Neuroimmuno

Inflammatory Cortical Demyelination in Early Multiple Sclerosis

2011-12-08T00:42:00.000-08:00

Background

Cortical disease has emerged as a critical aspect of the pathogenesis of multiple sclerosis, being associated with disease progression and cognitive impairment. Most studies of cortical lesions have focused on autopsy findings in patients with long-standing, chronic, progressive multiple sclerosis, and the noninflammatory nature of these lesions has been emphasized. Magnetic resonance imaging studies indicate that cortical damage occurs early in the disease.

Methods

We evaluated the prevalence and character of demyelinating cortical lesions in patients with multiple sclerosis. Cortical tissues were obtained in passing during biopsy sampling of white-matter lesions. In most cases, biopsy was done with the use of stereotactic procedures to diagnose suspected tumors. Patients with sufficient cortex (138 of 563 patients screened) were evaluated for cortical demyelination. Using immunohistochemistry, we characterized cortical lesions with respect to demyelinating activity, inflammatory infiltrates, the presence of meningeal inflammation, and a topographic association between cortical demyelination and meningeal inflammation. Diagnoses were ascertained in a subgroup of 77 patients (56%) at the last follow-up visit (at a median of 3.5 years).

Results

Cortical demyelination was present in 53 patients (38%) (104 lesions and 222 tissue blocks) and was absent in 85 patients (121 tissue blocks). Twenty-five patients with cortical demyelination had definite multiple sclerosis (81% of 31 patients who underwent long-term follow-up), as did 33 patients without cortical demyelination (72% of 46 patients who underwent long-term follow-up). In representative tissues, 58 of 71 lesions (82%) showed CD3+ T-cell infiltrates, and 32 of 78 lesions (41%) showed macrophage-associated demyelination. Meningeal inflammation was topographically associated with cortical demyelination in patients who had sufficient meningeal tissue for study.

Conclusions

In this cohort of patients with early-stage multiple sclerosis, cortical demyelinating lesions were frequent, inflammatory, and strongly associated with meningeal inflammation. (Funded by the National Multiple Sclerosis Society and the National Institutes of Health.)

N Engl J Med 2011; 365:2188-2197 December 8, 2011

Immunologist Awarded Nobel Prize

2011-10-20T10:30:00.000-07:00

Ralph Steinman's discovery and continuing investigation of a "missing link" in the human immune system have changed the field, colleagues say. This year, his work also has earned him the Nobel Prize.

"He discovered this important cell type, dendritic cells," says Max Cooper at the University of Alabama at Birmingham, a fellow immunologist who has known Steinman and his work for more than 30 years. "Back at a time when no one really believed him, he pushed the idea very hard and backed it up by isolating this very small fraction of cells to show that they were important in activating our T‑ and B‑cells in immune responses."His digging them out, isolating them, characterizing them in a painstaking way really did change the way we view how microbes enter the body and get recognized and responded to. And that changed our view of the way we understand how immunity is kicked off."Steinman, the director of the Chris Browne Center for Immunology and Immune Diseases at The Rockefeller University, is practiced at describing the cells he studies."The dendritic cell is, so to speak, a missing link in the immune system," he says. "The immune system has a number of different cells that provide resistance to infection and are involved in other disease conditions like cancer, allergy, transplantation and autoimmunity. Immune cells are like musicians in a symphony, each very talented and specialized, but they need a conductor and composer, and that's what dendritic cells are.""The cells sit up at surfaces of our body, along airways and along our intestine and in our skin, ready to pick up infections that enter the body. If they find one, they pick it up to display to the immune system. The cells migrate in the body, and when they get to the immune organs—lymphoid tissues—they find the musicians, so to speak, and then orchestrate the immune response. They tell the immune cells to grow and to develop into functioning protective cells."In the 1970s, when Steinman started his research career, researchers knew about the "musician" cells and they knew about infections. But in their laboratories, they could not seem to energize the immune cells to react to the infections. A link was missing, some cell in the immune-system soup that flipped the immune system cells on, and on in the right direction. They called the cells they were looking for "accessory cells."Steinman was working in the lab of the late Zanvil Cohn at Rockefeller University, an expert in the physiology of macrophages, which were considered to be a leading candidate for the missing accessory cells. "We looked at the populations [of cells] that were the source of the accessory cells," Steinman says. Using spleen tissue from mice.,"we found unusual cells that had never been seen before; they were tree-like in shape. Hence the name we gave them, dendritic, from the Greek word for tree."Exacting workMichel Nussenzweig was the first student to work with Steinman on dendritic cells, in 1977, shortly after the purification method for dendritic cells had been developed. He, Steinman and Cohn found that dendritic cells were very potent immune stimulators. Still, almost nobody believed dendritic cells were that special or significant, he says.For the next nearly 15 years, a good deal of the research published on dendritic cells came from Steinman and his colleagues. The cells are rare, making up less than one percent of white blood cells, and separating them out was an onerous process until Steinman and his colleagues devised a new method in the 1990s. "Once it became easy to work on the cells, then the field exploded, and there are now thousands of labs who study dendritic cells," Nussenzweig says.Steinman wasn’t afraid to argue that something so rare could be so important, says Antony Rosen, who worked in the Cohn lab with Steinman in the late 1980s and now is at Johns Hopkins University. "He was confident enough that it didn't matter to him that nobody had ever seen it before. It didn't matter to him that everybody rejected what he said. And he stayed at it and he knew he was right, and he proved that he was right."Vast potentialResearchers are studying the properties of dendritic cells and how to control their activities for many potential uses. Helping dendritic cells to dampen immune response could ease symptoms of autoimmune disorders and allergies. Helping dendritic cells to boost immune response could help fight infections like AIDS and cancers.Steinman is currently concentrating his work on investigating and designing vaccines. "I just feel the vaccines we already have are medical miracles, but the scope and potential of new vaccines that target dendritic cells is just enormous," he says. "I also find that when I try to think about making a vaccine against HIV or against cancer, that I start asking some interesting scientific questions about how our immune systems operate."He also continues to call for research that aims to study science in people, to help people and to extend scientific understanding. "We need to build a new kind of research network, one that develops and supports researchers who can study the immune system in patients," he says. "I think the natural deductive instinct of most scientists is to keep figuring out how the cells work in simpler systems like mice, learn more about mechanism, and that's obviously going to be very productive."But what I'd like to see is that we set our standards on the medical conditions that involve the immune system. I think that's where the biggest scientific challenges are, and if we don't direct ourselves to these conditions, we won't have the standards high enough for what we need to know."Part of that work is his role as a consultant for the Dana Foundation's immunology grants program, which targets patient-oriented research.Steinman has been a vociferous spokesman for such research, says Cooper at the University of Alabama. "He's just been unwavering in his insistence on trying to translate basic findings about how the immune system functions in a very basic way to see that that information gets translated into something that has relevance to people and their diseases."Of all the systems in the body, the immune system is the one "you can really teach, really make better," said Steinman during a forum at the Dana Center in 2006. But, he added, we don't yet know all the rules.Curiosity and collaborationThe discovery of dendritic cells was a co-discovery, shared with Zanvil Cohn and other colleagues, Steinman says. He continues to collaborate, with colleagues at Rockefeller University and across the world."He has been a mentor to a lot of people," says Madhav Dhodapkar, who worked in his lab a decade ago and now runs his own lab at Rockefeller. "People who haven't necessarily trained in his lab, I know, have benefited from interacting with him."Professionally, if one looks at how often his papers have been cited, thousands of times, it's easy to see how influential he is, says Rosen of Johns Hopkins. Most citations are for his work with dendritic cells, of course, but also for work on endocytosis and on basic cell biological progresses. "And some of those were classics in their time as well," he says."He has this great knowledge of science, and it's like he's a child discovering something for the first time every time he reads it," Rosen says. "He's just got a hunger for it. That's why I think he made a big discovery.""He would come into the lab with a journal or with a paper that he thought you would be interested in, and he told you about it as if it were the coolest thing that he could ever have come across."It's a reciprocal pleasure, Steinman says. "I think young people, especially in a complex, intricate science like immunology, need support and discussion time. It's just too intricate to do everything yourself. They certainly reciprocate, and you know, the young, energetic mind has the best ideas."The two Lasker prizes for medical research, nicknamed "America's Nobels," are given each year. Steinman received the Lasker award for basic medical research. This year's award for clinical medical research was shared by Alain Carpentier and Albert Starr, who developed replacement heart valves.

