Avi Cohen

Posttraumatic therapeutic vaccination with modified myelin self-antigen prevents complete paralysis while avoiding autoimmune disease

Spinal cord injury results in a massive loss of neurons, and thus of function. We recently reported that passive transfer of autoimmune T cells directed against myelin-associated antigens provides acutely damaged spinal cords with effective neuroprotection. The therapeutic time window for the passive transfer of T cells was found to be at least 1 week. Here we show that posttraumatic T cell-based active vaccination is also neuroprotective. Immunization with myelin-associated antigens such as myelin basic protein (MBP) significantly promoted recovery after spinal cord contusion injury in the rat model. To reduce the risk of autoimmune disease while retaining the benefit of the immunization, we vaccinated the rats immediately after severe incomplete spinal cord injury with MBP-derived altered peptide ligands. Immunization with these peptides resulted in significant protection from neuronal loss and thus in a reduced extent of paralysis, assessed by an open-field behavioral test. Retrograde labeling of the rubrospinal tracts and magnetic resonance imaging supported the behavioral results. Further optimization of nonpathogenic myelin-derived peptides can be expected to lead the way to the development of an effective therapeutic vaccination protocol as a strategy for the prevention of total paralysis after incomplete spinal cord injury.

Tumor necrosis factor facilitates regeneration of injured central nervous system axons

The results of this study attribute to tumor necrosis factor (TNF) a role in regeneration of injured mammalian central nervous system (CNS) axons which grow into their own degenerating environment. This is the first time that a specific factor involved in axonal regeneration has been identified. The axonal environment is occupied mostly by glia cells, i.e., astrocytes and oligodendrocytes. Previous studies have shown that mature oligodendrocytes are inhibitory to axonal growth. Therefore, it seemed likely that application of a factor such as TNF, which has been shown to be cytotoxic to oligodendrocytes, would contribute to the creation of permissive conditions for axonal regeneration. In the present work, injured adult rabbit optic nerves were treated with human recombinant TNF (rhTNF). As a result, abundant newly growing axons (circa 9000, about 4% of the total estimated number of axons in an intact adult rabbit) were observed traversing the site of injury.

Molecular imaging of cell death in vivo by a novel small molecule probe

Apoptosis has a role in many medical disorders, therefore assessment of apoptosis in vivo can be highly useful for diagnosis, follow-up and evaluation of treatment efficacy. ApoSense is a novel technology, comprising low molecular-weight probes, specifically designed for imaging of cell death in vivo. In the current study we present targeting and imaging of cell death both in vitro and in vivo, utilizing NST-732, a member of the ApoSense family, comprising a fluorophore and a fluorine atom, for both fluorescent and future positron emission tomography (PET) studies using an 18F label, respectively. In vitro, NST-732 manifested selective and rapid accumulation within various cell types undergoing apoptosis. Its uptake was blocked by caspase inhibition, and occurred from the early stages of the apoptotic process, in parallel to binding of Annexin-V, caspase activation and alterations in mitochondrial membrane potential. In vivo, NST-732 manifested selective uptake into cells undergoing cell-death in several clinically-relevant models in rodents: (i) Cell-death induced in lymphoma by irradiation; (ii) Renal ischemia/reperfusion; (iii) Cerebral stroke. Uptake of NST-732 was well-correlated with histopathological assessment of cell-death. NST-732 therefore represents a novel class of small-molecule detectors of apoptosis, with potential useful applications in imaging of the cell death process both in vitro and in vivo.

Vaccination with Dendritic Cells Pulsed with Peptides of Myelin Basic Protein Promotes Functional Recovery from Spinal Cord Injury

Injury-induced self-destructive processes cause significant functional loss after incomplete spinal cord injury (SCI). Cellular elements of both the innate (macrophage) and the adaptive (T-cell) immune response can, if properly activated and controlled, promote post-traumatic regrowth and protection after SCI. Dendritic cells (DCs) trigger activation of effector and regulatory T-cells, providing a link between the functions of the innate and the adaptive immune systems. They also initiate and control the bodys response to pathogenic agents and regulate immune responses to both foreign and self-antigens. Here we show that post-injury injection of bone marrow-derived DCs pulsed with encephalitogenic or nonencephalitogenic peptides derived from myelin basic protein, when administered (either systemically or locally by injection into the lesion site) up to 12 d after the injury, led to significant and pronounced recovery from severe incomplete SCI. No significant protection was seen in DC recipients deprived of mature T-cells. Flow cytometry, RT-PCR, and proliferation assays indicated that the DCs prepared and used here were mature and immunogenic. Taken together, the results suggest that the DC-mediated neuroprotection was achieved via the induction of a systemic T-cell-dependent immune response. Better preservation of neural tissue and diminished formation of cysts and scar tissue accompanied the improved functional recovery in DC-treated rats. The use of antigen-specific DCs may represent an effective way to obtain, via transient induction of an autoimmune response, the maximal benefit of immune-mediated repair and maintenance as well as protection against self-destructive compounds.

