Yoshio Okura

Upregulation of monocyte chemotactic protein-1 and CC chemokine receptor 2 in the central nervous system is closely associated with relapse of autoimmune encephalomyelitis in Lewis rats

Experimental autoimmune encephalomyelitis (EAE) is a disease model of multiple sclerosis (MS) that is characterized by remittance and relapse of the disease and autoimmune and demyelinating lesions in the central nervous system (CNS). To better understand the mechanism of disease relapse, we induced acute and chronic relapsing (CR)-EAE in Lewis rats and examined the differences between the two groups. An immunohistochemical study revealed that significantly higher numbers of macrophages infiltrated the spinal cord during the first and second attacks of CR-EAE than at the peak of acute EAE, whereas the number of infiltrating T cells was essentially the same in acute and CR-EAE. In accordance with this finding, monocyte chemoattractant protein-1 (MCP-1) mRNA, but not MIP-1alpha and RANTES mRNA, increased significantly in CR-EAE lesions rather than in acute EAE lesions. More importantly, the level of MCP-1 during the remission of CR-EAE was significantly higher than during the recovery phase of acute EAE, suggesting that this high level of MCP-1 in CR-EAE is associated with relapse of the disease. CC chemokine receptor 2 (CCR2), the main receptor for MCP-1, was expressed on astrocytes, macrophages and T cells and the number of positive cells was higher in CR-EAE than in acute EAE. Collectively, these findings suggest that high expression of MCP-1 and its receptor, CCR2, in the CNS play important roles in relapse of EAE.

Analysis of neurotrophic effects of hepatocyte growth factor in the adult hypoglossal nerve axotomy model.

Recent studies have shown that hepatocyte growth factor (HGF) promotes the survival of embryonic motor neurons. However, it remains unclear whether HGF has trophic effects on mature motor neurons. In the present study, we examined the effects of HGF on adult motoneurons using the hypoglossal nerve transection model. In adult rats, neurons in the hypoglossal nucleus show a dramatic loss of choline acetyltransferase (ChAT) protein and mRNA after the axotomy. This reduction of ChAT was markedly prevented when HGF was administered continuously at the cut end of the nerve using an osmotic pump. The HGF receptor, c‐met, protein and mRNA, which were faintly expressed in hypoglossal neurons under normal conditions, gradually increased and reached maximal levels 2 weeks after the axotomy. Administration of HGF reduced this c‐met upregulation almost to normal levels. We also quantified HGF mRNA in the tongue and hypoglossal nucleus. The tongue contained abundant HGF mRNA, whereas the nucleus contained only low levels. Interestingly, the HGF mRNA level in the nucleus did not increase after the axotomy. These findings suggest that HGF is principally produced in the tongue and contributes to maintain ChAT expression in the nucleus. HGF produced in the hypoglossal nucleus alone after disconnection from the tongue may not be sufficient for the maintenance of the motor neuron function. Thus, exogenously applied HGF was effective to prevent the downregulation of ChAT activities. These findings provide a strong rationale for the potential clinical use of HGF for the treatment of motor neuron degenerative disease.

Treatment of rat hemiparkinson model with xenogeneic neural transplantation: Tolerance induction by anti-T-cell antibodies

To obtain basic knowledge for the application of xenogeneic neural transplantation to patients with Parkinsons disease, the rejection process of xenogeneic neural grafts in rats was examined and a therapy to control it was developed. Tissues including the ventral mesencephalon were taken from mouse embryos and transplanted into the right lateral ventricle of mature male rats. Transplanted xenografts were usually rejected by day 15. To prevent the graft rejection, host rats were treated with anti‐T‐cell receptor αβ (anti‐TCRαβ) or anti‐CD2 monoclonal antibody (mAb) or by a combination of the two. Anti‐TCRαβ (1 mg/kg) and anti‐CD2 (7 mg/kg) mAb were administered for 3 consecutive days (day ‐2, ‐1, and 0 of transplantation). Although the administration of mAb against either CD2 or TCRαβ did not induce tolerance, the combination therapy with anti‐CD2 and anti‐TCR αβ mAb produced graft survival for more than 100 days. The tolerance induced by this combined antibody therapy is antigen specific because rats with long‐term surviving neural xenograft accepted a second neural graft from the same donor strain C3H/He mouse, but not from a third‐party strain BALB/c mouse, without additional treatment. In addition, T cells isolated from these rats did not respond to cultured C3H/He brain cells, but did respond vigorously to BALB/c brain cells in mixed lymphocyte reaction. More importantly, the finding that xenograft transplantation with the proper treatment reduced the rotation rate of 6‐OHDA‐lesioned rats confirmed that surviving grafts functioned properly. The results of the present study suggest that xenogeneic neural transplantation in combination with T‐cell‐targeted immunotherapy is an effective approach for treatment of Parkinsons disease. J. Neurosci. Res. 48:385–396, 1997.