Neurological and autoimmune disorders after vaccination against pandemic influenza A (H1N1) with a monovalent adjuvanted vaccine: population based cohort study in Stockholm, Sweden

2011-10-14T12:48:00.001-07:00

Objective To examine the risk of neurological and autoimmune disorders of special interest in people vaccinated against pandemic influenza A (H1N1) with Pandemrix (GlaxoSmithKline, Middlesex, UK) compared with unvaccinated people over 8-10 months.

Design Retrospective cohort study linking individualised data on pandemic vaccinations to an inpatient and specialist database on healthcare utilisation in Stockholm county for follow-up during and after the pandemic period.

Setting Stockholm county, Sweden.

Population All people registered in Stockholm county on 1 October 2009 and who had lived in this region since 1 January 1998; 1 024 019 were vaccinated against H1N1 and 921 005 remained unvaccinated.

Main outcome measures Neurological and autoimmune diagnoses according to the European Medicines Agency strategy for monitoring of adverse events of special interest defined using ICD-10 codes for Guillain-Barré syndrome, Bell’s palsy, multiple sclerosis, polyneuropathy, anaesthesia or hypoaesthesia, paraesthesia, narcolepsy (added), and autoimmune conditions such as rheumatoid arthritis, inflammatory bowel disease, and type 1 diabetes; and short term mortality according to vaccination status.

Results Excess risks among vaccinated compared with unvaccinated people were of low magnitude for Bell’s palsy (hazard ratio 1.25, 95% confidence interval 1.06 to 1.48) and paraesthesia (1.11, 1.00 to 1.23) after adjustment for age, sex, socioeconomic status, and healthcare utilisation. Risks for Guillain-Barré syndrome, multiple sclerosis, type 1 diabetes, and rheumatoid arthritis remained unchanged. The risks of paraesthesia and inflammatory bowel disease among those vaccinated in the early phase (within 45 days from 1 October 2009) of the vaccination campaign were significantly increased; the risk being increased within the first six weeks after vaccination. Those vaccinated in the early phase were at a slightly reduced risk of death than those who were unvaccinated (0.94, 0.91 to 0.98), whereas those vaccinated in the late phase had an overall reduced mortality (0.68, 0.64 to 0.71). These associations could be real or explained, partly or entirely, by residual confounding.

Conclusions Results for the safety of Pandemrix over 8-10 months of follow-up were reassuring —notably, no change in the risk for Guillain-Barré syndrome, multiple sclerosis, type 1 diabetes, or rheumatoid arthritis. Relative risks were significantly increased for Bell’s palsy, paraesthesia, and inflammatory bowel disease after vaccination, predominantly in the early phase of the vaccination campaign. Small numbers of children and adolescents with narcolepsy precluded any meaningful conclusions.

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Anti-Aβ Drug Screening Platform Using Human iPS Cell-Derived Neurons for the Treatment of Alzheimer's Disease

2011-10-04T11:05:00.000-07:00

Background

Alzheimer's disease (AD) is a neurodegenerative disorder that causes progressive memory and cognitive decline during middle to late adult life. The AD brain is characterized by deposition of amyloid β peptide (Aβ), which is produced from amyloid precursor protein by β- and γ-secretase (presenilin complex)-mediated sequential cleavage. Induced pluripotent stem (iPS) cells potentially provide an opportunity to generate a human cell-based model of AD that would be crucial for drug discovery as well as for investigating mechanisms of the disease.

Methodology/Principal Findings

(A) Time-dependent morphological changes of cells reseeded in a 24-well plate. Neuronal and glial cells were stained by anti-Tuj1 (left; red), anti-synapsin I (left; green), anti-MAP2 (right; red), and anti-GFAP (right; green) antibodies and DAPI (right; blue) at 38, 45, and 52 days. Scale bar, left; 20 µm, right; 50 µm. Expression levels of Tuj1 (B), synapsin I (C), MAP2 (D), and GFAP (E) at days 0, 24, 38, 45, and 52 were measured by qPCR and normalized by that of GAPDH. “Fold expression” is the ratio of expression at each day compared to day 0. Each point represents mean ± SD of 3 assays. ^*p<0.05, ^**p<0.01, ^***p<0.001, significantly different from day 0 by Dunnett's test. (F–H) Neurotransmitter phenotypes at day 52. PAG (red)- and GAD (green)-positive (F), vGlut1 (green)- and Tuj1 (red)-positive (G), and GABA (green)- and Tuj1 (red)-positive cells (H). Blue, DAPI. Scale bar, 50 µm.

We differentiated human iPS (hiPS) cells into neuronal cells expressing the forebrain marker, Foxg1, and the neocortical markers, Cux1, Satb2, Ctip2, and Tbr1. The iPS cell-derived neuronal cells also expressed amyloid precursor protein, β-secretase, and γ-secretase components, and were capable of secreting Aβ into the conditioned media. Aβ production was inhibited by β-secretase inhibitor, γ-secretase inhibitor (GSI), and an NSAID; however, there were different susceptibilities to all three drugs between early and late differentiation stages. At the early differentiation stage, GSI treatment caused a fast increase at lower dose (Aβ surge) and drastic decline of Aβ production.

Conclusions/Significance

These results indicate that the hiPS cell-derived neuronal cells express functional β- and γ-secretases involved in Aβ production; however, anti-Aβ drug screening using these hiPS cell-derived neuronal cells requires sufficient neuronal differentiation.

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Interferon β-1b–neutralizing antibodies 5 years after clinically isolated syndrome

2011-09-27T01:04:00.000-07:00

The objective of this is study is to determine the frequency and consequences of neutralizing antibodies (NAbs) in patients with a first event suggestive of multiple sclerosis (MS) treated with interferon β-1b (IFNβ-1b).

In the Betaseron/Betaferon in Newly Emerging MS For Initial Treatment (BENEFIT) study, patients were randomly assigned to 250 μg IFNβ-1b (Betaferon) or placebo subcutaneously every other day for 2 years or until diagnosis of clinically definite MS (CDMS). Patients were then offered open-label IFNβ-1b for up to 5 years. NAb status was assessed every 6 months by the myxovirus protein A induction assay. A titer >20 NU/mL was considered NAb-positive, with low (≥20–100 NU/mL), medium (≥100–400 NU/mL), and high (≥400 NU/mL) titer categories. Here we examine early-treated patients, who received IFNβ-1b for up to 5 years.

NAbs were measured in 277 of 292 early-treated patients and detected at least once in 88 (31.8%) patients, with 53 (60.2%) reverting to NAb negativity by year 5. Time to CDMS, time to confirmed disability progression, and annualized relapse rate did not differ between NAb-positive and NAb-negative patients or between periods of NAb positivity vs NAb negativity within patients. Increases in newly active lesion number and T2 lesion volume and conversion to McDonald MS were associated with NAb positivity and were more pronounced with higher titers.

