Studies of Bone Marrow Mesenchymal (Stromal) Cells and Neural Precursor Stem Cells transplantations in experimental models of Multiple Sclerosis and Stroke
In multiple sclerosis (MS), a major direction of current research is to devise strategies that will remyelinate axons and protect them against subsequent degeneration which leads to chronic disability (Bjartmar et al., 2003). On a relatively similar basis, stroke-oriented therapies aim either to replenish the lost neuronal structures or implement a kind of neuroprotection to the penumbra zone in order to ameliorate the neuropathology and thus the final clinical impairment.
The concept of cell-based therapies has been considered among the most challenging areas of investigation during the last years. Several cell types have been experimentally used to remyelinate axons in demyelinating CNS areas of rodent models. Among them, neural stem/precursor cells (NPCs) and bone marrow mesenchymal stem cells or stromal cells (BMSCs), have extensively been studied in various animal models of either the experimental autoimmune encephalomyelitis (EAE) or models of experimental focal cerebral ischemia, such as the model of Middle Cerebral Artery Occlusion (MCAO).

Studies in our Laboratory indicate for the first time, that there are important safety issues concerning the use of BMSCs grafts, since these cells were found capable of inducing mass formation after their intracerebroventricular (ICV) transplantation within the CNS. This phenomenon was correlated with local density of grafted cells after their periventricular migration and was strictly related to BMSCs and not NPCs.

Neuropathological collaborative studies with Research Groups from Israeli UniversitiesThe ENNI Lab has been collaborating with distinguished Research Groups from the Hadassah Hebrew University of Jerusalem and Tel-Aviv Universities since 1999. Our collaborations include the study of underlying Neuropathology in several experimental models conducted by the Israeli Groups. These models include tauopathies, Traumatic Brain Injury (TBI), Amyotrophic Lateral Sclerosis (ALS), Neuromyelitis Optica (NMO) and EAE. These collaborations lead to publish papers in Peer-reviewed Journals and participation in various research Grants/Programs.

Studies of Nogo-A/NgR signaling and kinetics in Experimental Autoimmune Encephalomyelitis
Nogo-A protein belongs to a family of myelin-associated neurite outgrowth inhibitors such as myelin-associated glycoprotein (MAG), oligodendrocyte myelin glycoprotein (OMgp) and CSPG proteoglycans that are partially responsible for the limited ability of axonal regeneration in the adult central nervous system (CNS) of higher vertebrates. These inhibitors exert their effect through a common receptor, the Nogo-receptor (NgR) and its complex with several other molecules such as TROY and Lingo-1. Studies indicate an abundant Nogo-A mRNA expression in brain and spinal cord of rat, mouse and human CNS, contrasted to a more specific NgR expression.

Studies in our Laboratory have elucidated the neuroanatomical distribution of Nogo-A specific mRNA in the healthy adult mouse CNS. Moreover, we are studying the temporal and spatial expression profiles of Nogo-A and its signaling cascade in the model of EAE, in an attempt to illustrate and clarify the exact association of Nogo-A to the axonal injury and regeneration in different disease phases.

Experimental Stroke studies
Experimental models of transient or temporary ischemic stroke apply various types of arterial occlusion with different results on infarct volume, brain edema and clinical and behavioral outcome. Among the models used, the one of transient Middle Cerebral Artery Occlusion (MCAO) with the aid of an intraluminal suture is considered the most common. The most common types of intraluminal suture used for the rat MCAO has long the ones of Koizumi et al (1986) and Longa et al (1989) initially described almost 20 years ago. However, lack of almost 100% efficacy in both types and the side effect of Subarachnoid hemmorhage (SAH) are the major disadvantages of these techniques.

In our Laboratory we have developed a modification of the known Koizumi suture which efficiently produces ischemic infarction in Wistar rats with no SAH complications, irrespective of the animal’s weight. Moreover, through the use of this model we have built a simple mathematical model (based solely on the animal’s clinical score) to predict new stereotactic coordinates for intracerebroventricular (ICV) infusions during the acute phase of stroke (6-24 hours post stroke). Though this approach is not yet highly preferred in the literature (compared to intravenous and selective intraarterial or intraparenchymal routes), various data highlight significant advantages of the direct ICV route such as bypassing of the blood brain barrier, achieving high drug concentrations in the CSF compartment and avoiding the periphery.

Studies of the effect of Helicobacter pylori (Hp) – related immune reactions in Experimental Autoimmune Encephalomyelitis
Existing evidence from epidemiological studies highlight numerous infections as potential environmental MS triggers; infection can prime autoimmune T cells specific for CNS antigens, if the pathogen has molecular mimicry with CNS proteins. Several pathogens have been under investigation, including Epstein-Barr virus, Chlamydia pneumoniae and Human Herpes Virus-6.

Helicobacter pylori (Hp), a spiral gram-negative bacterium that colonizes human gastric mucosa, has recently also been linked to a number of central and peripheral nervous system disorders. Hp has long been described to induce gastrointestinal disease by means of molecular mimicry. However, the concept of a role of Hp infection in MS is based on a rather limited number of clinical studies yet lacking the support from experimental studies investigating the relative effect of Hp systemic immunization.

In our Laboratory, in collaboration with the group of Dr. D. Sgouras in the Hellenic Pasteur Institute in Athens and Professor I. Kountouras group at the Aristotle University, worldwide known for their work on the Hp-neurodegeneration relationship, we currently study the hypothesis whether Hp modifies immunological reaction in mice with EAE.

Studies of Altered Peptide Ligands in Experimental Autoimmune Encephalomyelitis (Patent protected)

Experimental Autoimmune Encephalomyelitis (EAE) is an experimental model of demyelination, inflammatory processes and axonopathy within the Central Nervous System (CNS) of susceptible animals, using the triggering of various CNS antigens such as MBP, PLP and MOG proteins or their peptides. EAE is induced to susceptible animals using linear peptides (antigens) of myelin proteins of the CNS. The ability of these peptides to trigger immune reactions is in great extent determined by the properties of the MHC-II haplotype of the animal. In other words, specific peptide motifs within the antigen-binding groove of the MHC complex determine the affinity of the antigen in each animal and the consequent T-cell receptor (TCR) matching and activation of the cell. This MHC-antigen-TCR trimolecular complex is the key step to induce EAE with antigenic peptides and is possibly a key factor to MS immune-pathology as well. Thus, any modification to the antigenic peptide and any caused deviation from a tight MHC-antigen-TCR match can lead to reduced T-cell activation and different profiles of secreted cytokines afterwards.

Based on this concept, there have been several attempts to alter the trimolecular complex affinities and render autoreactive T-cells in MS and EAE inactive or eliminated. This concept is known as “Altered Peptide Ligand” (APL) immunomodulation.

In our Laboratory, in collaboration with the Chemical Department of Patra’s University (group of Prof. J. Matsoukas), we used a rather “different” APL therapeutic approach to ameliorate EAE in C57/bl mice. Prof. Matsoukas’ group have synthesized the MOG35-55 peptide and altered its 3-dimensional conformation to make it a cyclic one, thus rendering it less encephalitogenic and potentially immunomodulating EAE.