Antigen-specific therapy of experimental allergic encephalomyelitis by soluble class II major histocompatibility complex-peptide complexes.

Experimental allergic encephalomyelitis is a T-cell-mediated, major histocompatibility complex (MHC) class II gene-linked autoimmune demyelinating disease of the central nervous system. To develop therapies that will specifically inactivate only the autoantigen-reactive T cells, mice were treated with soluble MHC class II molecules that had been complexed with encephalitogenic peptides. Intravenous injections of 300 micrograms of complexes consisting of encephalitogenic peptide 91-103 of myelin basic protein plus I-As protein on day 0, 4, and 7 were effective in preventing experimental allergic encephalomyelitis. Similarly, administration of 45 micrograms of I-As protein complexed to peptide 139-151 from proteolipoprotein on day 1, 4, and 7 prevented mortality and significantly reduced paralysis induced by immunization with the encephalitogenic proteolipoprotein peptide. Histological examination of sections of animal brains revealed that treatment with I-As protein plus myelin basic protein 91-103 peptide prevents the development of inflammatory lesions characteristic of experimental allergic encephalomyelitis. Thus, treatment with MHC-self-peptide complexes could serve as a highly specific therapeutic modality in treating autoimmune disease when the putative autoantigen and the MHC restricting elements are known.     

Time-course changes of nerve growth factor, corticotropin-releasing hormone, and nitric oxide synthase isoforms and their possible role in the development of inflammatory response in experimental allergic encephalomyelitis

In this paper we report a time-course study of development of experimental allergic encephalomyelitis in Lewis rats, by monitoring neuroendocrine regulation of the hypothalamus–pituitary–adrenal axis through corticotropin-releasing hormone mRNA expression, inflammatory cellular infiltrate, macrophagic and neuronal nitric oxide synthase, nerve growth factor (NGF), and NGF p75 and trkA receptors in the brain and spinal cord. We analyzed animals during 20 days after immunization, a time interval that corresponds to the acute immunological phase. We have described a severe, early fall of corticotropin-releasing hormone mRNA expression, which could account for the decreased response of the hypothalamus–pituitary–adrenal axis to inflammatory stress. During this period, an increase of neuronal nitric oxide synthase was observed in the cerebral cortex and spinal cord, and macrophagic nitric oxide synthase positive cells were found in the inflammatory cellular infiltrate, which was abundant in perivascular and submeningeal areas 20 days after immunization. Concomitantly, we found a dramatic up-regulation of NGF receptors on the wall of blood vessels and adjacent neurons in perivascular areas. NGF content also had increased in some brain areas, such as the thalamus, while it had decreased in others, like the spinal cord and medulla oblongata, at time points in which the most serious cellular infiltrate was found.     

Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization.

We compared the antigen-specific antibody isotypes and lymphokine secretion by CD4+ T cells in BALB/c mice immunized intradermally with either Escherichia coli beta-galactosidase (beta-gal) or plasmid DNA (pDNA) encoding beta-gal in a cytomegalovirus-based expression vector (pCMV-LacZ). pCMV-LacZ induced mainly IgG2a, whereas beta-gal in saline or alum induced IgG1 and IgE beta-gal-specific antibodies. In addition, splenic CD4+ T helper (Th) cells isolated from pDNA-immunized mice secreted interferon-gamma but not interleukin (IL)-4 and IL-5, whereas Th cells from beta-gal-injected mice secreted IL-4 and IL-5 but not interferon-gamma after in vitro stimulation with antigen. Together these data demonstrate that pDNA immunization induced a T helper type 1 (Th1) response, whereas protein immunization induced a T helper type 2 (Th2) response to the same antigen. Interestingly, priming of mice with pCMV-LacZ prevented IgE antibody formation to a subsequent i.p. beta-gal in alum injection. This effect was antigen-specific, because priming with pCMV-LacZ did not inhibit IgE anti-ovalbumin antibody formation. Most importantly, intradermal immunization with pCMV-LacZ (but not pCMV-OVA) of beta-gal in alum-primed mice caused a 66-75% reduction of the IgE anti-beta-gal titer in 6 weeks. Also, pCMV-LacZ induced specific IgG2a antibody titers and interferon-gamma secretion by Th cells in the beta-gal in alum-primed mice. The data demonstrate that gene immunization induces a Th1 response that dominates over an ongoing protein-induced Th2 response in an antigen-specific manner. This suggests that immunization with pDNA encoding for allergens may provide a novel type of immunotherapy for allergic diseases.     

