHMV-induced demyelination, but indicate that the immune mechanisms which participate in demyelination and viral clearance are distinct. It may thus be possible to experimentally alter immunopathological responses without impairing antimicrobial immunity.

Immunotherapy in multiple sclerosis, Part 2.

The efficacies of corticosteroids and azathioprine (part 1) and of cyclophosphamide, immune globulin, cyclosporine, interferons, copolymer 1, and cladribine (part 2) in patients with multiple sclerosis (MS) are reviewed. MS is an inflammatory, demyelinating disease of the CNS that commonly affects young adults. The involvement of various immune mechanisms in MS suggests a role for immunomodulating therapy. The goals of immunotherapy vary with the clinical stage of the disease and include (1) improving recovery from exacerbations, (2) decreasing the number or severity of relapses, (3) preventing the development of chronic progressive disease from a relapsing-remitting course, and (4) decreasing further progression in patients with chronic progressive disease. In clinical trials, corticotropin and corticosteroids have been found to accelerate recovery from exacerbations. Tapering is often effective after high-dose induction therapy. Long-term maintenance regimens do not alter disease progression and are not recommended. Azathioprine produces modest benefits with respect to relapse rates and disease progression after two or more years of treatment; adverse effects are mild to moderate. Azathioprine should not be used in patients with aggressive disease who may approach severe disability in 6-18 months. Cyclophosphamide, because of its modest impact on disease progression and its potentially severe adverse effects, including cancer, should be reserved for patients with aggressive relapsing-remitting or chronic progressive disease in whom other treatments have failed to work; maintenance therapy is necessary after induction. Intravenous immune globulin may benefit patients with severe relapses; however, its efficacy remains unproven. Cyclosporine also cannot be recommended because of its modest efficacy, marked adverse effects, and high cost. Interferon beta-1b is a more specific immunotherapy that has been found to decrease the number and severity of relapses. This treatment should be considered in patients with relapsing-remitting disease who are having two or more exacerbations per year. Copolymer 1 and cladribine have shown some promising early results. Although various immunotherapeutic drugs can provide relief in patients with MS, none is capable of reversing disease progression, and some can cause serious adverse effects. Better understanding of the immunologic basis of MS may lead to more specific immunotherapies with more lasting benefits.

Immunotherapy in multiple sclerosis, Part 1.

The efficacies of corticosteroids and azathioprine (part 1) and of cyclophosphamide, immune globulin, cyclosporine, interferons, copolymer 1, and cladribine (part 2) in patients with multiple sclerosis (MS) are reviewed. MS is an inflammatory, demyelinating disease of the CNS that commonly affects young adults. The involvement of various immune mechanisms in MS suggests a role for immunomodulating therapy. The goals of immunotherapy vary with the clinical stage of the disease and include (1) improving recovery from exacerbations, (2) decreasing the number or severity of relapses, (3) preventing the development of chronic progressive disease from a relapsing-remitting course, and (4) decreasing further progression in patients with chronic progressive disease. In clinical trials, corticotropin and corticosteroids have been found to accelerate recovery from exacerbations. Tapering is often effective after high-dose induction therapy. Long-term maintenance regimens do not alter disease progression and are not recommended. Azathioprine produces modest benefits with respect to relapse rates and disease progression after two or more years of treatment; adverse effects are mild to moderate. Azathioprine should not be used in patients with aggressive disease who may approach severe disability in 6-18 months. Cyclophosphamide, because of its modest impact on disease progression and its potentially severe adverse effects, including cancer, should be reserved for patients with aggressive relapsing-remitting or chronic progressive disease in whom other treatments have failed to work; maintenance therapy is necessary after induction. Intravenous immune globulin may benefit patients with severe relapses; however, its efficacy remains unproven. Cyclosporine also cannot be recommended because of its modest efficacy, marked adverse effects, and high cost. Interferon beta-1b is a more specific immunotherapy that has been found to decrease the number and severity of relapses. This treatment should be considered in patients with relapsing-remitting disease who are having two or more exacerbations per year. Copolymer 1 and cladribine have shown some promising early results. Although various immunotherapeutic drugs can provide relief in patients with MS, none is capable of reversing disease progression, and some can cause serious adverse effects. Better understanding of the immunologic basis of MS may lead to more specific immunotherapies with more lasting benefits.

Evolution of mouse hepatitis virus (MHV) during chronic infection: quasispecies nature of the persisting MHV RNA.

Coronavirus infection of mice has been used extensively as a model for the study of acute encephalitis and chronic demyelination. To examine the evolution of coronavirus RNA during chronic demyelinating infection, we isolated RNA from intracerebrally inoculated mice at 4, 6, 8, 13, 20, and 42 days postinfection and used reverse transcription-polymerase chain reaction amplification methods (RT-PCR) to detect viral sequences. RNA sequences from two viral structural genes, the spike gene and the nucleocapsid gene, were detected throughout the chronic infection. In contrast, infectious virus was not detectable from brain homongenates beyond 13 days postinfection. These results indicate that coronavirus RNA persists in the brain at times when infectious virus is not detected. To determine if genetic changes were occurring during viral replication in the host, we cloned and sequenced the RT-PCR products from the spike and nucleocapsid regions and analyzed the sequences for mutations. Sequencing of the cloned products revealed that a variety of mutant forms of viral RNA persisted in the CNS, including point mutants, deletion mutants, and termination mutants. The mutations accumulated during persistent infection in both the spike and the nucleocapsid sequences, with greater than 65% of the mutations encoding amino acid changes. These results show that a diverse population or quasispecies consisting of mutant and deletion variant viral RNAs (which may not be capable of producing infectious virus particles) persists in the central nervous system of mice during chronic demyelinating infection. The implications of these results for the role of persistent viral genetic information in the pathogenesis of chronic demyelination are discussed.

