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.

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.

Experimental neuropathology of chronic demyelination induced by a JHM virus variant (DS).

A small plaque variant of JHM virus has a markedly reduced ability to kill mice following intracerebral inoculation. Spinal cords of mice surviving 13 to 16 months following acute infection with this variant were examined ultrastructurally. Multiple subpial areas of demyelination in the anterior and lateral white matter were found in five of 13 mice. The lesions had more gliosis, fewer oligodendrocytes, and less remyelination than has been described following other infections with JHM virus. No conclusive evidence of active demyelination or viral-like particles was found. The pathogenesis of the lesions observed may be due to a persistent, attenuated infection of oligodendrocytes or to immunologic processes. These lesions were similar to chronic multiple sclerosis plaques. Therefore, this variant should prove to be a useful tool for studying the long-term effects of viral-induced demyelinating diseases.

Antigenic assessment of coronaviruses isolated from patients with multiple sclerosis.

Many studies have either supported or discounted the role of coronaviruses as etiologic agents in multiple sclerosis (MS). Two new approaches were applied to investigate this controversy. First, monoclonal antibodies specific for either murine coronaviruses (mouse hepatitis viruses) or human coronaviruses were used to characterize the antigenic features of MS-derived coronaviruses SK and SD. Both isolates were found to have a mouse hepatitis virus-type profile. Second, serum and cerebrospinal fluid antibodies to different coronaviruses, including SD, were measured in MS and control groups. No significant difference in antibody level to coronaviruses was found between MS and control samples. The results of these antigenic studies do not support a specific association between MS and coronaviruses.

Multiple sclerosis: measurement and validation of central nervous system IgG synthesis rate.

An empirical formula has been developed to calculate the de novo rate of synthesis of IgG in the central nervous system (CNS), based on physiologic principles that govern the passage of albumin and IgG across the blood-brain barrier (BBB). To validate the formula, radiolabeled IgG and albumin from pooled normal sera were followed from the blood to the cerebrospinal fluid (CSF) over 21 days in nine patients with definite multiple sclerosis (MS). IgG synthesis rates were calculated by the isotope exchange method and compared to values obtained with the empirical formula. There was excellent concordance, from a low rate of synthesis of 5 mg per day to a high rate of 120 mg per day. A double radiolabeled IgG experiment in two patients showed that the MS BBB processed normal serum IgG in the same way as IgG derived from autologous MS serum. Accordingly, the empirical formula, which requires only one sample of CSF and matched serum, can reliably and validly estimate the de novo rate of IgG synthesis in CNS of patients with MS.

Measurement of the concentration of murine IgG monoclonal antibody in hybridoma supernatants and ascites in absolute units by sensitive and reliable enzyme-linked immunosorbent assays (ELISA).

We have investigated an enzyme-linked immunosorbent assay (ELISA) for mouse IgG using affinity-purified goat anti-mouse antibodies for capture and detection. This assay was used to measure the absolute or weight/volume concentration of murine monoclonal antibody in hybridoma supernatants. Bovine or subclasses except IgG3 in the 1-20 ng/ml range. Antibody capture was essentially complete in the optimized assay. In combination with an antigen-dependent ELISA, the assay allowed estimation of the absolute concentration of specific monoclonal antibody in ascites. These rapid and relatively simple assays may be applicable in many situations in which a practical means of measuring murine monoclonal antibodies in weight/volume units is needed.

Demyelination induced by murine hepatitis virus JHM strain (MHV-4) is immunologically mediated.

The neurotropic mouse hepatitis viruses (MHV), in particular strain JHM (JHMV or MHV-4), cause experimental central nervous system demyelination that pathologically resembles multiple sclerosis, an important human demyelinating disease. The mechanism of JHMV-induced demyelination remains unclear, though its tropism for oligodendrocytes had led to the belief that JHMV causes demyelination by direct lysis of these myelin-producing cells. However, several studies have also implicated the involvement of immune responses in the demyelinating process. In this communication, we present evidence that generalized immunosuppression with gamma irradiation prevents JHMV-induced demyelination, a finding that was not limited to a particular strain of JHMV or to one strain of mouse. In addition, significant paralytic-demyelinating disease was restored to infected, irradiated mice after the adoptive transfer of nylon wool nonadherent splenic cells and appeared to be restricted by the major histocompatibility complex (MHC). These observations indicate that the principal mechanisms of JHMV-induced demyelination are most likely immunopathological.

Improvements in obtaining and characterizing mouse cerebrospinal fluid. Application to mouse hepatitis virus-induced encephalomyelitis.