Plasmid DNA immunization against Japanese encephalitis virus: immunogenicity of membrane-anchored and secretory envelope protein.

Plasmid DNA synthesizing membrane-anchored or secretory Japanese encephalitis virus (JEV) envelope (E) protein and premembrane protein was delivered to mice by intramuscular injection or gene gun. Intramuscular plasmid immunization induced anti-E antibody responses similar to those associated with commercial JEV vaccine. The gene gun induced less antibody response. The 2 forms of the E protein induced similar antibody titers when administered by the same delivery mode. Both plasmids generated high titers of JEV-neutralizing antibodies, although the titers were lower than those induced by the vaccine. Intramuscular DNA immunization induced T helper 1 (Th1) immune responses, and the gene gun induced Th2 responses. Compared with secretory E protein, the membrane-anchored protein heavily skewed the immune response toward either Th1 or Th2, depending on the route of immunization. In an intracerebral JEV challenge model, plasmid-immunized mice had approximately 60% protection; this was not affected by the form of the E protein or by immunization route.     

Production of lethal infection that resembles fatal human disease by intranasal inoculation of macaques with Japanese encephalitis virus.

Twelve rhesus macaques (Macaca mulatta) challenged intranasally with a wild-type Japanese encephalitis virus (JEV) developed clinical signs 11-14 days later. Tissues from the cerebral cortex, cerebellum, brainstem, thalamus, meninges, and all levels of the spinal cord were stained for JEV antigen with hyperimmune mouse ascitic fluid and streptavidin-alkaline phosphatase; immunofluorescent staining was also done on frozen sections. Viral antigen was found in all cell layers of the cerebellum, the gray matter of the thalamus and brainstem, and the ventral horn of all levels of the spinal cord. Staining was limited to neurons and their processes. Histopathologic changes were limited to the nervous system and characterized by nonsuppurative meningoencephalitis. These results were comparable with those of previous studies done with human autopsy tissues. Intranasal inoculation of rhesus monkeys with JEV was effective in producing clinical disease comparable with natural disease in humans and may serve as a model to evaluate protective efficacy of candidate JEV vaccines.     

Short report: absence of protective neutralizng antibodies to West Nile virus in subjects following vaccination with Japanese encephalitis or dengue vaccines

Protection of individuals against West Nile (WN) encephalitis is an emerging concern in the United States and Europe. We investigated whether immunization with licensed inactivated Japanese encephalitis (JE) vaccine or experimental live attenuated dengue vaccines resulted in induction of cross-neutralizing antibodies against WN virus. Protective neutralizing antibody titers to WN virus were not detected in any volunteer despite successful immunization to related flaviviruses. Vaccination against JE or dengue is unlikely to prevent WN virus infection but may still protect against disease.     

火把花根片对EAE大鼠模型VCAM-1和L-选择素的影响

目的探讨火把花根片对实验性自身免疫性脑脊髓炎(EAE)大鼠模型的治疗作用以及对VCAM-1和L-选择素的影响。方法模型组采用豚鼠髓鞘蛋白匀浆和福氏完全佐剂诱发大鼠急性EAE模型;治疗组在模型组基础上予以火把花根(300mg·kg-1·d-1,每日2次),观察动物表现并评分,HE染色和Loyez氏髓鞘染色观察病理和髓鞘改变,免疫组化技术测定脑和脊髓的VCAM-1和L-选择素(L-selectin)水平。结果治疗组未出现临床症状,VCAM-1为(2.13±0.99),L-selectin为(1.88±0.64),与模型组比较差异有统计学意义(P<0.05);脑和脊髓小血管周围炎症细胞浸润明显减少,髓鞘结构完整。结论火把花根片对EAE有治疗作用,其作用机制与抑制VCAM-1及L-selectin表达有关。     