Table 1 Cross-sectional analyses for risk of CDMS, confirmed EDSS progression,and McDonald MS in NAb negative vs eventually NAb positive patients with 2consecutively positive NAb measurements

	Risk of event
	CDMS HR (95% CI) p Value^†	EDSS progression HR (95% CI) p Value^†	McDonald MS HR (95% CI) p Value*
Positive (≥20 NU/mL) vs negative	0.77 (0.05–1.18) p = 0.24	0.88 (0.50–1.54) p = 0.28	1.54 (1.15–2.08) p = 0.0044
*Single model*
Low titer (20–100 NU/mL) vs negative	0.73 (0.41–1.31) p = 0.29	1.05 (0.51–2.15) p = 0.99	1.41 (0.94–2.12) p = 0.09
Medium titer (100–400 NU/mL) vs negative	0.91 (0.46–1.82) p = 0.80	0.57 (0.18–1.83) p = 0.34	1.71 (1.10–2.77) p = 0.03
High titer (≥400 NU/mL) vs negative	0.71 (0.32–1.56) p = 0.39	0.91 (0.05–1.18) p = 0.85	1.62 (0.99–2.65) p = 0.06

Abbreviations:CDMS = clinically definite multiple sclerosis; EDSS = expanded disabilitystatus scale; NAb = neutralizing antibody; HR = hazard ratio, CI = confidenceinterval.

*By Cox proportional hazards regression adjustedfor age, gender, number of T2/gadolinium-enhancing lesions, mono-/multifocalpresentation, and use of steroids at the time of a first clinical eventsuggestive of MS. Hazard ratios above 1.0 indicate increased risk.

In conclusion, although NAb positivity was associated with increased brain MRI activity, no discernible effects on clinical outcomes were found. This finding may reflect the greater power of MRI compared with clinical outcomes to detect the treatment effects of IFNβ-1b and may also result from temporal changes in NAb titers and biology.

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Immunotherapy for Alzheimer's Disease: Rational Basis in Ongoing Clinical Trials

2011-07-03T01:37:00.001-07:00

Amyloid-β (Aβ) immunotherapy has recently begun to gain considerable attention as a potentially promising therapeutic approach to reducing the levels of Aβ in the Central Nervous System (CNS) of patients with Alzheimer's Disease (AD). Despite extensive preclinical evidence showing that immunization with Aβ(1-42) peptide can prevent or reverse the development of the neuropathological hallmarks of AD, in 2002, the clinical trial of AN-1792, the first trial involving an AD vaccine, was discontinued at Phase II when a subset of patients immunized with Aβ(1-42) developed meningoencephalitis, thereby making it necessary to take a more refined and strategic approach towards developing novel Aβ immunotherapy strategies by first constructing a safe and effective vaccine.
This review describes the rational basis in modern clinical trials that have been designed to overcome the many challenges and known hurdles inherent to the search for effective AD immunotherapies. The precise delimitation of the most appropriate targets for AD vaccination remains a major point of discussion and emphasizes the need to target antigens in proteins involved in the early steps of the amyloid cascade. Other obstacles that have been clearly defined include the need to avoid unwanted anti-Aβ/APP Th1 immune responses, the need to achieve adequate responses to vaccination in the elderly and the need for precise monitoring.

Proposed mechanisms of action of immunotherapy in amyloid reduction. Panel A describes the mechanism of microglial phagocytosis. Amyloid fibers opsonized by antibodies enter the brain from the bloodstream where microglial cells recognize the antibodies and phagocytose the amyloid via the Fcg receptor. Panel B represents the mechanism of catalytic disaggregation. Amyloid fibers are bound by antibodies that disrupt the tertiary structure of the amyloid deposit. This results in solubilization of the Ab and exit of the brain. Panel C shows the peripheral sink mechanism. In this case, monomeric soluble Ab circulating in the bloodstream is bound by the circulating antibodies. This sequestration of circulating Ab produces a shift in the concentration gradient of Ab between the brain and the blood causing an efflux of Ab out of the brain.

Molecular basis of active and passive immunization. A. Vaccination (active immunization) activates the body’s immune system to produce antigen-specific antibodies. In AD, full-length Aβ or a fragment of Aβ conjugated to a foreign T cell epitope carrier protein can be used as an antigen, which is delivered into the body along with an immune system booster (adjuvant). The humoral immune response is generated when APCs, which internalize and process the antigen, present T cell epitopes to naive Th lymphocytes. Binding of co-stimulatory molecules on the surfaces of APCs and T cells provides a secondary signal that enhances T cell activation. Meanwhile, the soluble antigen binds to B cell receptors via the B cell epitope, and this antigen is presented to activated T cells to help the B cell make antibodies against the antigen. Activated T cells also produce cellular immune responses. A Th1 cellular immune response leads to the release of pro-inflammatory cytokines, whereas a Th2 response causes release of anti-inflammatory cytokines. b. Passive immunization bypasses the need activate the immune system to initiate an immune response to produce antigen-specific antibodies. In both active and passive Aβ immunization, anti-Aβ antibodies bind Aβ, targeting the peptide for clearance. Abbreviations: Aβ, amyloid-β; APC, antigen presenting cell

Schematic representation of the two types of immunotherapy for Alzheimer's disease used on experimental animal models. Panel A describes active immunization. Fibrillar Ab1−42 is combined with an adjuvant by emulsification and the product of the reaction is then injected into the mouse. The mouse produces anti-Ab antibodies in response to the vaccination. Panel B describes passive immunization. In this case, mice are immunized with Ab as with active immunization. Hybridomas are then produced and selected for optimal antibody properties. The antibodies are then harvested, purified and administered to another mouse for treatment.

Novel strategies have been implemented to overcome these problems including the use of N-terminal peptides as antigens, the development of DNA based epitope vaccines and vaccines based on passive immunotherapy, recruitment of patients at earlier stages with support of novel biomarkers, the use of new adjuvants, the use of foreign T cell epitopes and viral-like particles and adopting new efficacy endpoints. These strategies are currently being tested in over 10,000 patients enrolled in one of the more than 40 ongoing clinical trials, most of which are expected to report final results within two years.

Ongoing clinical trials based on active or passive immunotherapy for AD.

