MS Abstracts 4e-2g

Since the PathoGenesis of Multiple Sclerosis (MS) lesions is not yet fully understood, we investigated the potential of Dynamic Susceptibility Contrast (DSC) Magnetic Resonance (MR) perfusion imaging for a better characterization of Lesion Pathology.

Twenty-five MS patients were examined on a 1.5 T scanner. A single dose of Gadolinium (Gd)-DOTA contrast agent was injected, and EchoPlanar images were acquired every 0.5 seconds for 1 minute.

From the signal intensity-versus-time curves, the relative Cerebral Blood Volume (rCBV) was evaluated for regions in Plaques and in Gray Matter and White Matter.

The rCBV calculated for acute, Gd-enhancing Plaques was corrected for the effects of Blood-Brain Barrier leakage, using a new correction algorithm.

1. Acute Plaques had significantly higher blood volumes than Normal-Appearing White Matter (P < = 0.01).

2. Chronic Plaques that appeared HypoIntense on T₁-weighted images had lower rCBV than T1-IsoIntense Plaques (P < = 0.03).

Our results indicate that the acute phase in MS is accompanied by Vasodilation. In later stages of Gliosis, the perfusion decreases with increasing Axonal injury.

Although the DSC technique is less sensitive than conventional MR imaging, the information provided is essentially different from that obtained with any other MR method.

J. Magn. Reson. Imaging 2000;11:495-505. Copyright 2000 Wiley-Liss, Inc.

One of the actions of GlucoCorticoids (GC) in Multiple Sclerosis (MS) is an inhibitory effect on DeMyelination.

This can be caused by a reduction in the number of infiltrating Macrophages and/or by an effect on the Phagocytosis of Myelin. Here we investigate the effect of GC on the Phagocytosis of Myelin.

Contrary to what was expected, we found that incubation of human Monocytes with Dexamethasone (DEX) for 48 h augmented (approximately threefold) the Phagocytosis of Myelin.

This enhancement of Phagocytosis by human Monocytes was not restricted to Myelin. Phagocytosis of various particles mediated by different Macrophage Receptors was increased by DEX.

We found that not only the Phagocytosis of Staphylococcus Aureus Bacteria was augmented, but also the killing of these Bacteria was at least twice as effective after culture with DEX.

Tumor Necrosis Factor- production of human Monocyte-derived Macrophages induced by LipoPolySaccharide and S. Aureus was suppressed by DEX.

Together our results show that DEX promotes the Phagocytosis of particles by human Monocytes and thereby may contribute to tissue repair after Immune-mediated tissue damage or infection. These data shed a new light on the clinical application of GC.

Multiple Sclerosis is a DeMyelinating Disorder of the Central Nervous System (CNS), in which an Immune attack directed against Myelin constituents causes Myelin destruction and death of Oligodendrocytes, the Myelin-producing cells.

Here, the efficacy of Nerve Growth Factor (NGF), a growth factor for Neurons and Oligodendrocytes, in promoting Myelin repair was evaluated using the DeMyelinating model of Experimental Allergic EncephaloMyelitis (EAE) in the common marmoset.

Surprisingly, we found that NGF delayed the onset of clinical EAE and, pathologically, prevented the full development of EAE lesions.

We demonstrate by ImmunoCytoChemistry that NGF exerts its AntiInflammatory effect by downregulating the production of Interferon-gamma by T-Cells infiltrating the CNS, and upregulating the production of InterLeukin-10 (IL-10) by Glial Cells in both Inflammatory lesions of EAE and Normal-Appearing CNS White Matter.

Thus, NGF, currently under investigation in human clinical trials as a Neuronal Trophic Factor, may be an attractive candidate for therapy of AutoImmune DeMyelinating Disorders.

Increasing evidences show a Global Immune Disregulation in Multiple Sclerosis (MS).

The possible involvement of Myelin and Non-Myelin AutoAntigens in the AutoAggressive process as well as the disregulation of both Adaptive and Innate Immunity challenge the concept of specific ImmunoTherapy.