Cytotoxic T lymphocyte antigen-4 (CTLA-4) limits the expansion of encephalitogenic T cells in experimental autoimmune encephalomyelitis (EAE)-resistant BALB/c mice

We and others previously reported that cytotoxic T lymphocyte antigen-4 (CTLA-4) regulates the severity of peptide-induced experimental autoimmune encephalomyelitis (EAE) in mouse strains that are inherently susceptible to the disease. In this report, we show that CTLA-4 engagement also controls disease susceptibility in BALB/c mice, a strain considered to be resistant to EAE induction. Although immunization of BALB/c mice with syngeneic spinal cord homogenate or an I-A(d)-binding myelin peptide antigen failed to result in EAE, immunization with either antigen preparation in conjunction with anti-CTLA-4 resulted in both clinical and histological EAE. CTLA-4 blockade also resulted in a preferential increase in the frequency of antigen-specific T cells secreting IFN-gamma. We conclude that CTLA-4 controls susceptibility in BALB/c mice by limiting the expansion of autoreactive T cells present in the periphery, suggesting a mechanism whereby CTLA-4 contributes to the maintenance of peripheral T cell tolerance to self antigens.     

Disappearance and reappearance of B cells after in vivo treatment with monoclonal anti-I-A antibodies.

Previous studies have shown that treatment with antibodies to the murine I-A antigen encoded in the major histocompatibility complex attenuates experimental allergic encephalitis and experimental autoimmune myasthenia gravis. These studies were conducted with SJL mice, an inbred strain that is highly susceptible to the induction of these diseases. Here we show that injection of monoclonal anti-I-A antibody in the amounts used for the above studies rapidly depletes B cells. Fluorescence-activated cell sorter (FACS) multiparameter analysis of the B-cell subpopulations in treated animals shows that maximum depletion occurs around 5 days after treatment and that recovery of some subpopulations i still incomplete 1 month later. SJL mice are more sensitive to this B-cell depletion and recover more slowly than putatively normal C3H.Ighb (CKB) mice. Some components of the primary, secondary and tertiary IgG antibody responses are reduced in anti-I-A-treated SJL animals immunized after the first and second anti-I-A injections. The persistence of some antibody response impairment well beyond the time when anti-I-A disappears raises a note of caution concerning human therapy protocols based on the injection of anti-Ia antibodies.     

Differential suppression of experimental allergic diseases in rats infected with trypanosomes

PVG/c rats, infected 3 days previously with 10(3) Trypanosoma brucei brucei S.42 organisms failed to develop adjuvant disease in response to an intradermal inoculation of mycobacterial adjuvant. By contrast, similarly infected rats, immunized with heterologous brain and spinal cord in Freund's complete adjuvant with pertussis vaccine as a secondary adjuvant, developed clinical signs of allergic encephalomyelitis (EAE) at least as severe as those in uninfected rats. Delayed hypersensitivity reactions to PPD were depressed in trypanosome-infected, adjuvant-injected rats, as were the reactions to myelin basic protein in infected rats developing EAE. There appeared to be no cross-reactivity between trypanosomal antigen and myelin basic protein which could account for the lack of suppression of EAE. It is suggested that the different extent to which autoimmunity is involved in these two experimental allergic diseases may account for the differential suppressive activity of trypanosome infections upon them.     

Embryonic expression patterns of the neural cell adhesion molecule gene are regulated by homeodomain binding sites.