NAME	FDA PHASE	MECHANISM OF ACTION
ACC-001	Phase II/IIa/IIb	ACC-001 is a short amino-terminal Aβ (1-6) fragment that is derived from the N-terminal B cell epitope of Aβ while avoiding T cell activation. Antibodies specific for this Aβ peptide can cross the blood-brain barrier and act directly in the central nervous system to induce plaque clearance. Note: this trial was temporarily suspended due to vasculitis detected in one patient.
CAD-105	Phase II	CAD-105 is Aβ_1–5coupled to Qb virus-like particles. Antibodies specific for this Aβ peptide can cross the blood-brain barrier and act directly in the central nervous system to induce plaque clearance.
V950	Phase I	V950 is an Aβ amino-terminal peptide conjugated to ISCO-MATRIX®.
UB311	Phase I	UB311 uses the peptide Aβ_1–14 with UBITh®. The UBITh AD immunotherapeutic vaccine has been engineered to elicit anti-N-terminal Aβ (1-14) antibodies while minimizing potential for the generation of adverse anti-Aβ immune responses. The vaccine has been further designed for minimization of inflammatory responses through the use of a proprietary vaccine delivery system that favors Th2 type regulatory T cell responses over Th1 pro-inflammatory T cell responses.
Affitope AD02 / Mimotope Aβ(1-6)	Phase II/IIa/IIb	Affitope AD02 is a short amino-terminal Aβ fragment (Aβ1-6) that is derived from the N-terminal B cell epitope of Aβ while avoiding T cell activation.
AAB-001 / Bapineuzumab	Phase III	Bapineuzumab is an anti-Aβ antibody that binds specifically to soluble amyloid-β and therefore may act to draw the peptide away from the brain through the blood to be cleared in the periphery.
CAD106	Phase II/IIa/IIb	CAD106 is a vaccine based on an epitope that contains multiple copies of the Aβ 1-6 peptide, that avoids T cell activation, coupled to the Qβ virus-like particle.
Intravenous Immunoglobulin /Gammagard, IVIg	Phase III	Intravenous Immunoglobulin (IVIg) is obtained from the pooled plasma of healthy human blood donors, and contains natural anti-amyloid antibodies. In vitro data have demonstrated that human anti-Aβ antibodies inhibit fibril formation and diminish neurotoxicity.
MABT5102A	Phase I	MABT5102A is a humanized monoclonal antibody that binds Aβ.
Solanezumab / LY2062430	Phase III	Solanezumab binds specifically to soluble amyloid-β and therefore may act to draw the peptide away from the brain to be cleared in the periphery.
PF-04360365	Phase II	Multiple IV Dose Study of anti-Aβ mAb in patients with mild To moderate AD. PF-04360365 binds to Aβ.
R1450	Phase II	R1450 is a fully humanized monoclonal antibody that binds Aβ.
GSK933766A	Phase I	Anti-Aβ antibody that binds Aβ.

Summary of the main strategies and rational to overcome the challenges raised from the AN1792 clinical trial:

Strategy

Rational

Challenge: Unwanted anti-Aβ/APP Th1 immune responses

Using N terminal peptide (Ab_1–15) as antigen

The Ab_1–15 peptide is the major B cell epitope. Antibodies raised to amino acids 1–11, 1–7, and 1–5 of Ab₄₂ bind to b-amyloid plaques with higher affinity and initiate immune responses much stronger than antibodies raised to amino acids 3–7, 5–11, and 11–26. The smallest domain in the Ab peptide to which the antibodies bind with high affinity is that encoding amino acids 4-10.

Using DNA based epitopes

DNA-based vaccination induces prolonged, endogenous antigen synthesis and processing within the subject’s own cells infected with virus encoding the desired epitopes and adjuvants.

Adjuvants facilitate the internalization of antigen into the antigen presenting cells to enhance the efficiency of processing and presentation of the antigens.

Using new adjuvants

Mannan is as a potentially optimal molecular adjuvant due to its ability to enhance both B- and T-cell immune responses. This adjuvant has the advantage of inducing a Th2 rather than Th1 response

Using passive immunotherapy

Vaccines using monoclonal antibodies or Immunoglobulins do not show adverse reactions related to cellular immunity

Challenge: The need of early treatment

Early diagnosis with support of biomarkers

Repeated vaccination at regular intervals since early stages would increase the probability of a good response before the toxic forms of β-amyloid begin to accumulate.

Challenge: Hypo-responsiveness to vaccines in the elderly

Recruiting previously generated memory T cells

A vaccine based on Ab peptide fused with Th epitopes from conventional vaccines or common pathogens produced during childhood vaccination or during prior exposure to human pathogens could potentially induce a rapid expansion of pre-existing memory T cells and their differentiation into effector T cells

Using Foreign T cell epitope

Foreign T cell epitopes as synthetic, non-natural Pan HLA DR-binding epitopes produce more potent responses on a molar basis than a tetanus-derived universal epitope.

Using Viral-like particles

Incorporation of the Ab B cell epitope into a viral capsid protein allows the expression of this epitope on the surface of VLP in a repetitive and ordered array. Such organization of the epitope may induce T cell-independent B cell activation and production of high titers of anti-Ab antibodies

Challenge: The need of more precise monitoring

Adopting new efficacy endpoints

Besides traditional endpoints, new clinical endpoints such as neuroimaging and biomarkers provide a more detailed picture of basal status and response of the patients.

Published at: Curr Pharm Des. 2011 Mar 4

Dr Frank Longo discusses exciting MS research results

2010-04-03T02:42:00.000-07:00

Stanford Department of Neurology and Neurosciences Chair Frank Longo talks about the groundbreaking Multiple Sclerosis research of Stanford scientist Lawrence Steinman.

Two kinds of multiple sclerosis, two different responses to beta-interferon, study shows

2010-04-03T02:39:00.001-07:00

There may be two distinct versions of multiple sclerosis, a study in both animal models and human blood samples suggests. What’s more, a patient’s responsiveness to the most popular first-line drug for this episodic and all-too-often recurring autoimmune condition seems to depend on which version that patient has.

If these findings are confirmed in larger human studies and by other laboratories, people with multiple sclerosis might someday be able to take a simple blood test to see whether they are likely to respond to treatment with the standard multiple-sclerosis therapy, said senior study author Lawrence Steinman, MD, the George A. Zimmerman Professor of Neurology and Neurological Sciences at the Stanford University School of Medicine.

Public health may benefit, too, Steinman said, as the cost savings from being able to predict in advance which patients will benefit from beta-interferon, a costly bioengineered drug whose global sales come to some $4 billion a year, could be considerable.

Beta-interferon’s overall efficacy is only fair, he said, with perhaps half of all multiple-sclerosis patients experiencing an average one-third reduction in recurrences. Plus, its discomfiting side effects — flulike symptoms — can make compliance an issue for patients, especially given the drug’s iffy efficacy.

In a study published online March 28 in Nature Medicine, Steinman and his colleagues used an established animal model of multiple sclerosis called experimental autoimmune encephalitis, or EAE, which they induced by injecting the animals with myelin in a way that caused the immune system to inappropriately attack the animals’ own myelin nerve-cell coatings.

Many nerve cells in mammalian brains and peripheral tissues must convey electrochemical impulses over great distances, and quickly. Long, wirelike projections that transmit these cells’ signals to other nerve or muscle cells are coated by myelin, a natural substance whose insulating properties sustain the impulses’ strength and increase their speed.

Multiple sclerosis is triggered when, for reasons that are not yet clear, immune cells called T cells attack the myelin sheathing, causing symptoms including paralysis and blindness. The condition affects 400,000 people in the United States, according to the National Multiple Sclerosis Society.

A few years ago while still a PhD student at the University of Alabama, the study’s first author, Robert Axtell, had shown that, as in people with multiple sclerosis, beta-interferon can reverse paralysis in mice with EAE. But it turns out that EAE can be induced by two different autoimmune pathways, characterized by different patterns of secretion by T cells.

Like nerve cells, immune cells also communicate with one another across long distances, but they accomplish this through various chemicals called cytokines that they secrete into the blood. Immune cells on the receiving end of a cytokine “signal” may respond quite differently, depending on the particular type of cytokine to which they are exposed. Two cytokines called gamma-interferon and IL-17, for example, tend to induce the kinds of inflammatory immune-system arousal that can trigger multiple sclerosis.

Axtell (now a postdoctoral scholar in Steinman’s lab), Steinman and their colleagues were able to induce two superficially similar forms of EAE in mice by directing the myelin-attacking T cells to predominantly secrete either gamma-interferon or IL-17, respectively. The researchers found that beta-interferon improved the condition of animals whose EAE had been induced by gamma-interferon-secreting T cells, but exacerbated symptoms in those whose EAE had been induced by IL-17-secreting T cells.

