A method to isolate and cryopreserve cerebrospinal fluid mononuclear cells from external ventricular drains to investigate immunological processes in acute brain injuries.

The inflammatory response to acute brain injuries is a key contributor to subsequent outcome. The study of local central nervous system inflammatory responses is hindered by raised intracranial pressure precluding cerebrospinal fluid sampling by lumbar puncture. External ventricular drains are sited in some acute brain injury patients to divert cerebrospinal fluid and thus reduce intracranial pressure, and represent a potential route to safely gather large volumes of cerebrospinal fluid for immunological studies. In this manuscript we show that mononuclear cells can be isolated from cerebrospinal fluid collected from external ventricular drains, and that the large volumes of cerebrospinal fluid available yield sufficient mononuclear cells to allow cryopreservation. Prolonged storage of cerebrospinal fluid in the external ventricular drain collection bag can alter the phenotype of cells recovered, but the predicted effect of this can be estimated for a given flow cytometry panel by assessing the changes in peripheral blood mononuclear cells exposed to the same conditions. The described method will allow clinical studies of acute brain injuries to investigate the immunological processes occurring within the central nervous system compartment, rather than relying on changes in the peripheral circulation.

The immunological response to traumatic brain injury.

Traumatic brain injury (TBI) is the leading cause of death and disability in young adults in the developed world. The accuracy of early outcome-prediction remains poor even when all known prognostic factors are considered, suggesting important currently unidentified variables. In addition, whilst survival and neurological outcomes have improved markedly with the utilisation of therapies that optimise physiology, no treatments specifically modulate the underlying pathophysiology. The immunological response to TBI represents both a potential contributor to outcome heterogeneity and a therapeutically tractable component of the acute disease process. Furthermore, chronic inflammation has been linked with neurodegeneration, and may mark a bridge between acute brain injury and the subsequent neurodegenerative process seen in a proportion of patients following TBI. Given the complexity of the immune response and its varying functions ranging from repair of injury to bystander damage of healthy tissue, attempts at immunomodulatory intervention must necessarily be highly targeted towards the maladaptive facets of the inflammatory process. In this review we aim to provide an integrated description of the immunological processes triggered by TBI in both humans and animal models, in particular considering the interplay between the innate immune system, danger-associated molecular patterns and loss of self-tolerance leading to adaptive autoimmunity.

First use of alemtuzumab in Balo's concentric sclerosis: a case report.

Balo's concentric sclerosis (BCS) is a rare demyelinating disorder of the central nervous system. The humanised monoclonal antibody alemtuzumab has shown efficacy in another demyelinating disorder, relapsing-remitting multiple sclerosis. We aimed to explore its efficacy in treatment-refractory BCS. A 52-year-old male with radiologically confirmed progressive BCS resistant to steroids, plasmapharesis and cyclophosphamide was administered a standard protocol of alemtuzumab. Treatment failed to slow his decline; he died 6 months after administration. Why alemtuzumab induced no clinical or radiological impact may be multifactorial. We review the evidence directing BCS therapy and propose the next steps for exploring this potentially fatal condition.

Alemtuzumab more effective than interferon ß-1a at 5-year follow-up of CAMMS223 clinical trial.

OBJECTIVE: To report the long-term safety and efficacy results from CAMMS223 comparing alemtuzumab with interferon ß-1a in early, active relapsing-remitting multiple sclerosis (RRMS). What are the long-term effects of alemtuzumab treatment, received 36 to 48 months previously, on relapse and disability in early, active RRMS? This study provides evidence of the effectiveness of alemtuzumab in reducing the relapse rate and accumulation of disability compared with interferon ß-1a (IFNß-1a) through extended follow-up (up to 60 months from baseline). METHODS: Of 334 patients originally randomized, 198 participated in the extension phase (151 [68%] alemtuzumab and 47 [42%] IFNß-1a). Disability, relapses, and safety were assessed as in the original study period. Efficacy outcomes were analyzed from baseline of the original trial period to 60 months. Safety data extended beyond 60 months. RESULTS: Over 5 years, alemtuzumab lowered the risk of sustained accumulation of disability by 72% and the rate of relapse by 69% compared with IFNß-1a (both p < 0.0001). The annualized relapse rate from baseline to month 60 was 0.11 for alemtuzumab and 0.35 for IFNß-1a. Complete safety follow-up reflected 988 and 376 person-years for alemtuzumab and IFNß-1a patients, respectively. Serious infections were seen in 7% of alemtuzumab patients and 3% of IFNß-1a patients, and thyroid disorders were seen in 30% of alemtuzumab patients vs 4% of IFNß-1a patients. Immune thrombocytopenia occurred in 3% of alemtuzumab patients and 0.9% of IFNß-1a patients during the initial study period; no additional events were reported during the extension phase. One alemtuzumab patient developed Goodpasture disease 39 months after the second annual cycle of alemtuzumab. CONCLUSIONS: Through extended follow-up, alemtuzumab remained significantly more efficacious than IFNß-1a, with a safety profile consistent with previous reports. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that alemtuzumab is more effective than interferon ß-1a in reducing relapses and disability in patients with RRMS in a long-term follow-up of a rater-blinded, randomized clinical trial with 59.5% of patients participating in the extended follow-up period.