T-Cells at the boundary between Innate and Adaptive Immunity, whose ImmunoRegulatory role is becoming increasingly clear, have recently been shown to bear relevance for MS PathoGenesis.

Global Immune interventions (and Type I Interferons may be considered as such) aimed at interfering with both Innate and Acquired Immune Responses seem to be a most promising therapeutic option in MS.

Experimental Allergic EncephaloMyelitis (EAE) is a T-Cell-mediated, AutoImmune Disorder characterized by Central Nervous System (CNS) Inflammation and DeMyelination, features reminiscent of the human disease, Multiple Sclerosis (MS).

In addition to the signal the Encephalitogenic T-Cell receives through the T-Cell Receptor (TCR), a second signal, termed CoStimulation, is required for complete T-Cell activation.

The B7 family of cell surface molecules expressed on Antigen Presenting Cells (APCs) is capable of providing this second signal to T-Cells via two receptors, CD28 and CTLA-4.

Our studies have shown that CoStimulation provided by B7 molecules to its Ligand CD28 is important in the initiation of the AutoImmune Response in EAE.

Further, it appears the CoStimulation provided by B7-1 is important in disease development, while B7-2 may play an important regulatory role. We and others later showed that B7/CTLA-4 interaction plays a critical role in down-regulating the Immune Response.

Previous work has shown that activated T-Cells and T-Cells of a Memory phenotype are less dependent on CoStimulation than naive T-Cells.

T-Cells reactive with Myelin components that are involved in the PathoGenesis of EAE and possibly MS would be expected to have been activated as part of the disease process.

Building upon our prior work in the EAE model, we have tested the hypothesis that Myelin-Reactive T-Cells, which are relevant to the PathoGenesis of CNS Inflammatory DeMyelination, can be distinguished from naive Myelin-Reactive T-Cells by a lack of dependence upon CoStimulation for activation and that the CoStimulatory requirements of these Myelin-Reactive T-Cells change during the course of disease.

Our studies in the EAE model have also addressed the mechanisms of ExtraThymic (Peripheral) T-Cell Tolerance following IntraVenous (I.V.) administration of high dose Antigen.

It is believed that TCR signaling in the absence of CoStimulation is a vital component of peripheral Tolerance mechanisms.

However, recent evidence suggests that peripheral Tolerance of Antigen-Specific T-Cells induced in vivo may require CTLA-4 engagement of the Tolerized T-Cells.

We have begun to examine the molecular mechanisms of Tolerance induction following IntraVenous and intraperitoneal administration of Myelin Antigens in the EAE model and test the hypothesis that Tolerance induction is dependent on the B7:CD28/CTLA-4 pathway.

The results from our studies will enhance our understanding of the role that Myelin-Reactive T-Cells may play in the PathoGenesis of MS.

We have determined that MBP-Reactive T-Cells in MS patients are less dependent upon CD28 CoStimulation than in normal controls, suggesting that these T-Cells were previously primed in vivo.

Characterization of these CD28-independent Myelin-Specific T-Cells will have broad implications for a variety of Immunologically based therapies in diseases such as MS.

Pro-Inflammatory Cytokines play a crucial role in the regulatory and Effector phase of the Immune-mediated mechanism sustaining Multiple Sclerosis PathoGenesis (MS) thus supporting the use of Anti-Inflammatory Cytokines as a therapeutic option.

Systemic administration of Cytokines shows, however, limited therapeutic efficacy and undesirable/unpredictable side-effects.

We have developed a non-toxic system to deliver Cytokines within the Central Nervous System (CNS) based on the Intrathecal (i.c.) administration of non-replicative Herpes Simplex (HSV) type-1-derived Viral vectors engineered with heterologous Cytokine Genes.

Compared to controls, mice affected by Experimental AutoImmune EncephaloMyelitis (EAE) and i.c. injected with an HSV-1-derived vector containing the Gene of the Anti-Inflammatory Cytokine IL-4 showed a significant amelioration of clinical and pathological EAE signs.

A decreased mRNA expression of the Monocyte ChemoAttractant protein-1 (MCP-1) by Mononuclear CNS-infiltrating cells was also observed.

