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Recently, it has been shown that inflammatory T-cells in human idiopathic myositis only very rarely undergo spontaneous apoptosis. The animal model of experimental autoimmune myositis (EAM) in the Lewis rat was chosen to investigate whether similar findings hold true in rat muscle and if glucocorticosteroids act by induction of T-cell apoptosis in inflammatory lesions. The rate of spontaneous T-cell apoptosis in rat EAM was low, even in muscle specimens with extensive inflammation. After intravenous glucocorticosteroid pulse therapy we found a dramatic increase in the rate of apoptotic T-cells in the inflamed muscles. Up to 50% of these apoptotic T-cells were CD8 positive apoptotic T-cells. T-cell apoptosis was significantly lower in similarly inflamed muscle specimens of the control group. We suggest that glucocorticosteroids induce apoptosis of endomysial T-cells in human idiopathic polymyositis. Glucocorticosteroid-induced apoptosis may be a candidate mechanism in the termination of inflammatory activity.

To elucidate the factor(s) accelerating the autoimmune disease processes, we induced two types of experimental autoimmune encephalomyelitis (EAE), severe and very mild, in F344 rats by immunization with myelin basic protein (MBP) plus pertussis toxin (PT) (PT+) or with MBP alone (PT-) and compared the differences between the two. Immunohistochemical examinations showed that although the nature of inflammation was essentially the same between the two groups, the proportion of Vbeta8.2(+) T cells in the CNS lesion of PT (+) rats was larger than that of PT (-) rats. Cytokine analysis by competitive PCR revealed that IL-10 mRNA in the lymphoid organ was significantly suppressed in the PT(+) group, whereas levels of IFN-gamma,TNF-alpha and TGF-beta mRNA were insignificantly different after PT administration. In addition, T cells taken from PT (+) rats proliferated well in response to MBP, while those from PT (-) rats showed a marginal response to the same antigen. However, this finding does not indicate the switching of non-encephalitogenic to encephalitogenic T cells upon PT administration because PT (-) rats contained encephalitogenic T cells and/or their precursor cells as revealed by adoptive transfer experiments. Taken together, these findings suggest that suppression of IL-10 by PT administration is the major factor contributing to the exacerbation of EAE in PT(+) rats.

Experimental autoimmune encephalomyelitis is a disease induced by neuroantigen-reactive T cells bearing particular types of T cell receptor (TCR). Although the nature of TCRs of encephalitogenic T cells has been partially delineated using encephalitogenic T cell clones established in vitro, the entire TCR repertoire formed in situ after immunization with neuroantigen remains unclear. In the present study, we immunized Lewis rats with myelin basic protein (MBP) and its fragment peptides and determined the TCR repertoire of spinal cord T cells formed after the immunization by CDR3 spectra-typing. It was revealed that the oligoclonal expansion of Vbeta2, Vbeta8.2, and Vbeta17 spectratypes was detectable after immunization with guinea pig MBP and its immunodominant epitope, the 68-88 sequence, whereas immunization with a peptide containing a minor epitope induced Vbeta10 expansion. Immunization with rat MBP induced much broader TCR Vbeta expansion (all of the above Vbetas plus Vbeta3). These findings suggest that TCRs activated by immunization with guinea pig MBP used as heteroclitic immunogen recognize autoantigen, rat MBP. Furthermore, the strategy used in this study gives insight into the pathogenesis of autoimmune disease and provides useful information for designing TCR-based immunotherapy.

The identification of TCRs of autoimmune disease-inducing T cells within a short period of time is a key factor for designing TCR-based immunotherapy during the course of the disease. In this study, we show that experimental autoimmune carditis-associated TCRs, Vβ8.2 and Vβ10, were determined by complementarity-determining region 3 (CDR3)-spectratyping analysis and subsequent sequencing of the CDR3 region of spectratype-derived TCR clones. Immunotherapy targeting both Vβ8.2 and Vβ10 TCRs using mAbs and DNA vaccines significantly reduced the histological severity of experimental autoimmune carditis and completely suppressed the inflammation in some animals. Since depletion or suppression of one of two types of effector cells does not improve the severity of the disease significantly, combined TCR-based immunotherapy should be considered as a primary therapy for T cell-mediated autoimmune diseases. TCR-based immunotherapy after rapid identification of autoimmune disease-associated TCRs by CDR3 spectratyping can be applicable, not only to animal, but also to human autoimmune diseases whose pathomechanism is poorly understood.

To characterize experimental autoimmune neuritis (EAN)-inducing T cells in more detail, we performed CDR3 spectratyping analysis and found oligoclonal expansion of several Vbeta spectratypes in nerve-infiltrating T cells. Vbeta5 expansion was observed all the stages examined, whereas Vbeta8.2 and Vbeta17 expansion was mainly found at the peak and preclinical stages, respectively. Since Vbeta5 expansion persists throughout the course of the disease, Vbeta5+ T cells are judged to be the main effector cells. Vbeta8.2+ and Vbeta17+ T cells may also be pathogenic but are not the main effectors because expansion of these spectratypes was found at a limited period of time. Sequence analysis revealed that Vbeta5, Vbeta8.2 and Vbeta17 spectratype-derived TCR clones possess their own dominant sequences in the CDR3 region with no homology among the clones. These findings suggest that polyclonally activated T cells are involved in the formation of the nerve lesion. Furthermore, vaccination with Vbeta5 DNA, but not with Vbeta10 DNA, suppressed the development of EAN significantly. Collectively, these findings indicate that determination of autoimmune disease-associated TCR by CDR3 spectratyping provides useful information for designing TCR-based immunotherapy for the disease.