During development of the vertebrate nervous system, the neural cell adhesion molecule (N-CAM) is expressed in a defined spatiotemporal pattern. We have proposed that the expression of N-CAM is controlled, in part, by proteins encoded by homeobox genes. This hypothesis has been supported by previous in vitro experiments showing that products of homeobox genes can both bind to and transactivate the N-CAM promoter via two homeodomain binding sites, HBS-I and HBS-II. We have now tested the hypothesis that the N-CAM gene is a target of homeodomain proteins in vivo by using transgenic mice containing native and mutated N-CAM promoter constructs linked to a beta-galactosidase reporter gene. Segments of the 5' flanking region of the mouse N-CAM gene were sufficient to direct expression of the reporter gene in the central nervous system in a pattern consistent with that of the endogenous N-CAM gene. For example, at embryonic day (E) 11, beta-galactosidase staining was found in postmitotic neurons in dorsolateral and ventrolateral regions of the spinal cord; at E14.5, staining was seen in these neurons throughout the spinal cord. In contrast, mice carrying an N-CAM promoter-reporter construct with mutations in both homeodomain binding sites (HBS-I and HBS-II) showed altered expression patterns in the spinal cord. At E11, beta-galactosidase expression was seen in the ventrolateral spinal cord, but was absent in the dorsolateral areas, and at E 14.5, beta-galactosidase expression was no longer detected in any cells of the cord. Homeodomain binding sites found in the N-CAM promoter thus appear to be important in determining specific expression patterns of N-CAM along the dorsoventral axis in the developing spinal cord. These experiments suggest that the N-CAM gene is an in vivo target of homeobox gene products in vertebrates.     

Monoclonal antibodies distinguish antigenically discrete neuronal types in the vertebrate central nervous system.

Eight hundred hybridoma lines were generated from mice immunized with the fixed gray matter of cat spinal cord. Of these lines, 47 were positive when screened immunohistochemically against sections of the cat spinal cord. Twenty-nine lines secreted antibodies that bound to neuronal antigens. Of these, 16 bound to axons only, 8 bound to axons and cell bodies, and 5 bound to cell bodies only. Eighteen lines secreted antibodies that bound to glial cells. Five lines that secreted antibodies that intensely stained spinal cord sections were cloned and screened against other parts of the central nervous system. Each of these five antibodies bound to specific subsets of neurons. For example, in the spinal cord, one antibody (Cat-301) recognized a surface determinant on the dendrites and cell bodies of neurons that, in morphology and location, resemble long-distance projection neurons. A second antibody (Cat-201) recognized an antigen in axons and in the cytoplasm of neuronal cell bodies that may be a subset of those recognized by Cat-301. A third antibody (Cat-101) recognized only axons. The subcellular localization of the antigen recognized by each antibody is the same in all areas of the central nervous system we have examined. The fact that each of the antibodies described here has a restricted distribution in the central nervous system shows that there is a high degree of molecular diversity among vertebrate neurons and that hybridoma technology can be used to explore this diversity. This class of reagents should be a useful addition to the many established techniques for studying the organization of the vertebrate central nervous system.     

Induction of immune responses in vivo with small numbers of veiled (dendritic) cells.

The role of dendritic or veiled cells (VC) from lymph nodes or spleens of rats and mice in initiating immune responses in vivo has been investigated. Host-versus-graft responses were induced by injection of VC from spleens of (C57BL/10 X CBA) F1 mice into the footpads of parental strain (CBA) animals and measured by the increase in the weight of the draining popliteal lymph nodes. The potency of VC to induce the responses was 100-fold greater than that of unseparated spleen cells. The in vivo effect of VC was not limited to this direct allogeneic stimulation because autologous VC could also be used in the induction of an experimental autoimmune disease. In these studies, experimental allergic encephalomyelitis was produced in Lewis rats by injection of guinea pig brain and spinal cord material emulsified in Freund's complete adjuvant. Small numbers of VC from spleens or lymph nodes of rats showing clinical signs of experimental allergic encephalomyelitis induced a mild form of the disease when injected intravenously into normal Lewis rats. Thus, VC carrying antigen, either as an integral part of their surface membrane or acquired during exposure to antigenic substances, appeared to be very potent agents for the induction of immune responses.     