Peripheral T-Cells from IL-4-treated mice were not affected both in their Antigen-specific proliferative response and in the Cytokine secretion pattern.

Our results indicate that CNS Cytokine delivery with HSV-1-derived vectors is a feasible therapeutic strategy and might represent an alternative approach for the treatment of Immune-mediated DeMyelinating diseases.

Advantages of this approach over systemic Cytokine administration are the high Cytokine level reached within the CNS and the absence of side-effects on the peripheral Immune System.

The short-lasting Cytokine production in the CNS after a single vector administration (4 weeks) is the limiting factor of this novel technology which, although promising, has to be improved.

Although AutoReactive T-Cells have a pivotal role in initiating the Inflammatory process in Experimental AutoImmune EncephaloMyelitis (EAE) and Multiple Sclerosis (MS), recent evidence suggests a relevant role for AutoAntiBodies specific for Myelin proteins as well.

To examine the role of B-Cells in the CerebroSpinal Fluid of patients with MS, we analyzed the V(H) Gene usage in ten MS patients by PCR technologies. Analysis of HCDR3 length revealed an OligoClonal accumulation of B-Cells.

Sequence analysis of the V(H)3 and V(H)4 gamma transcripts of two MS individuals demonstrated that this accumulation was related to the expansion and Somatic diversification of a limited groups of B-Cell clones.

These findings are indicative of a chronic and intense Antigenic stimulation occurring in the CNS. Animal models, such as EAE, are of particular importance in order to elucidate the PathoGenetic effector mechanisms in AutoImmune DeMyelination.

In a non-human primate model of EAE, we describe that the ImmunoDominant T-Cell Epitope is presented exclusively by a Monomorphic DRB1 allele, suggesting that susceptibility to EAE may be linked to this unique restriction and, therefore, providing a possible mechanism for MHC linkage to diseases.

Moreover, we report on the presence of Inflammation, sharp DeMyelination and Axonal damage in EAE induced with whole Myelin as well as with recombinant Myelin Oligodendrocyte Glycoprotein (MOG), but not with Myelin Basic Protein alone.

The presence of Axonal pathology was supported by ImmunoHistoChemistry with Anti-Amyloid Precursor Protein and Anti-Non Phosphorilated NeuroFilaments MonoClonal AntiBodies within early active DeMyelinated Plaques.

These findings suggest that Axonal damage may be an early event in the PathoGenesis of AutoImmune DeMyelinating Diseases of the CNS and highlights the importance of animal models in which therapies targeting repair and Axonal survival may be exploited.

Phagocytosis removes Pathogens and tissue debris during Inflammatory reactions, but also plays an important role in AutoImmune reactions. The main Phagocytes in the Central Nervous System (CNS) are Microglial Cells that are activated during CNS Inflammation.

In the treatment of Inflammatory DeMyelinating Diseases like Multiple Sclerosis (MS), administration of IntraVenous ImmunoGlobulins (IVIg) has become a promising ImmunoModulatory therapy.

Although a large number of potential mechanisms for the effects of IVIg has been suggested, the precise mode of action in CNS Inflammation is unknown.

We assessed the influence of IVIg on Phagocytosis and Endocytosis in Microglia in vitro.

IVIg had little effect on Non-Specific Phagocytosis of latex particles in untreated Microglia, while there was a dose-dependent inhibition in Microglia activated with LPS and IFN-γ.

Endocytosis of soluble Myelin Basic Protein (MBP) was downregulated by IVIg in both untreated and activated Microglia.

The effect was mediated by an F(ab')(2) preparation of ImmunoGlobulins, suggesting that Fc receptor-mediated Phagocytosis is not involved.

Intact IVIg, but not F(ab')(2) fragments also suppressed Fc receptor-mediated Phagocytosis of opsonised Erythrocytes in both untreated and activated Microglia.

These results show that IVIg can inhibit the Phagocytic activity of Microglia via different mechanisms. Such an effect could contribute to the ImmunoModulatory capacity of IVIg in Inflammatory CNS Diseases.