To elucidate the mechanisms of relapses of the clinical signs in experimental autoimmune encephalomyelitis (EAE), the cytokine profile of chronic relapsing EAE (CR-EAE) in rats was determined by competitive polymerase chain reaction (PCR). By immunization with guinea pig spinal cord homogenate and treatment with low-dose cyclosporin A (CsA), rats developed two attacks of EAE with remission in between. Cytokine analysis revealed that the level of TNF-alpha mRNA increased at the first and second attacks with transient disappearance at the remission phase. In contrast, the level of IFN-gamma mRNA was suppressed at the first attack by CsA and peaked at the second attack. Intraventricular administration of IFN-gamma prior to onset of disease signs induced more relapses, or a severe lethal form. In addition, the intraventricular injection of TNF-alpha caused the persistence of the clinical signs. These findings suggest that TNF-alpha contributes to the first and second attacks of CR-EAE, while IFN-gamma is not required for the first attack but is closely related to the relapse of the disease. With regard to anti-inflammatory cytokines, the levels of both TGF-beta1 and IL-10 mRNA at the second attack were higher than those at the first attack. Taken together, differential involvement of TNF-alpha and IFN-gamma is closely associated with the clinical features of CR-EAE.

We studied the effect of nitric oxide (NO) on experimental autoimmune myocarditis (EAC) in rats. We examined the role of inducible nitric oxide synthase (iNOS), an enzyme that produces NO, on hearts affected with EAC, by testing the effects of aminoguanidine (AG), a selective iNOS inhibitor, on the course of EAC. Western blotting detected iNOS in the affected cardiac tissues, but not in CFA immunized cases. Immunohistochemically, the majority of ED1+ macrophages in the EAC lesions were positive for iNOS and nitrotyrosine. A high dose of AG (200 mg/kg/day) significantly reduced the incidence of EAC (p

In the present study, we examined the therapeutic effects of T-614 (3-formylamino-7-methylsulfonylaminoxy-4H-1-benzopyran-4-one), a new anti-rheumatic drug, on a T cell-mediated autoimmune disease, experimental autoimmune encephalomyelitis (EAE). T-614 dose-dependently suppressed the development of active EAE induced in Lewis rats by immunization with myelin basic protein (MBP) when administered for 2 weeks starting on the day of immunization (day 0 to 14). Amelioration of clinical signs was also obtained by the treatment at the effector phase (day 7 to 14) of the disease. Furthermore, T-614 treatment of recipient rats that had received MBP-sensitized lymphoid cells resulted in suppression of the clinical severity of EAE. Immunohistological examination revealed that the number of TCR alpha beta-expressing T cells and the extent of MHC class II expression in the spinal cord of rats treated with T-614 was markedly reduced. In vitro study using MBP-specific T cells showed that the addition of T-614 inhibited the proliferative responses of T cells and the production of pro-inflammatory cytokines such as IFN-gamma, IL-6 and TNF produced by T and accessory cells. Taken together, these findings imply that T-614 suppresses the development of EAE by inhibiting the proliferation of autoreactive T cells and pro-inflammatory cytokine production not only by T cells but also by macrophages/microglia. This may be attributable to the result that T-614 is more effective at the effector phase rather than the induction phase. Thus, this drug has a potential value for the treatment of various T cell-mediated autoimmune diseases including multiple sclerosis (MS) as well as rheumatoid arthritis.

To characterize the nature of autoimmune disease-inducing T cells in the target organ, oligoclonal expansion of spinal cord T cells of Lewis rats with experimental autoimmune encephalomyelitis (EAE) was examined by complementarity-determining region 3 (CDR3) size spectratyping. It is known that TCR of in vitro-established myelin basic protein-specific T cell clones and lines have a short CDR3 and that the amino acid sequence in this region is highly preserved. On the basis of these findings, we analyzed 22 spectratypes of the TCR β-chain (Vβ1–20). Among them, only Vβ8.2 and Vβ17 showed oligoclonal expansion of TCR with a short CDR3 at the early stage of EAE. More interestingly, the spectratype profile of Vβ8.2 seen at the early stage was preserved throughout the course of EAE, whereas that of Vβ17 became more diverse at the peak stage of the disease. Analysis of nucleotide and predicted amino acid sequences of Vβ8.2 CDR3 derived from the spectratypes revealed that the clones with CASSDSSYEQYFGPG, which is one of the representative sequences of encephalitogenic T cell clones, constituted the predominant population not only at the early stage but also at the peak and recovery stages (71, 71, and 60%, respectively). These findings imply that although the phenotype of T cells in the target organ diversifies as the autoimmune disease progresses, disease-associated TCR spectratype(s) are preserved throughout the course of the disease. Thus, CDR3 size spectratyping is a powerful tool for the screening of disease-inducing T cells in an autoimmune disease of unknown pathomechanism.