Intrigued, the investigators turned to humans. Another postdoctoral scholar in the Steinman lab, Brigit deJong, MD, the study’s second author, had previously been involved in research in Amsterdam in which multiple-sclerosis patients were treated with beta-interferon and meticulously followed up. The Stanford group obtained blood samples taken from 26 of these patients both before and about two years after the initiation of treatment. Without knowing which samples came from patients who had responded well or poorly to beta-interferon treatment, they went about measuring IL-17 levels in those samples.

Eventually, patients’ follow-up histories were revealed to the researchers and their measured IL-17 levels were paired with their post-treatment progress. A clear pattern emerged. Measurements of a particular variety of IL-17, called IL-17F, clustered at either very high or very low levels in individual patients’ blood. Those with very low detectable blood levels of IL-17F responded well to beta-interferon treatment, experiencing no relapses or instances of required steroids (to quickly shut down a malfunctioning immune system). But patients with very high IL-17F levels — about one out of three subjects — responded poorly by the same criteria. In fact, said Steinman, there is some evidence that beta-interferon actually worsened these patients’ conditions.

Steinman cautioned that the results need to be confirmed in larger patient groups, in his lab as well as in others. But, he said, “I think this has the potential to transform the way we take care of people with multiple sclerosis.” He said a simple, already available blood test could spare many patients the inconvenience and side effects — and spare the health-care system the expense — of a drug that most likely won’t do any good. “The other side of the coin is that beta-interferon, if it’s given only to those who are predisposed to respond to it, could turn out to be a far better drug than we ever imagined.”

Although Steinman and his colleagues do not stand to benefit in any direct way from this work, Stanford University’s Office of Technology Licensing has filed a patent application on the use of the blood test. Earlier work by Steinman, proceeding from animal models to clinical trials, led to the development of another blockbuster multiple-sclerosis drug, natalizumab, marketed under the trade name Tysabri.

Several other scientists from Stanford and elsewhere co-authored the Nature Medicine study, which was funded by the National Multiple Sclerosis Society. Axtell’s former PhD advisor, Chander Raman, PhD, of the University of Alabama-Birmingham’s Department of Medicine shares senior authorship with Steinman. More information about Stanford’s Department of Neurology and Neurological Sciences, which supported the work, is available at http://neurology.stanford.edu/.

B cells as therapeutic targets in autoimmune neurological disorders

2008-10-03T10:46:00.000-07:00

Summary
B cells have a fundamental role in the pathogenesis of various autoimmune neurological disorders, not only as precursors of antibody-producing cells, but also as important regulators of the T-cell activation process through their participation in antigen presentation, cytokine production, and formation of ectopic germinal centers in the intermeningeal spaces. Two B-cell trophic factors—BAFF (B-cell-activating factor) and APRIL (a proliferation-inducing ligand)—and their receptors are strongly upregulated in many immunological disorders of the CNS and PNS, and these molecules contribute to clonal expansion of B cells in situ. The availability of monoclonal antibodies or fusion proteins against B-cell surface molecules and trophic factors provides a rational approach to the treatment of autoimmune neurological diseases. This article reviews the role of B cells in autoimmune neurological disorders and summarizes the experience to date with rituximab, a B-cell-depleting monoclonal antibody against CD20, for the treatment of relapsing–remitting multiple sclerosis, autoimmune neuropathies, neuromyelitis optica, paraneoplastic neurological disorders, myasthenia gravis, and inflammatory myopathies. It is expected that ongoing controlled trials will establish the efficacy and long-term safety profile of anti-B-cell agents in several autoimmune neurological disorders, as well as exploring the possibility of a safe and synergistic effect with other immunosuppressants or immunomodulators.

Introduction
During the past three decades, investigations into neuroimmunological diseases of the CNS—and to a lesser degree the PNS—have centered predominantly on the roles of activated, cytotoxic and immunoregulatory T cells rather than B cells. The decision to focus on T cells can probably be attributed to the long-standing observation that the main lymphocytic subset within the lesions in the two most common autoimmune disorders, multiple sclerosis (MS) and Guillain–Barré syndrome, are dominated by T-cell infiltrates. In addition, myelin-specific T cells are responsible for disease transfer in the respective animal models for these conditions. The contribution of activated B cells to these disorders has traditionally been viewed as a secondary consequence of the breakdown of T-cell tolerance. Over the past few years, however, compelling data on the roles of B cells as sensors, coordinators and regulators of the immune response1 have strengthened the view that B cells and autoantibodies are fundamental for activating T cells and/or mediating tissue injury in several disorders of the CNS and PNS. The observation that B-cell depletion is an effective therapy in autoimmune disorders such as rheumatoid arthritis has provided the impetus to explore the functions of B cells in neurological diseases, and has triggered an interest in conducting clinical trials in this area.
This Review focuses on B-cell homeostasis, addresses the roles of B-cell functions in autoimmune neurological disorders, and summarizes the experience to date with anti-B-cell therapies, in particular the B-cell-depleting monoclonal antibody rituximab.
Roles of B cells in the immune response: neurological aspects
In the context of autoimmune neurological disorders, B cells have traditionally been associated with the production of autoantibodies from plasma cells, the end products of B-cell differentiation.2, 3, 4, 5 In several neurological diseases, including myasthenia gravis and certain neuropathies, the autoantibodies are pathogenetic, exerting a direct effect on self antigens either by functioning as neutralizing antibodies or by activating and fixing complement on the targeted tissues (Figure 1A). Autoantibodies and immune complexes can also activate Fc receptors on macrophages or dendritic cells, leading to the production of cytokines, which cause further tissue injury (Figure 1A). In most autoimmune neurological disorders, however, the autoantibodies are directed against cytosolic antigens and might not be directly involved in tissue injury. In such cases, B cells might still participate in the autoimmune process through antibody-independent mechanisms that include antigen presentation, costimulation, cytokine production, and coordination of T-cell functions (Figure 1A–D).4, 6

A proof-of-principle that activated B cells are fundamental for coordinating T-cell functions was provided by the observation that B-cell-depleted mice exhibit a dramatic decrease in numbers of CD4+ and CD8+ T cells, and a tenfold inhibition of memory CD8+ T cells.7, 8 An important function of B cells is their ability to present antigenic peptides, in the context of major histocompatibility complex class II molecules on their surface, to the T-cell receptors of CD4+ cells, leading to expansion of antigen-specific T cells (Figure 1B).1, 2, 3, 4, 5, 6 B cells are 100–1,000 times more potent in antigen presentation than are the other antigen-presenting cells, such as macrophages or dendritic cells,9 and they are especially effective at presenting low concentrations of antigen. Activated B cells are also as efficient as T cells at producing cytokines—most notably interleukins (IL-1, IL-4, IL-6, IL-10, IL-12, IL-23 and IL-16), tumor necrosis factor (TNF) and the chemokines macrophage inflammatory protein 1 (MIP1) and MIP1.10, 11 These inflammatory mediators modulate the migration of dendritic cells, activate macrophages, exert a regulatory role on T-cell functions, and provide feedback stimulatory signals for further B-cell activation (Figure 1C). Some cytokines might theoretically exert an inhibitory role in the immune process, although this has not been clearly established in human diseases.
An additional role of B cells that is relevant to neurology is their involvement in de novo formation and maintenance of ectopic lymphoid structures, a process termed neolymphogenesis.6 This is accomplished through the actions of -lymphotoxin, a TNF family member molecule that is expressed on the surface of B cells (Figure 1D). Ectopic follicular structures are found in the meningeal compartment in patients with various neuroinflammatory conditions, as discussed below. The multiple contributions of B cells to the complexity of the autoimmune process make B cells attractive targets for therapeutic interventions that extend beyond the traditional effects on antibody production.
B-cell maturation and homeostasis
B lymphocytes arise from hematopoietic stem cells in the bone marrow. These cells mature independently of an antigen first into pro-B cells, then into pre-B cells and immature B cells (Figure 2).4, 5 They subsequently enter the antigen-dependent phase in the peripheral lymphoid tissues, where mature-but-naive B cells, after encountering their antigen in the extrafollicular regions of the lymphoid organs, become activated B cells and migrate to the follicular regions. From here, they exit to differentiate into memory B cells, late plasmablasts and plasma cells (Figure 2).1, 2, 3, 4, 5, 6 Specific markers, such as CD20, CD27, BAFF-R (B-cell-activating factor receptor), CD38 and CD138, identify the transitional phases of B cells from stem cells to plasma cells (Figure 2).