ApoSense: a novel technology for functional molecular imaging of cell death in models of acute renal tubular necrosis

PurposeAcute renal tubular necrosis (ATN), a common cause of acute renal failure, is a dynamic, rapidly evolving clinical condition associated with apoptotic and necrotic tubular cell death. Its early identification is critical, but current detection methods relying upon clinical assessment, such as kidney biopsy and functional assays, are insufficient. We have developed a family of small molecule compounds, ApoSense, that is capable, upon systemic administration, of selectively targeting and accumulating within apoptotic/necrotic cells and is suitable for attachment of different markers for clinical imaging. The purpose of this study was to test the applicability of these molecules as a diagnostic imaging agent for the detection of renal tubular cell injury following renal ischemia.MethodsUsing both fluorescent and radiolabeled derivatives of one of the ApoSense compounds, didansyl cystine, we evaluated cell death in three experimental, clinically relevant animal models of ATN: renal ischemia/reperfusion, radiocontrast-induced distal tubular necrosis, and cecal ligature and perforation-induced sepsis.ResultsApoSense showed high sensitivity and specificity in targeting injured renal tubular epithelial cells in vivo in all three models used. Uptake of ApoSense in the ischemic kidney was higher than in the non-ischemic one, and the specificity of ApoSense targeting was demonstrated by its localization to regions of apoptotic/necrotic cell death, detected morphologically and by TUNEL staining.ConclusionApoSense technology should have significant clinical utility for real-time, noninvasive detection of renal parenchymal damage of various types and evaluation of its distribution and magnitude; it may facilitate the assessment of efficacy of therapeutic interventions in a broad spectrum of disease states.

Molecular Imaging of Neurovascular Cell Death in Experimental Cerebral Stroke by PET

Clinical molecular imaging of apoptosis is a highly desirable yet unmet challenge. Here we provide the first report on 18F-labeled 5-fluoropentyl-2-methyl-malonic acid (18F-ML-10), a small-molecule, 18F-labeled PET tracer for the imaging of apoptosis in vivo; this report includes descriptions of the synthesis, radiolabeling, and biodistribution of this novel apoptosis marker. We also describe the use of 18F-ML-10 for small-animal PET of neurovascular cell death in experimental cerebral stroke in mice. Methods: 18F-ML-10 was synthesized by nucleophilic substitution from the respective mesylate precursor, and its biodistribution was assessed in healthy rats. Permanent occlusion of the middle cerebral artery (MCA) was induced in mice, and small-animal PET was performed 24 h later. Results: Efficient radiolabeling of ML-10 with 18F was achieved. Biodistribution studies with 18F-ML-10 revealed rapid clearance from blood (half-life of 23 min), a lack of binding to healthy tissues, and rapid elimination through the kidneys. No significant tracer metabolism in vivo was observed. Clear images of distinct regions of increased uptake, selectively in the ischemic MCA territory, were obtained in the in vivo small-animal PET studies. Uptake measurements ex vivo revealed 2-fold-higher uptake in the affected hemisphere and 6- to 10-fold-higher uptake in the region of interest of the infarct. The cerebral uptake of 18F-ML-10 was well correlated with histologic evidence of cell death. The tracer was retained in the stroke area but was cleared from blood and from intact brain areas. Conclusion: 18F-ML-10 is useful for noninvasive PET of neurovascular histopathology in ischemic cerebral stroke in vivo. Such an assessment may assist in characterization of the extent of stroke-related cerebral damage and in the monitoring of disease course and effect of treatment.

Dichotomy of the glial cell response to axonal injury and regeneration.