To obtain basic knowledge for the application of xenogeneic neural transplantation to patients with Parkinson's 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 alphabeta (anti-TCR alphabeta) or anti-CD2 monoclonal antibody (mAb) or by a combination of the two. Anti-TCR alphabeta (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 TCR alphabeta did not induce tolerance, the combination therapy with anti-CD2 and anti-TCR alphabeta 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 Parkinson's disease.

It has recently been determined that not only Aβ oligomers, but also other Aβ species and amyloidogenic peptides are neurotoxic in Alzheimer disease (AD) and play a pivotal role in AD pathogenesis. In the present study, we attempted to develop new DNA vaccines targeting a wide range of Aβ species. For this purpose, we first performed in vitro assays with newly developed vaccines to evaluate Aβ production and Aβ secretion abilities and then chose an IgL-Aβx4-Fc-IL-4 vaccine (designated YM3711) for further studies. YM3711 was vaccinated to mice, rabbits and monkeys to evaluate anti-Aβ species antibody-producing ability and Aβ reduction effects. It was found that YM3711 vaccination induced significantly higher levels of antibodies not only to Aβ1-42 but also to AD-related molecules including AβpE3-42, Aβ oligomers and Aβ fibrils. Importantly, YM3711 significantly reduced these Aβ species in the brain of model mice. Binding and competition assays using translated YM3711 protein products clearly demonstrated that a large part of antibodies induced by YM3711 vaccination are directed at conformational epitopes of the Aβ complex and oligomers. Taken together, we demonstrate that YM3711 is a powerful DNA vaccine targeting a wide range of AD-related molecules and is worth examining in preclinical and clinical trials.

Inflammation of the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE) starts in the subarachnoid space (SAS) and spreads later to the adjacent CNS parenchyma. To characterize the nature of lesion-forming T cells in situ in more detail, T cells were isolated from the SAS and their surface phenotype and the nucleotide sequence of the junctional region of the T cell receptor (TCR) was determined and compared with those of the lymph node (LN) and spinal cord (SC) T cells. Characteristically, more than 70% of SAS TCR alpha beta + T cells isolated at the early stage of EAE lacked both CD4 and CD8 molecules, whereas those from LN and SC were either CD4+ or CD8+. Analysis of nucleotide sequences of the junctional region of TCR revealed that T cells bearing a sequence identical to that for encephalitogenic T cell clones were found in both SAS and SC. Furthermore, purified CD4-CD8- T cells expressed CD4 molecules after culture. At the same time, these T cells acquired reactivity to myelin basic protein and induced passive EAE in naive animals after adoptive transfer. Our results suggest that CD4-CD8- T cells in the SAS are precursors of lesion-forming T cells in the SC and that phenotype switching takes place during the process of T cell infiltration into the CNS parenchyma. The double-negative nature of these T cells may explain an escape of encephalitogenic T cells from negative selection in T cell differentiation.

Microglia form a regularly spaced network of resident glial cells throughout the central nervous system (CNS). They are morphologically, immunophenotypically and functionally related to cells of the monocyte/macrophage lineage. In the ultimate vicinity of the blood-brain barrier two specialized subsets of macrophages/microglia can be distinguished: firstly, perivascular cells which are enclosed within the basal lamina and secondly juxtavascular microglia which make direct contact with the parenchymal side of the CNS vascular basal lamina but represent true intraparenchymal resident microglia. Bone marrow chimera experiments indicates that a high percentage of the perivascular cells undergoes replacement with bone marrow-derived cells. In contrast, juxtavascular microglia like other resident microglia form a highly stable pool of CNS cells with extremely little turnover with the bone marrow compartment. Both the perivascular cells and the juxtavascular microglia play an important role in initiating and maintaining CNS autoimmune injury due to their strategic localization at a site close to the blood-brain barrier, their rapid inducibility for MHC class II antigens and their potential scavenger role as phagocytic cells. The constantly replaced pool of perivascular cells probably represents an entry route by which HIV gets access to the brain. Microglia are the first cell type to respond to several types of CNS injury. Microglial activation involves a stereotypic pattern of cellular responses, such as proliferation, increased or de-novo expression of immunomolecules, recruitment to the site of injury and functional changes, e.g., the release of cytotoxic and/or inflammatory mediators. In addition, microglia have a strong antigen presenting function and a pronounced cytotoxic function. Microglial activation is a graded response, i.e., microglia only transform into intrinsic brain phagocytes under conditions of neuronal and or synaptic/terminal degeneration. In T-cell-mediated autoimmune injury of the nervous system, microglial activation follows these lines and occurs at an early stage of disease development. In experimental autoimmune encephalomyelitis (EAE), microglia proliferate vigorously, show a strong expression of MHC class I and II antigens, cell adhesion molecules, release of reactive oxygen intermediates and inflammatory cytokines and transform into phagocytic cells. Due to their pronounced antigen presenting function in vitro, activated microglia rather than astrocytes or endothelial cells are the candidates as intrinsic antigen presenting cel of the brain. In contrast to microglia, astrocytes react with a delay, appear to encase morphologically the inflammatory lesion and may be instrumental in downregulating the T-cell-mediated immune injury by inducing T-cell apoptosis.(ABSTRACT TRUNCATED AT 400 WORDS)