The memory B cells, late plasmablasts and long-lived plasma cells migrate not only to the bone marrow, spleen and lymphoid tissues, but also to the brain, where they transform into antibody-secreting cells after encountering their antigen (Figure 3).12 Interactions between the homeostatic chemokines CXC-chemokine ligand (CXCL) 13, CXCL10 and CXCL12 secreted from the endothelial cell wall and their respective receptors on B cells3, 13 are fundamental for B-cell homeostasis not only within the lymphoid follicles but also within the brain. These molecules are upregulated in the brains of patients with MS, allowing the recruitment and transmigration of antibody-producing B cells into the brain.14, 15 B-cell transmigration into the brain is also facilitated by the adhesion molecules very late antigen-4 (VLA-4; also known as integrin -4 or ITA4) and lymphocyte function-associated antigen-1 (LFA-1; also known as integrin -L or ITAL) and their counter-receptors vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1) on the endothelial cells.13 In secondary progressive MS, activated B cells form germinal centers not only in the lymphoid tissues but also within the intermeningeal spaces, where they undergo the same stages of differentiation as in the periphery (Figure 3).16, 17 Within these structures, which are observed in 41.4% of patients with secondary progressive MS,17 B cells generate inflammatory mediators that can stimulate plasma cells for in situ production of immunoglobulins. The production of intrathecal immunoglobulins (i.e. the life-long persistent oligoclonal bands) in all forms of MS indicates a central role for activated B cells and plasma cells in this disease.

Two members of the TNF family, BAFF (B-cell-activating factor) and APRIL (a proliferation-inducing ligand), have emerged as crucial factors for B-cell survival, differentiation, germinal center formation and immunoglobulin production.3, 18, 19 BAFF and APRIL are produced by monocytes, macrophages and dendritic cells, and they circulate in trimeric forms. They bind to B cells through three different receptors (Table 1): BAFF-R, BCMA (B-cell-maturation antigen) and TACI (transmembrane activator and calcium modulator and cytophilin ligand interactor). Levels of BAFF-R and APRIL mRNA are increased in the monocytes and B cells of patients with MS20 and in the muscles of patients with inflammatory myopathies (Raju R and Dalakas MC, unpublished data). In MS lesions, BAFF and APRIL are produced by astrocytes, and they promote the in situ survival and clonal expansion of B cells (Figure 3).21, 22 Agents that target BAFF or APRIL might, therefore, exert therapeutic effects in various neurological disorders by suppressing B-cell proliferation.

Anti-B-cell therapy in neurology: present and future
Various immunomodulatory drugs that are currently used in neurology, such as intravenous immunoglobulin (IVIg), alemtuzumab, cyclophosphamide, mitoxanthrone and natalizumab, can affect some aspects of B-cell function that are relevant to the pathogenesis of neurological disease. New monoclonal antibodies or fusion proteins that specifically target B-cell survival or proliferation are, however, now becoming available. The evidence that B cells have a role in autoimmune neurological disorders is summarized here:

Observations supporting a role for B cells in the pathogenesis of autoimmune neurological disorders.

B cells are clonally expanded within the CNS, producing intrathecal
immunoglobulin (IgG), in various CNS disorders such as multiple sclerosis (MS),
paraneoplastic CNS disorders and stiff-person syndrome
B cells, plasma
cells, myelin-specific IgG and complement are present in the active and chronic
plaques of MS
IgGs specific for myelin oligodendrocyte glycoprotein and
myelin basic protein are detected in the brains of individuals with MS
Memory B cells, along with early, late or short-lived plasmablasts, are
detected in the cerebrospinal fluid and ectopic germinal centers in the meninges
of patients with MS
B cells are required for disease induction by antigenic
peptides in some experimental autoimmune encephalomyelitis and experimental
autoimmune neuritis models, a requirement consistent with the unique ability of
B cells to recognize antigenic conformation
B cells have a role in the
regulation of CNS inflammation
Autoantibodies against glycolipids and
glycoproteins can induce demyelination within the PNS
T-cell-dependent
B-cell activation leads to production of pathologic autoantibodies in myasthenia
gravis
Several antibody-mediated neurological disorders have been
successfully treated using plasmapheresis or intravenous immunoglobulin, which
remove autoantibodies or modify the idiotypic repertoire
New therapeutic
monoclonal antibodies such as rituximab that deplete B cells can result in
clinical improvement when used in certain CNS or PNS disorders

Agents affecting B-cell survival
Drugs that target BAFF or APRIL, or their receptors BAFF-R, TACI or BCMA, affect B cell survival and differentiation, resulting in reduced numbers of mature B cells in the lymphoid tissues and the circulation (Figure 4).19 Blockade of BAFF-R, TACI or BCMA in mouse models of systemic lupus erythematosus (SLE) not only reduces antibody titers, but also improves animal survival.6, 23, 24 The targeting of BAFF, BAFF-R and APRIL is of therapeutic interest in the neurological context, because these molecules are upregulated in the tissues of patients with autoimmune diseases. A number of agents are currently in phase I–II clinical trials in rheumatoid arthritis and SLE.1, 2, 3, 4, 5, 6, 24 Agents that target BAFF include belimumab, a humanized monoclonal antibody against soluble BAFF, and the BAFF antagonist AMG G23. BR3-Fc is directed against BAFF-R, resulting in blockade of BAFF binding and, subsequently, B-cell reduction. BCMA-IgG is directed against APRIL. The TACI–IgG fusion protein neutralizes BAFF, APRIL and BAFF–APRIL heterodimers. Anti-lymphotoxin- receptor disrupts the architecture in the ectopic germinal centers.