Demyelination induced by murine coronavirus JHM infection of congenitally immunodeficient mice.

Mouse hepatitis virus JHM (JHMV or MHV-4) induces demyelination in rodents and has been studied as a model for the human disease, multiple sclerosis (MS). As is proposed in MS, the mechanism of subacute demyelination induced by JHMV appears to be primarily immunopathological, since demyelination in JHMV-infected mice is abrogated by immunosuppressive doses of irradiation and restored by adoptive transfer of splenocytes. Thy-1+ cells play a critical role in transmitting disease to these recipient mice. To further characterize cells which may mediate JHMV-induced immunopathology, we inoculated congenitally immunodeficient mice with JHMV. By 12 days post-inoculation, both immunocompetent C57BL/6J controls and athymic nude C57BL/6 mice had severe paralysis and demyelination. In marked contrast, C57BL/6 mice with the severe combined immune deficiency (SCID) mutation had little or no paralysis or demyelination. Adoptive transfer of immune spleen cells from nude mice to infected SCID mice produced paralysis and demyelination. These findings suggest that a cell population present in immunocompetent C57BL/6J and nude mice but absent or non-functional in irradiated and SCID mice is essential for JHMV-induced demyelination. Identification of cells which mediate demyelination in this experimental system may have implications for our understanding of coronavirus pathogenesis and human demyelinating diseases.

Mutations associated with viral sequences isolated from mice persistently infected with MHV-JHM.

Mouse hepatitis virus JHM (JHMV or MHV-4) induces subacute and chronic demyelination in rodents and has been studied as a model human demyelinating diseases, such a multiple sclerosis. However, despite intensive investigation, the state of JHMV during chronic disease is poorly understood. Using reverse transcription-polymerase chain reaction amplification (RT-PCR) to "rescue" viral RNA, we have found that JHMV-specific sequences persist for at least 787 days after intracerebral inoculation of experimental mice. Analysis of persisting viral RNA reveals that it is extensively mutated, and we hypothesize that the mutations observed reflect adaptation of the viral quasispecies to low-level intracellular replication during chronic disease.

Identification of an immunodominant linear neutralization domain on the S2 portion of the murine coronavirus spike glycoprotein and evidence that it forms part of complex tridimensional structure.

Numerous studies have demonstrated that the spike glycoprotein of coronaviruses bears major determinants of pathogenesis. To elucidate the antigenic structure of the protein, a panel of monoclonal antibodies was studied by competitive ELISA, and their reactivities were assayed against fragments of the murine coronavirus murine hepatitis virus strain A59 S gene expressed in prokaryotic vectors. An immunodominant linear domain was localized within the predicted stalk, S2, of the peplomer. It is recognized by several neutralizing antibodies. Other domains were also identified near the proteolytic cleavage site, in the predicted globular head, S1, and in another part of the stalk. Furthermore, competition results suggest that the immunodominant functional domain forms part of a complex three-dimensional structure. Surprisingly, some antibodies which have no antiviral biological activities were shown to bind the immunodominant neutralization domain.

Interaction of immune and central nervous systems: contribution of anti-viral Thy-1+ cells to demyelination induced by coronavirus JHM.

The murine coronavirus JHM (JHMV or MHV-4) has been intensively studied as an experimental model of viral-induced demyelination; nonetheless, the degree to which demyelination results from direct viral cytolysis of oligodendroglia or immunological mechanisms remains controversial. To examine the contribution of immunity to the pathogenesis of JHMV in the central nervous system (CNS), mice were exposed to immunosuppressive doses of x-irradiation 3 days post infection and observed for clinical and pathological evidence of acute and subacute demyelination. Irradiated mice were found to have a nearly thousand-fold increase in central nervous system virus titer, as well as the presence of both abundant virus and viral antigen in white matter cells with the morphological characteristics of oligodendrocytes. Nonetheless, infected, irradiated mice had little or no evidence of demyelination or destruction of CNS cells. Adoptive transfers of spleen cells from syngeneic JHMV-immunized donors into irradiated JHMV-infected mice were carried out in order to determine the effect of immune reconstitution on pathogenesis. Splenocytes from JHMV-immune donors, but not naive donors or donors immunized with irrelevant antigen, completely restored demyelination in irradiated, JHMV-infected recipients. Depletion of Thy-1+ cells by treatment with monoclonal antibody and complement abolished the ability to transfer demyelination. We conclude that: 1) JHMV infection of the CNS does not result in acute or subacute demyelination in the absence of an intact immune response, and 2) viral-specific Thy-1+ cells are an essential element in the induction of demyelinating CNS lesions that result from JHMV infection.