To characterize the phenotype of inflammatory cells in the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE), Lewis rats were immunized with guinea pig myelin basic protein and frozen sections of the spinal cord with EAE were examined immunohistochemically using a panel of monoclonal antibodies against T cells and adhesion molecules. In addition, double immunostaining was performed with glial and T cells markers to examine the interaction between infiltrating T cells and reactive brain cells during the course of EAE. In the early stage of EAE, inflammatory cells first appeared in the subarachnoid space (SAS) and infiltrated the subpial region. The majority of inflammatory cells in SAS expressed TCR alpha beta and either CD4 or CD8 molecules. However, only CD4+ T cells infiltrated the parenchyma while the majority of CD8+ cells remained in SAS. A similar differential localization of T cells was observed with regard to CD45RC molecules. Inflammatory cells in SAS consisted of both CD45RC+ and CD45RC- population, while those in the parenchyma were largely CD45RC-. With regard to adhesion molecules, the leptomeninges constitutively expressed fibronectin (FN) and intercellular adhesion molecule 1 (ICAM-1). Most SAS inflammatory cells expressed very late activation antigen 4 (VLA-4) and, to lesser extent, lymphocyte function-associated antigen 1 (LFA-1) in the early stage of EAE. On the other hand, parenchymal infiltrating cells expressed LFA-1 more strongly in the peak stage. Double staining for V beta 8.2 TCR and microglia demonstrated an increase in the number of microglia together with morphological changes into rod-shape cells in the vicinity of infiltrating T cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The rejection of a transplanted allograft is dependent on T cell activation, which requires T cell receptor engagement by antigen and costimulatory signals delivered by T cell surface molecules such as CD2. Anti-CD2 mAbs have been shown to suppress cell-mediated immunity. The effects of anti-CD2 mAbs OX34 and OX54 on rejection of BN (RT1n) rat hearts transplanted heterotopically to LEW (RT1l) rats were investigated. Administration of OX34 (7 mg/kg/day i.p.), either for 3 consecutive days immediately before or 8 consecutive days immediately after transplantation induced indefinite allograft survival (median survival time: 7, > 150, and > 150 days for control, preoperative treatment, and postoperative treatment, respectively). In contrast, pre- or postoperative treatment with OX54 (40 mg/kg/day) prolonged median survival time to only 28 and 11 days, respectively. Administration of OX34 or OX54 to naive rats induced a transient depletion of T cells in the peripheral immune organs. In vitro studies revealed that whereas OX54 had no effect on the allogeneic mixed lymphocyte reaction, OX34 partially inhibited both the allogeneic mixed lymphocyte reaction, in an IL-2-reversible manner, and T cell proliferation in response to immobilized mAb to either the T cell receptor or CD3. OX34-treated rats in which the cardiac allograft had survived > 100 days accepted a second heart from the donor strain. Treatment with OX34 induced an alloantigen-unresponsive state in T cells. These results suggest that treatment with an appropriate anti-CD2 mAb, especially postoperatively, may prove an effective approach for preventing cardiac allograft rejection.

Autoimmune-like hepatic lesions were induced by injection of CD4+ T cells from B10.Thy-1.1 mice into MHC class II-disparate (B10.Thy-1.1 x bm12)F1 mice. Hepatic lesions characterized by mononuclear cell accumulation in the portal area of the central vein and around interlobular bile ducts were observed in these recipients. The morphologic features of the lesions resembled primary biliary cirrhosis. The origin of T cells invading at the site of the hepatic lesions was immunohistochemically analyzed. It was shown that many recipient-derived T cells were present at the lesions and that some of them infiltrated the bile duct epithelia. Furthermore, the lesions were weakened when recipient-type T cells were depleted by thymectomy and the administration of anti-Thy-1.2 monoclonal antibody. Recipient-derived T cells were observed to take part in the formation of autoimmune hepatic lesions. These findings suggest the possibility that the tolerance of self-reactive T cells is abrogated by the graft-versus-host reaction.

Experimental autoimmune encephalomyelitis (EAE) in Lewis rats is an acute monophasic paralytic central nervous system disease, in which most rats spontaneously recover from paralysis. EAE in Lewis rats is induced by encephalitogenic antigens, including myelin basic protein. EAE is mediated by CD4(+) Th1 cells, which secrete pro-inflammatory mediators, and spontaneous recovery is mediated by regulatory T cells. Recently, it was established that classically activated macrophages (M1 phenotype) play an important role in the initiation of EAE, while alternatively activated macrophages (M2 phenotype) contribute to spontaneous recovery from rat EAE. This review will summarize the neuroimmunological aspects of active monophasic EAE, which manifests as neuroinflammation followed by neuroimmunomodulation and/or neuroprotection, with a focus on the role of alternatively activated macrophages.

SUMMARY We investigated the effects of anti-αβ T cell receptor antibody in rat experimental autoimmune myocarditis (HAM), using a new animal model of autoimmune myocarditis characterized by the appearance of multinucleated giant cells. KAM was induced by injecting Lewis rats subcutaneously in the footpads with 1.0 mg of human cardiac myosin in an equal volume of Freund's complete adjuvant (FCA) on days 0 and 7. In experiment 1, we evaluated the effect of long-term anti-αβ TCR antibody therapy on prevention of progression of EAM. Long-term administration of anti-αβ TCR antibody prevented progression of FAM in a dose-dependent manner. Flow cytometry performed at the time of sacrifice showed that the percentage of αβ T cells in lymph nodes and spleen was similar in the control group and the group in which almost no histologic evidence of myocarditis was found. In experiment 2, we examined the effects of short-term therapy. Rats were killed at different stages and pathologic specimens were examined. Short-term therapy delayed the onset of myocarditis. Results of flow cytometry suggested that impairment of antigen recognition or T cell function by occupancy of the TCR rather than depletion of TCR was the mechanism responsible for suppression of EAM.