Nonviral DNA Vaccination Augments Microglial Phagocytosis of β-Amyloid Deposits as a Major Clearance Pathway in an Alzheimer Disease Mouse Model

Immunotherapies markedly reduce &bgr;-amyloid (A&bgr;) burden and reverse behavioral impairment in mouse models of Alzheimer disease. We previously showed that new A&bgr; DNA vaccines reduced A&bgr; deposits in Alzheimer disease model mice without detectable side effects. Although they are effective, the mechanisms of A&bgr; reduction by the DNA vaccines remain to be elucidated. Here, we analyzed vaccinated and control Alzheimer disease model mice from 4 months to 15 months of age to assess which of several proposed mechanisms may underlie the beneficial effects of this vaccination. Immunohistochemical analysis revealed that activated microglial numbers increased significantly in the brains of vaccinated mice after DNA vaccination both around A&bgr; plaques and in areas remote from them. Microglia in treated mice phagocytosed A&bgr; debris more frequently than they did in untreated mice. Although microglia had an activated morphological phenotype, they did not produce significant amounts of tumor necrosis factor. Amyloid plaque immunoreactivity and A&bgr; concentrations in plasma increased slightly in vaccinated mice compared with controls at 9 but not at 15 months of age. Collectively, these data suggest that phagocytosis of A&bgr; deposits by microglia plays a central role in A&bgr; reduction after DNA vaccination.

Recent advance in immunotherapies for Alzheimer disease: with special reference to DNA vaccination.

Alzheimer’s disease (AD) is the most common cause of dementia characterized by progressive neurodegeneration. Based on the amyloid cascade hypothesis, several immunotherapies for AD have been developed as curative treatment. In 1999, Schenk et al. reported for the first time that amyloid beta (Aβ) deposits in AD model mice could be reduced by active vaccination with Aβ peptide. Although clinical trials with the Aβ peptide were halted due to the development of meningoencephalitis in some treated patients, the vaccine therapy was judged to be effective on the basis of clinical and pathological analyses. Passive immunization using humanized anti-Aβ monoclonal antibodies is also under clinical trials; however they have some problems to be solved. As other strategies, DNA vaccines have been developed as immunotherapies for AD, which is simple, easily modified and can be administered without adjuvant. DNA vaccines were developed by several groups including our laboratory, which induced Aβ reduction in AD model mice without side effects. DNA vaccination may be open up new avenue of vaccine therapies for AD in the near future.

DNA Vaccine Therapy for Alzheimer's Disease: Present Status and Future Direction

Alzheimers disease is the most common cause of dementia characterized by progressive neurodegeneration. Based on the amyloid cascade hypothesis, a vaccine therapy for Alzheimers disease (AD) was developed as a curative treatment. In 1999, the amyloid beta (Abeta) reduction in AD model transgenic mice with active vaccination with Abeta peptide was first reported. Although the clinical trials of active vaccination for AD patients were halted due to the development of meningoencephalitis in some patients, from the analysis of the clinical and pathological findings of treated patients, the vaccine therapy is thought to be effective. Based on such information, the vaccines for clinical application of human AD have been improved to control excessive immune reaction. Recently, we have developed non-viral DNA vaccines and obtained substantial Abeta reduction in transgenic mice without side effects. DNA vaccines have many advantages over conventional active or passive immunization. In this article, we review conventional vaccine therapies and further explain our non-viral DNA vaccine therapy. Finally, we show some data regarding the mechanisms of Abeta reduction after administration of DNA vaccines. DNA vaccination may open up new avenues of vaccine therapy for AD.