Human fetal neural precursor cells can up-regulate MHC class I and class II expression and elicit CD4 and CD8 T cell proliferation.

The use of allogeneic fetal neural precursor cells (NPCs) as a cell replacement therapy in neurodegenerative disorders holds great promise. However, previous studies concerning the possibility of alloimmune rejection of the transplanted cells have been inconclusive. Here, we used flow cytometry to quantify the expression of major histocompatibility complex (MHC) molecules by human NPCs, obtained from the cortex or ventral mesencephalon of fetuses with gestational ages between 7 and 11 weeks. MHC class I was undetectable on the surface of freshly isolated primary fetal tissue from either location, but increased over time in proliferating NPC cultures; after 7days in vitro, MHC class I was detectable on most cells. Following differentiation, MHC class I expression persisted on non-neuronal cells. MHC class II levels remained low at all time points but were inducible by pro-inflammatory cytokines, whereas the co-stimulatory molecules, CD80 and CD86, remained undetectable. Nonetheless, CD4+ and CD8+ T cells proliferated when peripheral blood mononuclear cells (PBMCs) were cultured with allogeneic NPCs. Weaker responses were obtained when NPCs were co-cultured with purified allogeneic responder T cells, suggesting that indirect allorecognition contributed significantly to PBMC responses. In conclusion, differentiating human NPCs are immunogenic in vitro, suggesting that they may trigger immune rejection unless transplant recipients are immunosuppressed.

Alemtuzumab in the treatment of IVIG-dependent chronic inflammatory demyelinating polyneuropathy.

Chronic inflammatory demyelinating polyneuropathy (CIDP) is an idiopathic immune mediated neuropathy causing demyelination and conduction block thought to occur as the result of an aberrant autoimmune response resulting in peripheral nerve inflammation mediated by T cells and humoral factors. Diagnosis commonly prompts initial treatment with steroids or intravenous immunoglobulin (IVIG) on which 5-35% subsequently become dependent to maintain function. Despite a number of small scale trials, the role for alternative long-term immunosuppression remains unclear. Alemtuzumab is a humanised monoclonal antibody targeting the CD52 antigen present on the surface of lymphocytes and monocytes. A single intravenous infusion results in rapid and profound lymphopoenia lasting >12 months. We report its use and clinical outcome in a small series of patients with severe IVIG-dependent CIDP. Seven patients (4 Males; 3 Females) who had failed to respond to conventional immunosuppression were treated in 5 centres receiving 9 courses of alemtuzumab (dose range 60-150 mg). Following treatment, mean monthly IVIG use fell 26% from 202 to 149 g and IVIG administration frequency from 22 to 136 days. Two patients had prolonged remission, two patients had a partial response and no clear benefit was observed in the remaining three patients (2 Males, 1 Females). Responding patients had a younger age at onset (19.5 years) and shorter disease duration than non-responders. Three patients developed autoimmune disease following treatment. Alemtuzumab may offer an alternative treatment for a subset of early onset IVIG dependent CIDP patients failing conventional immunosuppressive agents, but concerns about toxicity may limit its use.

Spotlight on alemtuzumab.

Alemtuzumab, formally known as Campath-1H, is a humanized monoclonal antibody directed against CD52, a protein on the surface of lymphocytes and monocytes with unknown function. A single dose of alemtuzumab leads to a rapid, profound and prolonged lymphopenia. A Phase II trial has shown that alemtuzumab reduces the risk of relapse and accumulation of disability by over 70% compared with interferon beta in patients with early relapsing-remitting multiple sclerosis (MS). Alemtuzumab has been used in Cambridge as an experimental treatment for MS since 1991. In this review we summarize our experience; describing how this prototypical, "bench-to-bedside" therapy continues to inform basic science, revealing aspects of the pathogenesis of MS and lymphopeniaassociated autoimmunity.