This report describes advances in techniques for analyzing cellular and humoral immune components in the cerebrospinal fluid (CSF) of the mouse that are applicable to other laboratory animals. CSF studies undertaken during experimental infection of mice with JHM strain virus (JHMV) of mouse hepatitis virus are presented. A critical pitfall which can lead to erroneous or invalid results is contamination of the CSF by even minute quantities of blood. Means of avoiding this contamination are attention to anatomical reference points, the use of a micropipet, and prior intracardiac perfusion of animals with phosphate-buffered saline. Cells in the CSF were typed as either B, T, polymorphonuclear, or mononuclear cells by the combination of a microcytotoxicity assay and histologic stains. A radioimmunoassay (RIA) allowed quantification of antibodies to JHMV in the CSF and indicated the presence of intrathecal synthesis of antibody in chronically infected mice. The combined use of these sensitive methods makes possible CSF analysis in individual mice rather than in pooled groups.

Synthesis of immunoglobulin within the central nervous system in multiple sclerosis and other neurological diseases. Detection by analysis of CSF/serum IgG ratio.

This study included 49 patients with clinical multiple sclerosis (MS), 105 patients with other neurological diseases (OND), and 30 controls. It compared seven assays for CNS IgG synthesis with the oligoclonal banding (agarose electrophoresis) method. A newly developed assay which determined the differences between the measured and calculated CSF/serum IgG ratio (M-C value), using albumin as a reference protein, was particularly sensitive to the diagnosis of MS. In 40/46 (87%) of patients with MS, the M-C value was 0.001 or more, while oligoclonal banding was found in CSF of 38/49 (78%). In the 30 controls, the M-C value was invariably less than 0.001 and oligoclonal banding was not found. In patients with OND, 26/104 (25%) had an M-C value of 0.001 of greater while 11/105 (11%) had oligoclonal banding in CSF. The M-C value also offers a convenient means of quantifying CNS IgG synthesis during disease activity of treatment. It is concluded that the combined use of the oligoclonal banding method and the M-C value determination gives the greatest predictive value for the diagnosis of MS.

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.

Viral infections of the nervous system.

Neurotropic viruses cause a number of important infectious syndromes including encephalitis, myelitis, meningitis, and radiculopathy. In this review, the biology of conventional and unconventional viruses is examined. The host immune response to viruses is discussed, and patterns of viral pathogenesis are explained. The clinical features, laboratory findings, management of important viral infections, such as herpes simplex encephalitis and epidemic encephalitis, are presented. Post-infection syndromes, such as the Guillain-Barré syndrome, and chronic viral infections, such as those causing progressive multifocal leukoencephalopathy and subacute sclerosing panencephalitis, are discussed. Current knowledge concerning the nature of unconventional virus-like agents of the spongiform encephalopathies, including kuru and Creutzfeldt-Jakob disease, is summarized. Finally, viral infections of immunocompromised patients and the possible role of viruses in the newly described acquired immunodeficiency syndrome (AIDS) are examined.

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.

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.

RNA recombination of coronavirus.

We have previously shown that Mouse hepatitis virus (MHV) can undergo RNA-RNA recombination at a very high frequency (S. Makino, et al., J. Virol. 57, 729-737, 1986). To better define the mechanism of RNA recombination, we have performed additional crosses involving different MHV strains. We have obtained recombinant viruses with multiple cross-overs. The isolation of such recombinants further indicates the high frequency of coronavirus RNA recombination. By using cell fusion as a selection marker, we have also obtained recombinants between MHV-2 and A59 strains. Some of these recombinants have cross-overs in the 3'-end genes of the genome, thus demonstrating that recombination could occur along the entire genome. Finally, we have obtained recombinants by selecting with neutralizing monoclonal antibodies. These recombinants have cross-overs within gene C which encodes the peplomer protein. The genetic structure of these recombinants allowed us to determine the important domains of the peplomer proteins.

Persistence of viral RNA in the central nervous system of mice inoculated with MHV-4.

In order to study the role that viral persistence may play in chronic central nervous system (CNS) disease induced by murine coronaviruses, we have used the reverse transcriptase-polymerase chain reaction (RT-PCR) to study viral RNA in the brains of mice after intracerebral inoculation of JHM virus (JHMV or MHV-4). Quantitative RT-PCR showed that JHMV RNA decreased from approximately 2 ng/ug total brain RNA at day 6 post-inoculation (PI) to 0.1 pg/ug total brain RNA at 360 days PI. Double-stranded viral RNA could be detected up to day 20 PI. By the selective use of upstream or downstream primers during the RT step, it was possible to measure negative sense and positive sense JHMV RNA respectively, and we found that there was a marked rise in the ratio of positive to negative sense JHMV RNA after day 13 PI. Analysis of amplified products by dideoxy DNA sequencing showed that the characteristic mutation of our input virus (at position 3340 of gene 3) is maintained to at least day 42 PI. Taken together, these results favor a model of JHMV persistence in vivo in which viral RNA is present as double stranded forms initially and predominantly as single stranded, positive sense forms at late timepoints. Further analysis of this model in quantitative terms may contribute to our understanding of the biological significance of coronavirus persistence in the CNS.

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.

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.