Acute and monophasic experimental autoimmune encephalomyelitis (EAE) was induced in rats by immunization with myelin basic protein (MBP). Proliferative responses of lymph node cells to major and minor encephalitogenic and nonencephalitogenic determinants of the MBP molecules were measured at various time intervals after the immunization. Results of these experiments revealed that additional responses to minor determinants which had been observed at the late stage of mouse EAE (Lehmann et al., Nature 356 , 155, 1992) were very weak and short-lived in the rat. Furthermore, the response to the minor encephalitogenic determinant was not recognized throughout the course of EAE. Coimmunization with synthetic peptides, corresponding to the major and minor determinants, induced T-cell response only to the major determinant. These findings suggest that poor generation of T cells reactive with the minor encephalitogenic epitope is attributable to peptide competition between these two determinants. The present results, together with those reported in mice, strongly suggest that differences in the clinical course of EAE, i.e., acute monophasic or chronic relapsing, is closely related to the presence or absence of "determinant spread" to minor encephalitogenic epitope(s).

Although T cell receptor (TCR) peptide therapy was initially reported to be a very effective method for prevention of the development of experimental autoimmune encephalomyelitis (EAE), it was recently demonstrated that the same peptide immunization led to enhanced and chronic EAE in some cases. In the present study, we examined the effect of the TCR peptide (V beta 8.2-39-59) vaccination on the development of EAE by employing several immunization protocols. We found that TCR peptide vaccination effectively prevented EAE development only when the peptide was injected with Mycobacterium tuberculosis-enriched CFA in the vicinity of the challenge site. Under such conditions, a sufficient number of peptide-reactive T cells were generated. Flow cytometry and immunohistochemical analyses using anti-peptide antibody and anti-V beta 8.2 mAb revealed that despite the presence of V beta 8.2+ cells, very few peptide-positive T cells appeared in the lymphoid organs throughout the course of EAE. These findings imply that antibodies that are generated after immunization with V beta 8P are hardly accessible to their specific epitopes in the native protein. Insufficient generation of both T cells and antibodies against V beta 8.2-positive T cells may be attributable to the outcome of the therapy. To establish effective TCR peptide immunotherapy, these disadvantages should be overcome by using other TCR sequences and/or by employing a more suitable adjuvant.

The fate of cultured keratinocyte (KC) allografts remains controversial. Although prolonged survival of cultured KC allografts in naive mice has been reported, the detailed mechanisms remain undetermined. Furthermore, it was also reported that in the human cultured KC allografts do not survive permanently, and they are rapidly replaced by recipient cells. In the present study, we have addressed this issue and obtained findings that cultured KC allografts survive for a prolonged period in naive mice under the conditions in which reepithelization by recipient cells is prevented. However, the same cultured KC allografts were rejected when they were grafted onto recipients primed with allogeneic spleen cells or full-thickness skin grafts. To clarify the mechanisms behind these findings, the allostimulatory ability of cultured KC and their susceptibility to alloreactive cytotoxic T cells were examined in vitro. In a mixed epidermal cell-lymphocyte reaction, cultured KC were unable to induce allospecific proliferative responses of naive T cells. On the other hand, primed T cells from presensitized mice showed weak but significant proliferative responses against allogeneic KC. It also was confirmed that cultured KC are susceptible to lysis by alloreactive cytotoxic T cells. These data indicate that prolonged survival of cultured KC allografts in naive mice is attributable to a defect in the afferent, but not the efferent, phase of the rejection process that is caused by the weak allostimulatory ability of cultured KC. This assumption was also supported by the finding that spleen cells from the recipient mice bearing long-surviving KC allografts retain in vitro responsiveness against stimulator cells syngeneic to the grafted KC. Taken together, these findings indicate that long-term survival of cultured KC allografts in naive mice may be due solely to the weak allostimulatory ability of cultured KC, but not to loss of susceptibility to alloreactive cytotoxic T cells after culture of KC or induction of tolerance in the recipient mice bearing KC allografts.

Staphylococcal enterotoxins (SEs) can bind major histocompatibility antigens and stimulate T cells which bear particular types of T cell receptor. Therefore, it has been postulated that SEs may trigger or modulate the development or autoimmune diseases caused by T cells. In the present study, we examined the effects of SEs on rat encephalitogenic T cells and the clinical manifestation of experimental autoimmune encephalomyelitis (EAE). SED, but not other SEs, stimulated encephalitogenic T cells. Furthermore, culture of lymphoid cells from myelin basic protein (MBP)-immunized rats with SED augmented the clinical manifestation of passively transferred EAE, whereas SEA and SEB showed no significant EAE-transfer ability. Flow cytometric analysis demonstrated that in vitro SED stimulation of T cells from MBP-immunized rats, but not from normal rats, resulted in selective expansion of Vβ8.2 + T cells. Consistent with in vitro findings, in vivo administration of SED modulated EAE elicited by immunization with MBP. SED given after the immunization augmented clinical manifestation, especially at low doses. On the other hand, SED given 7 days before the immunization suppressed the development of EAE in a dose-dependent manner. Interestingly, the same toxin given at a dose of 20 μg to thymectomized rats induced enhanced EAE regardless of the timing of administration. It has already been established that SEs stimulate T cells bearing a particular type of TCR Vβ chain and subsequently induce unresponsiveness of these T cells. The present results suggest that a similar mechanism may operate in rats after the toxin treatment and MBP immunization. However, in vitro assay showed that the proliferative responses of T cells from EAE-suppressed rats to MBP and SED were not eliminated, suggesting that SED-induced suppressor T cells may also play some roles in EAE suppression. The present study has shown that SED, one of the superantigens, modulates an autoimmune disease. More importantly, its effects are not uniform, but instead are closely related to the dose of the toxin, timing of toxin exposure, and the status hosts.