Rationale for cytotoxic monoclonal antibodies in MS.

Monoclonal antibodies (MAbs) have been used to treat human disease for over 20 years, and have found a range of applications, predominantly in inflammatory and neoplastic disorders. They are attractive therapies due to their unique specificity and capacity for production on an industrial scale. Cytotoxic MAbs, those that trigger lysis of their cellular target, were first used to probe experimental allergic encephalomyelitis, the standard animal model of MS. Since then, antibodies with specificity for a variety of lymphocytic antigens have been used in MS, with variable results. On the basis of experience with the anti-CD52 antibody, alemtuzumab (Campath-1H), it has been postulated that clinical efficacy of some MAbs in MS may not result from cellular depletion, but from the immunomodulatory effect of the subsequent immune system reconstitution.

An investigation of auto-reactivity after head injury.

Murine models of CNS injury show auto-reactive T cell responses directed at myelin antigens, associated with improved neuronal survival and functional recovery. This pilot study shows, for the first time, that similar immune responses against myelin occur in human traumatic brain injury (TBI), with an expansion of lymphocytes recognising myelin basic protein observed in 40% of patients studied. "Reactive" patients did not have greater contusion volume on imaging, but were younger than the "unreactive" subgroup and tended towards a more favorable outcome. These findings are consistent with the concept of "beneficial autoimmunity".

Pulsed monoclonal antibody treatment and autoimmune thyroid disease in multiple sclerosis.

BACKGROUND: Multiple sclerosis results from T-cell-dependent inflammatory demyelination of the central nervous system. Our objective was long-term suppression of inflammation with short-term monoclonal antibody treatment. METHODS: We depleted 95% of circulating lymphocytes in 27 patients with multiple sclerosis by means of a 5-day pulse of the humanised anti-CD52 monoclonal antibody, Campath-1H. Clinical and haematological consequences of T-cell depletion, and in-vitro responses of patients' peripheral-blood mononuclear cells were analysed serially for 18 months after treatment. FINDINGS: Radiological and clinical markers of disease activity were significantly decreased for at least 18 months after treatment. However, a third of patients developed antibodies against the thyrotropin receptor and carbimazole-responsive autoimmune hyperthyroidism. The depleted peripheral lymphocyte pool was reconstituted with cells that had decreased mitogen-induced proliferation and interferon gamma secretion in vitro. INTERPRETATION: Campath-1H causes the immune response to change from the Th1 phenotype, suppressing multiple sclerosis disease activity, but permitting the generation of antibody-mediated thyroid autoimmunity.

Monoclonal antibody treatment exposes three mechanisms underlying the clinical course of multiple sclerosis.

The elective treatment of patients with multiple sclerosis, using a humanized anti-leukocyte (CD52) monoclonal antibody (Campath-1H), has illuminated mechanisms that underlie the clinical course of the disease. Twenty-seven patients were studied clinically and by magnetic resonance imaging (MRI) before and for 18 months after a single pulse of Campath-1H. The first dose of monoclonal antibody was associated with a transient rehearsal of previous symptoms caused by the release of mediators that impede conduction at previously demyelinated sites; this effect remained despite selective blockade of tumor necrosis factor-alpha. Disease activity persisted for several weeks after treatment but thereafter radiological markers of cerebral inflammation were suppressed for at least 18 months during which there were no new symptoms or signs. However, about half the patients experienced progressive disability and increasing brain atrophy, attributable on the basis of MRI spectroscopy to axonal degeneration, which correlated with the extent of cerebral inflammation in the pretreatment phase. These data support the formulation that inflammation and demyelination are responsible for relapses of multiple sclerosis; that inflammatory mediators, but not tumor necrosis factor-alpha, cause symptomatic reactivation of previously demyelinated lesions; and that axonal degeneration, conditioned by prior inflammation but proceeding despite its suppression, contributes to the progressive phase of disability. These results provide evidence supporting the emerging view that treatment in multiple sclerosis must be given early in the course, before the consequences of inflammation are irretrievably established.

Quantitative MRI in patients with secondary progressive MS treated with monoclonal antibody Campath 1H.