Agents causing B-cell depletion
Drugs directed against the CD20 or CD22 B-cell-surface glycoproteins can coat B cells and thereby cause their depletion (Figure 4). These drugs include: epratuzumab, which blocks CD22 survival signals on immature and mature B cells, as well as on pro-B and pre-B cells; rituximab, which is directed against the CD20 molecule; and occrelizumab, the humanized version of rituximab. In contrast to agents against trophic factors and their receptors, as mentioned above, these drugs deplete B cells but not the antibody-producing plasma cells.1, 2, 3, 4, 5, 6
Anti-B-cell agents in neurology: the role of rituximab
Among all the aforementioned agents, only two, BCMA-IgG and rituximab, have been used in autoimmune neurological disorders. In mice with experimental autoimmune encephalomyelitis induced by myelin oligodendrocyte glycoprotein (MOG), BCMA-IgG prevented disease development, improved the strength of already weak animals, depleted the CD19+ B cells in the blood, spleen and lymph nodes, reduced anti-MOG-specific IgG antibody titers, and suppressed inflammation and ongoing demyelination in the brain and spinal cord.25
Rituximab is a chimeric mouse–human monoclonal antibody consisting of human IgG1 and kappa constant regions and a mouse variable region. It was derived from a hybridoma directed at human CD20, a 297-amino-acid transmembrane phosphoprotein that is present on all cells of the B-cell lineage except for stem cells, pro-B cells and plasma cells (Figure 2).1, 2, 3, 4, 5, 6 In contrast to BAFF and APRIL, CD20 is not secreted, and it is not shed or endocytosed when exposed to rituximab.26 The function of CD20 is unclear—it is thought to be involved in B-cell activation and proliferation,1, 2, 3, 4, 5, 6 although CD20 knockout mice do not exhibit B-cell deficits.1, 2, 3, 4, 5, 6, 26 Rituximab is approved for the treatment of rheumatoid arthritis,27 and, as outlined in the sections that follow, its use is currently being explored in a number of autoimmune neurological disorders in which B cells have a role.
Rituximab in autoimmune neurological disorders
Multiple sclerosis
In MS, B cells and antibodies are involved to varying degrees at different stages of the disease and in different subgroups of MS. In the I–IV classification of Lucchineti, for example, pattern II is characterized by prominent lymphocytic and macrophagic infiltrates, complement activation and deposits of immunoglobulins.28 Additional evidence supporting an antibody-mediated process in patients with MS includes accumulations of clonally expanded B cells in the MS plaques; intrathecal production of IgG bands from oligoclonal populations of B cells; autoantibodies against MOG in actively demyelinating lesions; ectopic lymphoid tissue in the intermeningeal spaces; and upregulation of BAFF and APRIL.17, 21, 22, 28, 29, 30, 31
In patients with MS, 24 weeks of treatment with rituximab was shown to deplete B cells from the cerebrospinal fluid (CSF) and to suppress B-cell activation, but it did not affect the intrathecal synthesis of oligoclonal IgG bands derived from long-lived plasma cells.32 In a phase II, controlled, multicenter clinical trial of 104 patients with relapsing–remitting MS, a 58% relative reduction in the proportion of patients who experienced a relapse was noted after 24 weeks of therapy. A significant reduction in the mean number of gadolinium-enhancing MRI lesions (the study's primary end point), was also observed in the treated patients compared with the placebo group (P <0.0001).33>Neuromyelitis optica
Neuromyelitis optica (NMO) is an inflammatory CNS disorder that affects the optic nerves and the spinal cord. It typically presents with myelitis and optic neuritis, and is characterized by varying degrees of sensory motor disturbances, bladder–bowel dysfunction and visual loss. In NMO, autoantibodies, collectively termed NMO-Ig, bind to cerebral microvessels.35 The main target antigen of these autoantibodies is the aquaporin-4 water channel. NMO-Ig is derived from peripheral B cells, activates complement, and has been implicated in the induction of inflammatory demyelination and necrosis in the endothelial cells of the spinal cord.35 Patients with NMO experience frequent relapses, and the disease is associated with high morbidity. Some acute flare-ups can respond to plasmapheresis, although the disease responds poorly to immunotherapies overall.
In an open-label study, six out of eight patients with NMO became relapse-free after a year of rituximab treatment, with a decline in relapse rate from 26 to zero attacks per year. In addition, seven of the patients showed a substantial improvement in their Expanded Disability Status Scale (EDSS) score.36 In a retrospective review of 34 patients from two different centers, rituximab significantly lowered the relapse rate compared with pretreatment data, and stabilized or improved the EDSS scores in 91% of the patients.37, 38
Paraneoplastic neurological disorders
Patients with paraneoplastic neurological disorders have circulating antibodies against a variety of antigens that are expressed in both brain and cancer cells. There is evidence that B cells, plasma cells and cytotoxic T cells cross the blood–brain barrier, and antibodies are synthesized intrathecally. In paraneoplastic opsoclonus–myoclonus, the number of clonally expanded B cells within the CSF correlates with clinical severity.39 Rituximab, as an add-on therapy to IVIg or adrenocorticotropic hormone (ACTH), improved the ataxia severity scores, ameliorated myoclonus and reduced the rate of clinical relapse in 81% of 16 children with opsoclonus–myoclonus, and selectively reduced the numbers of clonally expanded B cells in the CSF.40 In addition, the required ACTH dose was reduced by 51% after rituximab treatment.
Chronic autoimmune neuropathies
The chronic autoimmune neuropathies include a spectrum of predominantly demyelinating neuropathies, the most common of which are chronic inflammatory demyelinating neuropathy (CIDP), multifocal motor neuropathy (MMN) and IgM anti-myelin-associated glycoprotein (IgM-MAG) neuropathies. Evidence for a role for B cells in the pathogenesis of these conditions includes the deposition of immunoglobulins and complement on the patients' nerves, and the presence of complement-fixing antibodies against MAG and gangliosides.41, 42 In an open series of 21 patients with IgM antibodies to gangliosides, rituximab improved symptoms in 61% of the patients 6 months after therapy, and the benefits were maintained for up to 2 years with repeated infusions.43 The IgM antibody titers dropped by 36% in the first year and by 57% in the second. Rituximab has also been reported to be effective in some patients with MMN or CIDP.44, 45 In another study, rituximab improved the symptoms in six out of nine patients with IgM-MAG neuropathy, and reduced IgM-MAG titers by a mean of 52% from baseline,46 prompting a placebo-controlled study. In 26 randomized patients, rituximab significantly improved disability scores and reduced IgM-MAG titers after 8 months.47 Rituximab is the first drug to demonstrate efficacy in a randomized trial in this particular neuropathy.
Stiff-person syndrome
Stiff-person syndrome (SPS) is a rare but often misdiagnosed CNS disorder that is clinically characterized by stiffness and rigidity in the limbs and paraspinal muscles, intermittent superimposed muscle spasms, and heightened sensitivity to external stimuli. The majority of patients with SPS have antibodies against glutamic acid decarboxylase (GAD) or GABARAP, a linker protein responsible for -aminobutyric acid receptor clustering.48 Anti-GAD antibodies are synthesized intrathecally, and oligoclonal bands are commonly detected in the CSF.49 In one case report, rituximab was effective at reducing stiffness and increasing mobility 2 months after the treatment was initiated, and resulted in disappearance of GAD antibodies and normalization of the electromyogram.50 The first double-blind controlled study using rituximab to treat SPS has now been completed at the NIH, and the results are currently being analyzed.
Inflammatory myopathies
There are three main subsets of inflammatory myopathies: polymyositis, dermatomyositis and inclusion body myositis. In all these forms of the disease, B cells and plasma cells are present in the muscle tissues, and in dermatomyositis immunoglobulins are deposited on endomysial capillaries.51 In 10 patients with polymyositis or dermatomyositis who had responded poorly to current therapies, rituximab increased or normalized muscle strength in 8 cases. Serum levels of creatine kinase and the required prednisone dose were concurrently reduced.52, 53 A multicenter NIH-sponsored clinical trial of rituximab is now ongoing for the treatment of polymyositis and dermatomyositis. The drug has not yet been tested in inclusion body myositis.
Myasthenia gravis
Myasthenia gravis is a prototypic B-cell-mediated autoimmune disease caused by pathogenetic antibodies against the muscle acetylcholine receptors. Evidence from around 20 case reports suggests that rituximab is effective in most patients, but a controlled study has not yet been done.54, 55, 56, 57
Effect of rituximab on circulating B cells, autoantibodies and immunoglobulin levels
In general, 1 month after rituximab infusion, circulating B cells become undetectable, and their numbers remain low for at least 6 months. The cells start reappearing slowly thereafter, but even after 10 months their numbers remain below baseline.3 The circulating memory CD20+CD27+ B cells are also depleted, and their levels remain low until month 8 (Figure 5). The B cells in the follicular splenic regions are preferentially affected, being depleted by 90%, compared with 25% depletion of marginal-zone B cells.6 The germinal-center B cells are resistant to rituximab, even though they express CD20, possibly reflecting an inability of the antibody to access the intravascular spaces within the lymphoid tissues, or different sensitivities of B cells according to the local lymphoid microenvironment.6 Stem cells in the bone marrow that do not express CD20 are also spared, thereby allowing the generation of new naive B cells.58