Giant cell myocarditis is a serious and frequently fatal inflammatory heart disease of which the etiology remains unknown. In the present study, we investigated the origin of multinucleated giant cells in myocarditis with the use of an experimental model. We also examined the factors relating to the formation of giant cells in myocarditis. Severe myocarditis characterized by the appearance of multinucleated giant cells was induced in Lewis rats by immunization with cardiac myosin in complete Freund's adjuvant. Two types of giant cells, foreign body giant cell-like and myocytelike, were observed in this myocarditis. Immunohistochemical studies revealed that both types of multinucleated giant cells were stained with OX42 and ED1 (macrophage markers) and were not stained with anti-desmin antibody and HHF35 (markers for muscle fibers). Therefore, it is likely that multinucleated giant cells in this myocarditis are derived from macrophages. During the course of the disease, the appearance of multinucleated giant cells was restricted to a period corresponding with the fulminant phase of inflammation. When the severity of the disease was modulated by immunization with various doses of the antigen, multinucleated giant cells appeared only in severe myocarditis after inoculation of a large dose of the antigen. Administration of immunoadjuvants also affected the formation of giant cells. Most of the rats injected with cardiac myosin in complete Freund's adjuvant developed giant cell myocarditis.(ABSTRACT TRUNCATED AT 250 WORDS)

Alzheimer's disease is the most common cause of dementia characterized by progressive neurodegeneration. Recently, a vaccine therapy for Alzheimer's disease was developed as a curative treatment. Although clinical trials of active vaccination for Alzheimer's disease were halted due to the development of meningoencephalitis in some patients, the clinical and pathological findings of treated patients suggest that the vaccine therapy is effective. Hence, newly designed vaccines are being invented to control excessive T-cell immune reactions after the human clinical trial. In this article, we will review conventional vaccine therapies and newly developed vaccine therapies, mainly DNA vaccines, for possible clinical application in the near future.

Localization of bone marrow-originated cells in the central nervous system (CNS) of the rat was investigated by using bone marrow chimeras. In order to do this, Lewis rats which carry major histocompatibility complex (MHC) class I antigens haplotype 1 (RT1.A 1 ) were reconstituted with (Lew × PVG)F 1 ( RT1.A 1 c bone marrow cells after lethal irradiation. Transferred bone marrow cells were detected by immunohistochemical staining using a monoclonal antibody, OX27, specific for haplotype c of rat MHC class I antigens (RT1.A c ). The spleen and thymus of chimeric rats were fully reconstituted with transferred F 1 cells 4 weeks after bone marrow transplantation. At this stage, mononuclear cells in the subarachnoid space of the CNS expressed OX27 antigen indicating that they were of bone marrow origin. A few OX27-positive blood cells were scattered in the CNS parenchyma 4–12 weeks after reconstitution. Ramified microglia, however, remained OX27-negative. Bone marrow-derived microglia were not observed throughout the period of examination until 24 weeks. In addition, experimental allergic encephalomyelitis (EAE) was induced in chimeric rats in order to augment the expression of MHC class I antigens on microglia. Even under this condition, no OX27-positive microglia were observed. Taken together, ramified microglia might be of neuroectodermal origin and there is little possibility that the microglia are derived from the bone marrow. However, if the ramified microglia are derived from blood cells, the microglia may be expected to have characteristic cell kinetics from the following points: (1) the precursor cells of the microglia may enter the CNS only at the perinatal stage; and (2) even under the condition in which lymphocytes and macrophages enter the CNS as observed in EAE, the precursor cells of the microglia are not supplied from the blood.