Neurons in the mammalian central nervous system (CNS) have a poor capacity for regenerating their axons after injury. In contrast, neurons in the CNS of lower vertebrates and in the peripheral nervous system (PNS) of mammals are endowed with a high posttraumatic capacity to regenerate. The differences in regenerative capacity have been attributed to the different compositions of the respective cellular environments and to different responses to injury the nonneuronal cells display, which range from supportive and permissive to nonsupportive and hostile for regeneration. The same cell type may support or inhibit regeneration, depending on its state of maturity or differentiation. Astrocytes and oligodendrocytes are examples of cells in which such a dichotomy is manifested. In developing and in spontaneously regenerating nerves, these cells support (astrocytes) and permit (oligodendrocytes) growth. However, in nonregenerating adult mammalian nerves, astrocytes form the nonsupportive scar tissue; and the mature oligodendrocytes inhibit axonal growth. Maturation of these cells may be regulated differently during development than after injury. Among the putative regulators are factors derived from astrocytes, resident microglia; or cytokines produced by macrophages. During development, regulation leads to a temporal separation between axonal growth and maturation of the cellular environment, which might not occur spontaneously after injury in a nonregenerating CNS without intervention at the appropriate time. Data suggest that temporal intervention aimed at the glial cells might enhance the poor regenerative capacity of the mammalian CNS. Possible regulation of the nonneuronal cell response to injury via involvement of protooncogenes is proposed.— Schwartz, M.; Cohen, A.; Stein‐Izsak, C.; Belkin, M. Dichotomy of the glial cell response to axonal injury and regeneration. FASEB J. 3: 2371‐2378; 1989.

Oligodendrocyte cytotoxic factor associated with fish optic nerve regeneration: implications for mammalian CNS regeneration.

The limited capacity for regenerative axonal growth by adult mammalian central neurons has been attributed, at least in part, to the presence of mature oligodendrocytes, which are non-permissive for axonal growth. These cells do not interfere with growth during development, as developmental growth is largely completed before the maturation of the oligodendrocytes. Unlike mammals, fish central nervous system is endowed with a high regenerative capability. When soluble substances derived from regenerating fish optic nerves are applied to injured adult rabbit optic nerves, regenerative axonal growth is permitted. Therefore, in the present study, we tested whether the fish optic nerve, after injury, is endowed with a mechanism by which it avoids the possible inhibitory effect of the process-bearing mature oligodendrocytes. Specifically, we looked for the possible presence of soluble substances that can regulate the number of process-bearing mature oligodendrocytes. We found that soluble substances derived from regenerating fish optic nerve, when added to cultures of oligodendrocytes derived from newborn or injured adult rat optic nerves, caused a decrease in the number of process-bearing mature oligodendrocytes. Soluble substances derived from normal noninjured fish optic nerves, had a significantly lower effect. The observed decrease in the number of mature oligodendrocytes could not be mimicked by the addition of platelet-derived growth factor (PDGF), a known mitogen of oligodendrocyte progenitors which transiently inhibits their maturation. This study suggests a role to oligodendrocyte inhibitory/cytotoxic factor(s) in regeneration.

Monitoring of chemotherapy-induced cell death in melanoma tumors by N,N'-Didansyl-L-cystine.

Early assessment of the efficacy of anticancer agents is a highly desirable and an unmet need in clinical oncology. Clinical imaging of cell-death may be useful in addressing this need, as induction of tumor cell-death is the primary mechanism of action of most anticancer drugs. In this study, we examined the performance of N,N′-Didansyl-L-cystine (DDC), a member of the ApoSense family of novel small molecule detectors of cell-death, as a potential tool for monitoring cell-death in cancer models. Detection of cell-death by DDC was examined in fluorescent studies on B16 melanoma cells both in vitro and ex vivo following its in vivo administration. In vitro, DDC manifested selective uptake and accumulation within apoptotic cells that was highly correlated with Annexin-V binding, changes in mitochondrial membrane potential, and caspase activation. Uptake was not ATP-dependent, and was inducible by calcium mobilization. In vivo, DDC selectively targeted cells undergoing cell-death in melanoma tumors, while not binding to viable tumor cells. Chemotherapy caused marked tumor cell-death, evidenced by increased DDC uptake, which occurred before a detectable change in tumor size and was associated with increased animal survival. These data confirm the usefulness of imaging of cell-death by DDC as a tool for early monitoring of tumor response to anti-cancer therapy.

Glial Cell Differentiation in Regeneration and Myelination

Spontaneous growth of axons after injury is extremely limited in the mammalian central nervous system (CNS). It is now clear, however, that injured CNS axons can be induced to elongate when provided with a suitable environment.