Assessment of Non-Viral Amyloid-β DNA Vaccines on Amyloid-β Reduction and Safety in Rhesus Monkeys

We recently demonstrated that newly developed non-viral amyloid-β (Aβ) DNA vaccines are safe and effective in reducing Aβ burdens in the brains of Alzheimers disease (AD) model mice. The present study was undertaken to examine whether DNA vaccines effectively and safely reduce Aβ deposition in the brain of rhesus monkeys. For this purpose, DNA vaccines or empty vector at a dose of 3 mg were injected intramuscularly on a biweekly basis into rhesus monkeys (15-18 years old). Before and during vaccination, blood was drawn once a month and used for hematological and biochemical examinations. Six months after the first vaccination, it was demonstrated that anti-Aβ antibodies in plasma of vaccinated monkeys were significantly elevated than that of control monkeys. Immunohistochemical examinations revealed that DNA vaccination reduced the Aβ burden to approximately 50% of that found in control monkeys (p=0.026). There was neither inflammation nor microhemorrhage in the brain and no significant changes in cytokine and chemokine levels in the blood throughout the observation period. Taken together, DNA vaccination to monkeys is safe and effective in Aβ reduction and provides useful information for performing preclinical and clinical trials.

Immunohistochemical analysis on the rejection of xenogeneic brain grafts.

The central nervous system (CNS) of mammals has long been thought of as an immunologically privileged site. However, this concept is now changing because the rejection of histo-incompatible neural grafts has been frequently observed in the CNS. In neural transplantation used as therapy for some human neurodegenerative diseases, it is important to determine which factors are related to brain graft rejection. In this study, we examined immunological reactions in brains that had received isogeneic (rat to rat) and xenogeneic (mouse to rat) neural transplants. In the immunohistochemical analysis, antibodies against T cell receptor αβ (R73), macrophage and microglia (0X42), MHC class II antigens (0X6), CD4 (W3/25), CD8 (0X8), NK cell (3.2.3), B cell (RLN-9D3), T cell receptor (TCR) Vβ8.2 (R78), TCR Vβ8.5 (B73) and TCR Vβl0 (G101) were used. At the early stage of both isogeneic and xenogeneic transplantation, a nonspecific inflammatory reaction characterized by macrophage infiltration was observed along the needle track which was produced by the grafting procedure. From the day 7 stage onwards, the non-specific inflammatory reaction was replaced by the specific immune reactions of T cell infiltration, neovascularization and necrosis of xenogeneic grafts. Marked T cell infiltration was detected in the lesions, whereas NK and B cells were not. Quantitative analysis of T cell subsets revealed that both CD4+ and CD8+ T cells were found in the xenogeneic transplants. Microglia became activated and strongly expressed MHC class II antigens at the time of graft rejection. Isogeneic transplants, in contrast, showed no histological characteristics of rejection, and numerous dopaminergic neurons with several neurites were observed in the grafts. Based on these findings, we concluded that T cells are the principal effectors in the rejection of xenogeneic neural grafts, and that activated microglia may have some role in presenting antigens to the infiltrating T cells during the rejection process.

Analysis of neuronal death in the central nervous system using a new apoptosis model

To examine in detail neural apoptosis in the central nervous system (CNS) for the establishment of new therapies, we have developed an experimental in vitro model of neuronal death and analyzed the mechanism of apoptosis. Septal nuclei were dissected from embryonic brains (E16) of Wistar rats and cultured in chemically defined medium. Highly enriched neurons were obtained from the cultures at 4 days. Exposure to heat shock (43.0 degrees C, 60 min) between 24 and 36 h later, resulted in the death of approximately 70% of cells. Morphologically, dying neurons showed disruption of neurites, nuclear condensation, multiple nuclear fragments, condensation of cytoplasm and multiple cellular fragmentation. Agarose electrophoresis of chromosomal DNA revealed a typical ladder-pattern of fragmentation. Following heat treatment, incubation at 37 degrees C was necessary to detect DNA fragmentation. Quantitative analysis by in situ terminal deoxynucleotidyl transferase assay, revealed that the percentage of apoptotic cells markedly increased 8 h after heat treatment, and continued to gradually increase up until 30 h. Neuronal death and DNA fragmentation were prevented by inhibiting RNA and protein synthesis. Cell death and the DNA cleavage were also inhibited by cultivation in calcium-free medium. The addition of homogenous basic fibroblast growth factor to the medium markedly enhanced cell survival under these pathogenic conditions. These results suggest that neural cell death after mild heat treatment has apoptotic characteristics and may be useful for analyzing the mechanism of apoptosis. The clinical application of drugs acting against molecular components and as well as neurotrophic factors, may in the future prevent apoptosis in neural disease.