“99mTc-dynamics study” and computed tomography (CT) were analyzed on patients with ischemic lesions of the unilateral middle cerebral artery. Regions of interest (ROIs) were placed symmetrically on both hemispheres in frontal static scanning image, and their count ratios to the background were plotted on the time scale. Mode of transit time (MOTT) of the patients were prolonged bilaterally compared to the controls. The prolongation was more marked on the lesion side especially in acute cases, and it gradually became normal with the passage of time from onset. The plotted curves (count ratio dynamics) of the ROIs on the lesion side were clearly divided into the following four types: gradually increasing pattern (Type I); pattern of higher count ratio than contralateral side without gradual increase (Type II) ; decreasing pattern and lower count ratio than contralateral side (Type III) ; and gradually decreasing pattern almost identical to the contralateral side (Type IV). There is no correlation between MOTTs and the four types of count ratio dynamics. Therefore, the factors affecting the MOTT (the primary hemodynamics) seemed to be different from those of count ratio dynamics (pathological changes following ischemia). These types were very characteristically distributed by the duration from onset. The majority of Type I was distributed after the 4th week from onset and a small percentage in earlier phases. Type II was similar to Type I, but it seemed to be seen in relatively earlier phases. Type III was characteristically distributed in two phases; ultra-acute phase and late chronic phase. Type IV was distributed over all phases, except ultra-acute. The findings of CT also had close correlations to difference in type. The results from Type I indicated that the increasing pattern of gamma-ray ratio in acute cases seemed to reveal the accumulation of radio-isotope (RI) at the extravascular spaces probably due to disruption of blood-brain barrier (B.B.B.). In chronic cases, the accumulation of RI would be due to neovascularization without B.B.B. and/or to necrotic brain tissue. Type II suggested focal hyperemia (luxury perfusion). The ultra-acute cases in Type III usually showed normal CT findings but they frequently changed to Type I or Type II along the course of time. Type III seen in the ultra-acute phase was thought to reveal the state of low perfusion not associated with massive disruption of B.B.B. In late chronic phase having massive low density (LD) without mass effect (ME) or contrast enhancement (CE), may suggest the terminal stage of massive infarction. Type IV suggests mild or no change of cerebral circulation. It is concluded that Type I and Type II cases with ME and CE are contraindicative for surgical revascularization in acute phase, because they have disrupted B.B.B. massively. On the other hand, Type III and Type II cases with normal CT findings have an indication for vascular reconstructive surgery.

To determine in situ localization of cells bearing major histocompatibility complex (MHC) class I or II antigens in the central nervous system (CNS), immunohistochemical examination was performed on CNS sections of Lewis rats sensitized for experimental allergic encephalomyelitis (EAE). Class I antigens identified by OX18 were detected on endothelial cells (EC) and cells with dendritic morphology (DC) of normal rats. OX18+ DC increased in number as the clinical signs of EAE became more severe, while the number of OX18+ EC in clinical EAE rats was not different from that of normal control rats. Infiltrating lymphocytes were always observed around OX18+ vessels. Double staining showed that OX18+ DC was negative for glial fibrillary acidic protein (GFAP). Cells with morphological features of oligodendroglia were not detected with OX18 in both normal control and EAE rats. MHC class II antigens (Ia antigens) were detected using three MAbs: OX3, OX6 and OX17. These three different MAbs essentially showed the same staining pattern. In normal controls, mononuclear cells in the subarachnoid space were stained positively, but no Ia+ parenchymal cells were detected. In EAE rats, Ia+ DC were first detectable in the white matter of the spinal cord at the preclinical stage, and increased in number as the disease progressed. On the other hand, double-staining with OX6 and anti-factor VIII-related antigen antiserum, or with OX3 and anti-vimentin antiserum demonstrated that endothelial cells even with lymphocyte cuffing were negative for Ia antigens. Based on the data obtained in the present study, the possible role of MHC class I and II antigens in the development of EAE is discussed.

In order to examine developing lesions of experimental allergic encephalomyelitis (EAE) at the preclinical stage, adoptive transfer of EAE was achieved in Lewis rats using spleen cells obtained from rats previously immunized with 50 micrograms of guinea pig myelin basic protein (MBP) in complete Freund's adjuvant. After cell transfer, all the recipient rats developed severe paraparesis with minimal variation in the onset of clinical signs, which facilitated immunohistochemical examination of developing lesions of EAE in asymptomatic animals. The initial pathological finding detected in this system was an increased number of inflammatory cells (both OX19+ and OX8+ T cells and macrophages) in the subarachnoid space (SAS) which was observed on day 3 post-transfer (PT) when the recipient animals showed no neurological abnormalities. By day 4 PT, inflammatory cells were detected in the Virchow-Robin space of vessels which were continuous to the SAS. Perivascular lymphocytic infiltration was scarcely detected in the gray matter. On day 5 PT, the rats showed severe paraparesis. Inflammatory foci increased in number and were detected in both the white and gray matter. Diffuse parenchymal T-lymphocyte infiltration was also recognized at this stage. From day 6 PT onwards, inflammatory cells decreased gradually. On day 8 PT, all the rats recovered from EAE and few inflammatory cells were detected in the parenchyma and SAS. Immunohistochemical examination of T-cell subsets and Ia antigens revealed that the number of OX19+ cells was greater than that of OX8+ cells in the parenchyma and SAS at all stages examined, the distribution of OX19+ cells was almost the same as that of OX8+ cells, OX8+ cells decreased in number before clinical signs subsided, and that after the infiltration of T cells into the parenchyma, cells with dendritic morphology (microglia) expressed Ia antigens in close association with the infiltrating T cells. Adoptive transfer of MBP-sensitized spleen cells provides constant induction of EAE with minimal variation in clinical onset and severity, thus being a useful model for the examination of early events of EAE.