BACKGROUND: To assess the long-term effect of the lymphocyte-depleting humanized monoclonal antibody Campath 1H on MR markers of disease activity and progression in secondary progressive MS patients. METHODS: Twenty-five patients participated in a crossover treatment trial with monthly run-in MR scans for 3 months, followed (after a single pulse of Campath 1H) by monthly MR scans from months 1 to 6 and again from months 12 to 18. MR analysis was performed to provide measurements of the number and volume of gadolinium (Gd)-enhancing lesions as well as the hypointense lesion volume on a T1-weighted sequence. In addition, serial measurements of T2 brain lesion volume, brain volume, and spinal cord cross-sectional area were made over the duration of the study. The relationship between clinical and MR measures of disease evolution was also assessed. RESULTS: Treatment was associated with a reduction in the number and volume of Gd-enhancing lesions (p < 0.01). Despite this, a decrease in brain volume was seen in 13 patients during the 18 months post-treatment. The mean pretreatment Gd-enhancing lesion volume was predictive of subsequent reduction in brain volume (r = 0.77, p = 0.002). Reduction in brain volume also correlated with the change in T1 hypointense lesion volume after treatment (r = 0.53, p < 0.01). A reduction in spinal cord area was also seen throughout the study duration, and this correlated with an increase in disability (r = 0.65, p = 0.01). CONCLUSION: Campath 1H treatment was associated with a sustained and marked reduction in the volume of Gd enhancement, indicating suppression of active inflammation. Nevertheless, many patients developed increasing brain and spinal cord atrophy, T1 hypointensity, and disability. This study highlights the potential role for novel MR techniques in monitoring the effect of treatment on the pathologic process in MS.

Disease activity and the immune set in multiple sclerosis: blood markers for immunotherapy.

There is no established immunological marker of multiple sclerosis activity, which reflects the poor understanding of the immunopathogenesis of multiple sclerosis. Passive measurement of the levels of soluble inflammatory markers, whose half lives are usually measured in minutes and hours, can only indicate the extent of instantaneous inflammation, which is known to fluctuate in multiple sclerosis. We favour measurement of immune responses in vitro. As healthy individuals have T cell reactivities to myelin proteins that are postulated to be pathogenic in multiple sclerosis, we prefer non-antigen specific mitogen and recall antigen assays as immunological markers. We illustrate their use in the treatment of 27 patients with multiple sclerosis using a pulse of humanised anti-lymphocyte (CD52) antibody that caused prolonged T cell depletion. The mitogen-induced proliferation, and secretion of IFN-gamma, from peripheral blood mononuclear cells in vitro was significantly reduced after treatment, suggesting that immune responses had been modulated. Such observations will only gain credence as an outcome measure if they are shown to correlate with clinical or radiological measures of multiple sclerosis activity. Perhaps more importantly, aspects of the pathogenesis of multiple sclerosis may be revealed by close immunological surveillance of patients undergoing experimental treatments.

Secondary progressive multiple sclerosis: the relationship between short-term MRI activity and clinical features.

We report the findings in 60 patients with secondary progressive multiple sclerosis who had monthly brain MRI studies for 4 months (one baseline and three follow-up scans). The purpose was to define the short-term MRI natural history in a large cohort with secondary progressive disease and to ascertain its relationship with other clinical and MRI features. The patients were participating in either a natural history study or the placebo arm or non-treatment phase of a therapeutic trial. The cohort had clinical features typical of secondary progressive disease: thus, all had moderate or severe locomotor disabilities [Expanded Disability Status Scale (EDSS), score 3.5-8], with a median disease duration of 12 years. There was equal representation of males and females. During the 3 months of follow-up there was a total of 362 new enhancing lesions seen in 42 patients, and there were 24 relapses in 20 patients. There was no correlation between new enhancing lesions and age at study entry, age of disease onset, gender disease duration or EDSS, but there was a strong correlation with the number of enhancing lesions on the baseline scan (r = 0.65, P < 0.0001) and subsequent activity. There was a non-significant trend for higher numbers of new enhancing lesions in those having relapses during the 3 months of scanning (P = 0.14) or in the preceding 6 months (P = 0.06). The 34 patients who did not relapse in either period had significantly fewer new active lesions (P = 0.02) than those who relapsed at some stage during the 9 months. Nevertheless, considerable activity was seen in the non-relapsing cohorts: there was a mean of 3.5 (median 2) new enhancing lesions in those not relapsing during the 3 month study, and 5.5 (median 2) in those not relapsing in the previous 6 months. We conclude that short-term MRI activity is generally high in secondary progressive disease, confirming a useful role for the technique in exploratory trials. Further work should concentrate on elucidating the mechanisms of secondary progression by longer term follow-up studies of larger cohorts using multiple MRI and clinical measurements.