Rituximab is not expected to affect the levels of antibodies produced by plasma cells, although some reductions in these levels have been noted. In rheumatoid arthritis, for example, the titers of rheumatoid factor were shown to decrease two-to-threefold,58, 59 and in IgM-MAG neuropathy by 30–50%, after treatment with rituximab.45, 47 Such reductions can probably be attributed to depletion of CD27+ memory B cells, the precursors of short-lived plasma cells.4 As the CD27+ memory B cells reappear, so do the short-lived plasma cells.60 Given that the reconstituting B cells are naive cells with a new and diverse immunoglobulin rearrangement pattern,4, 58, 59 it might take some time for them to be restimulated by the original antigen, hence the slow re-emergence of serum antibody titers. Antibody titers might therefore fall after rituximab treatment, and rebound slowly at a rate controlled by the replenishment of memory and short-lived plasma cells.60 Interestingly, after several years of treatment, the antibody titers against anamnestic antigens, such as tetanus toxoid, remain stable.59 This finding suggests that rituximab might have differential effects on 'autoreactive' B cells and their corresponding short-lived plasma cells, compared with 'non-self-reactive' B cells and their corresponding longer lived plasma cells, which are responsible for post-vaccination responses.6 A recent study supports different roles for B cells and longer lived plasma cells in protective immunity.61
Dosing, tolerance, safety and combination therapy
Rituximab can be administered intravenously at a dose of 375 mg/m2, given weekly for 4 weeks, or in two 1 g infusions, given at fortnightly intervals (total 2 g). The average half-life of the drug after completion of an infusion is 21 days. The infusions can be repeated after 6–12 months, at a point when B cells start rebounding or when the patient has relapsed. The drug is generally very well tolerated, although mild hypotension can be observed in some patients, necessitating the discontinuation of antihypertensive drugs on the day of the infusions. Anaphylactic or skin reactions can occur in rare cases, but these respond to intravenous methylprednisolone. Premedication with antihistamine is desirable to prevent the occurrence of such reactions.
Rituximab has been used in combination with other immunosuppressants, such as corticosteroids, mycophenolate, cyclophosphamide, azathioprine or methotrexate,6, 62 for the treatment of vasculitis and rheumatoid arthritis, without additional complications. This experience differs from that with natalizumab, which requires discontinuation of the other immunosuppressants for 2–3 months before initiating therapy.63 It remains to be determined whether combination therapy will be more effective than monotherapy in difficult neurological cases. Rituximab has been also used effectively in some cases of pediatric SLE in two infusions of 750 mg/m2 administered 2 weeks apart, either alone or in combination with corticosteroids and cyclophosphamide,1 suggesting that it can be used in children with difficult autoimmune neurological disorders.
The resistance of long-lived plasma cells to rituximab probably explains its excellent safety profile, the absence of infections, and the patients' retained ability to produce immunoglobulins and mount an antibody response against anamnestic antigens. In spite of the apparent plasticity of the immune system, which enables it to compensate for the peripherally depleted B cells, vigilance is still required to guard against the possibility of infections in patients receiving repeated doses or concurrent immunosuppressants.3 Such infections might not be limited to common bacterial or viral agents, but might also extend to agents that cause latent infections, such as JC virus or herpesviruses, as has been experienced with natalizumab.63 Rare cases of progressive multifocal leukoencephalopathy have been reported in patients with SLE receiving rituximab, although a cause-and-effect relationship has not been established.64
A consistent observation in many series is the elevation of BAFF levels after rituximab treatment, probably as an inherent compensatory mechanism to drive B-cell production.1, 2, 3, 4, 5, 6 Theoretically, combining rituximab with one of the agents against BCMA or TACI-IgG, which reduce the survival of BAFF-dependent, immunoglobulin-producing, long-lived plasma cells, might have a prolonged effect on B cells and autoantibody levels. Such a combination therapy might be attractive in the future, in view of increased levels of BAFF in autoimmune neurological disorders.
Modes of action of rituximab
Rituximab depletes B cells through three mechanisms (Figure 6): antibody-dependent cellular cytotoxicity, whereby antibody-coated cells bind to the Fc receptors of macrophages or natural killer cells; activating the membrane attack complex on B cells (complement-dependent cytotoxicity); and inducing apoptosis by changing the lipid raft environment on the CD20+ B-cell membrane.1, 2, 3, 4, 5, 6

The most impressive observation in all published series, and from our own experience, is the long-lasting benefit of rituximab, sometimes exceeding 6–8 months after therapy. The degree of clinical response, however, varies from patient to patient, probably reflecting the varying degree of contribution of B cells to the autoimmune process, as discussed earlier. It is difficult to ascertain which of the B-cell functions depicted in Figure 1 is primarily influenced by the drug and is responsible for the noted benefit. Diminished production of pathogenetic autoantibodies might be a contributing factor, but this decrease in production might be insufficient to be clinically meaningful.27, 59 The effects of rituximab on other B-cell functions—effects that include blockade of costimulatory molecules required for clonal expansion of T cells, inhibition of the antigen-presenting role of B cells, suppression of the cytokine network, inhibition of immune complexes, and induction of immunoregulatory T cells—might be more important in explaining the noted clinical benefit. Accordingly, rituximab might be beneficial not only in antibody-mediated disorders of the CNS and PNS, but also in other autoimmune diseases where both B cells and T cells contribute to disease pathogenesis.

Conclusions and future prospects
Anti-B-cell therapy, in particular treatment with rituximab, is a promising approach for immunotherapy of neurological diseases, and it has the potential to produce long-lasting benefits. The reported excellent tolerance of rituximab administered in combination with other immunosuppressants is an important advantage, but close monitoring will be required to promptly identify any long-term sequelae or unforeseen adverse effects. Newer monoclonal antibodies designed to target B-cell survival factors might prove to be even more effective than rituximab, because they can also affect the production of immunoglobulin and antibodies by plasma cells. The B cell is an attractive target for immunotherapeutic interventions, and controlled trials with rituximab and other new agents that work through similar mechanisms are warranted for the treatment of neurological disorders.

Key points

B cells have a key role in the pathogenesis of various autoimmune neurological
disorders
A number of monoclonal antibodies or fusion proteins directed
against B-cell surface molecules and trophic factors are currently in clinical
trials
The anti-CD20 monoclonal antibody rituximab has shown promise in the
treatment of disorders such as relapsing–remitting multiple sclerosis,
autoimmune neuropathies, neuromyelitis optica, paraneoplastic neurological
disorders, stiff-person syndrome, myasthenia gravis and inflammatory myopathies
Circulating B cells, but not the antibody-producing plasma cells that do not
express CD20, become undetec Table
1 month after rituximab treatment, and levels remain low for at least 6
months
Despite B-cell depletion, patients do not seem to be prone to common
infections after rituximab treatment
Experience in conditions such as
vasculitis and rheumatoid arthritis indicates that rituximab can be used in
combination with other immunosuppressants

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