We have studied frozen sections of the developing and adult mouse central nervous system (CNS), with or without cold lesions, by immunohistochemical and histochemical methods. Using a monoclonal antibody against a macrophage differentiation antigen (Mac-1), we have shown that some neuroglia in the white matter of adult mice stained positively. In the developing CNS, with or without cold lesioning, Mac-1-positive glia were not detected. In the normal adult CNS, a small number of glia in the white matter stained faintly. After cold injury, the number of Mac-1-positive glia and their staining intensity increased for several months. Mac-1-positive glia were always negative for glial fibrillary acidic protein (GFA). Their morphology and distribution were similar to those of nucleoside diphosphatase-positive cells. Considering that the phagocytic activity of glia increases after injury to the CNS (Trachtenberg 1983) and that Mac-1 has been reported to be associated with the complement receptor (Beller et al. 1982), Mac-1-positive glia may play a role in phagocytosis in the damaged CNS.

Recently, several investigators have demonstrated that the MN blood group precursor antigens Thomsen-Friedenreich antigen (T-Ag) and Tn-antigen (Tn-Ag) are expressed on the cell surfaces of several cancers, including urinary bladder cancer. T-Ag is composed of a specific carbohydrate chain, galactose-beta-1-3-N-acetylgalactosamine (GalNAc), which is specifically detectable through the immunohistochemical binding of peanut agglutinin (PNA). In normal cells, T-Ag is cryptic (cT-Ag) and can be unmasked by treatment with neuraminidase. Tn-Ag is composed of another carbohydrate chain, alpha-GalNAc-serine/threonine, and binds specifically to Vicia villosa agglutinin (VVA). With the use of both these lectins, VVA and PNA, the presence or absence of Tn-Ag and T-Ag was examined in 24 specimens of normal bladder epithelium and specimens from 53 cases of human urinary bladder transitional cell carcinoma of various histologic grades by staining paraffin sections by means of the avidin-biotin-immunoperoxidase technique. The correlation between the expression of these antigens and the patient's clinical course was then estimated. Out of 21 patients in whom the tumors expressed the phenotype Tn-Ag(+), T-Ag(+), or cT-Ag(-), 17 suffered from invasive recurrence. Although patients with tumors expressing the phenotypes T-Ag(-) and cT-Ag(+) have been reported to show a good clinical course, in our studies some of them showed a switch to invasive recurrence. Thus it was not possible to estimate the patient's clinical course only by the presence or absence of T-Ag and cT-Ag. The expression of Tn-Ag was then examined with the use of VVA. Of 38 cases expressing the phenotypes T-Ag(-) and cT-Ag(+), 6 had tumors that carried Tn-Ag; 5 of them suffered from invasive recurrence. These results indicated that the detection of Tn-Ag with the use of VVA in combination with the examination of T-Ag and cT-Ag is useful for estimating the degree of malignancy of bladder cancer and the patient's clinical course.

A study on the localization of fetal and neonatal brain macrophages of mice from embryonic day 10 (E10) to postnatal day 21 (P21) was carried out immunohistochemically using a monoclonal antibody against a macrophage differentiation antigen (Mac-1) and the labeled avidin-biotin technique. In the central nervous system, the macrophages recognized first were mainly located in the choroid plexuses of the fourth and lateral ventricles at E14. Their number increased at E17-P3 and gradually decreased thereafter. In the cerebral parenchyma, a few macrophages appeared at E14 in the matrix cell layer. They were also detected in the migrating zone at E15, E17 and in the cortical plate at E19. Mapping of positive cells at the stage of neuroblast formation (E15, E17, E19) disclosed the precise distribution of cerebral macrophages. The macrophages that appeared first in the choroid plexuses at E15 may be derived from the subarachnoid vessels, which extend into the stroma of the choroid plexuses when the matrix cell layer invaginates into the lateral ventricle to form the choroid plexuses. Almost all of the macrophages recognized in the cerebral parenchyma disappeared at P9 when the cytoarchitecture seemed to be completed. In the cerebellum, which develops later than the cerebrum, macrophages appeared after birth and were located mainly in the internal granular layer. The brain macrophages always appeared in the regions where cell proliferation and brain remodeling are most active at each stage. These findings suggest that fetal and neonatal brain macrophages may play an important role in scavenging degenerated cells and cell debris during histogenesis of the central nervous system.

Pepleomycin is a derivative of bleomycin, which is less toxic to the host but possesses greater antitumor activity. Experiments are described here in which Chinese hamster V-79 cells in culture and a murine squamous cell carcinoma in vivo have been used to obtain survival curves for pepleomycin alone or in combination with radiation. In both systems the survival curves for pepleomycin alone are biphasic, and this drug proved to be twice as effective as bleomycin. Further, in contrast to bleomycin, which showed simple additivity with radiation, pepleomycin potentiated radiation injury to the tumor cells both in vivo and in vitro. Therefore, pepleomycin may be superior to bleomycin not only in drug therapy but also in combined modality therapy.

Virus-like particles with cylindrical form were found in cultured alveolar macrophages and lung fibroblasts of C3H/St mice, after treating these cells with a carcinogenic polycyclic hydrocarbon, 1, 2, 5, 6, -Dibenzanthracene (DBA). Their morphology looked identical to those observed in vivo in the reactive cells which engulfed DBA crystals implanted into the brain and muscle of the same mouse strain. These particles were not found in either the untreated cells of C3 H/St mice or the treated cells of BALB/c mice. In the alveolar macrophages, these particles appeared first at 3 days after DBA treatment and reached the maximum number around the 30th day. They still kept their morphology in the degenerating cells which had lost the cytoplasmic organelles. These findings suggest the possibility that DBA induced the expression of the viral genome endogeneous to C3H/St cell.