Immunohistochemical localization and mRNA expression of apolipoprotein A-I in rat spinal cord

Apolipoproteins in the cerebrospinal fluid (CSF) play important roles in lipid metabolism in the central nervous system. Although it has been demonstrated that apo E is synthesized in the neuron, the synthesis of apo A-I has only been determined in fish and chicken. It was demonstrated that apo A-I concentrations in the CSF were increased in poliovirus-infected macaques, however, the origin of the CSF apo A-I was not determined. The present immunohistochemical study provided evidence that apo A-I was localized within the nerve cell body of the rat spinal cord. In situ hybridization also showed that apo A-I mRNA was predominantly expressed in the neurons. As a further experiment, we compared apo A-I levels in the spinal cord from control rats and rats with experimental allergic encephalomyelitis (EAE), which was induced by sensitization with myelin basic protein. Although no significant changes in serum apo A-I levels were observed, apo A-I levels in the spinal cord were significantly elevated in EAE rats. Furthermore, apo A-I in the spinal cord of rats with EAE was not seen in the nerve cell body, but at the interstitium, particularly in lesions where inflammation had occurred. The current study clearly demonstrated that apo A-I is synthesized in the neurons of the rat spinal cord and the synthesis was suppressed in EAE rats.     

Thyroid hormone activates oligodendrocyte precursors and increases a myelin-forming protein and NGF content in the spinal cord during experimental allergic encephalomyelitis

Remyelination in the adult central nervous system has been demonstrated in different experimental models of demyelinating diseases. However, there is no clear evidence that remyelination occurs in multiple sclerosis, the most diffuse demyelinating disease. In this article, we explore the possibility of promoting myelination in experimental allergic encephalomyelitis, a widely used experimental model of multiple sclerosis, by recruiting progenitors and channeling them into oligodendroglial lineage through administration of thyroid hormone (T4). A large number of proliferating cells (BrdUrd uptake and Ki67-IR) and the expression of markers for undifferentiated precursors (nestin) increased in the subventricular zone and spinal cord of experimental allergic encephalomyelitis animals. T4 administration reduces proliferation and nestin-immunoreactivity and up-regulates expression of markers for oligodendrocyte progenitors [polysialylated-neural cell adhesion molecule (PSA-NCAM), O4, A2B5] and mature oligodendrocytes (myelin basic protein) in the spinal cord, olfactory bulb, and subventricular zone.     

Transverse myelitis extended to disseminated encephalitis in systemic lupus erythematosus: Histological evidence for vasculitis

A 42-year-old woman was admitted due to systemic lupus erythematosus complicated with glomerulonephritis and pulmonary hypertension. During the treatment for these complications, she presented motor paresis and sensory loss caused by transverse myelitis. In spite of methyl prednisolone pulse therapy, she further developed acute confusional state due to disseminated encephalitis and fell into respiratory arrest. On laboratory examination, elevation of anti-NR2 antibodies in serum as well as in cerebrospinal fluid was noted. Although she recovered from the disseminated encephalitis after extensive treatment with high doses of corticosteroid and intravenous cyclophosphamide, she suddenly died of pulmonary hypertension. Autopsy findings confirmed the presence of liquefaction necrosis in the entire circumference of the whole spinal cord along with intimal hyperplasia and obliteration of the small arteries, accompanied by mononuclear cell infiltration and disruption of internal elastic lamina. It is therefore most likely that our patient developed longitudinal transverse myelitis through spinal cord vasculitis, which extended to brainstem and brain parenchyma, leading to the development of disseminated encephalitis.     

丙型肝炎病毒核心基因免疫诱生细胞免疫应答研究

目的 研究丙型肝炎病毒（ＨＣＶ）核心（Ｃ）基因免疫在诱生特异性细胞免疫应答中的作用。方法 将包含ＨＣＶ Ｃ基因片段的重组真核表达质粒ｐｃＣＮＡ ＨＣＶ Ｃ,在主宰其可以在小鼠骨髓瘤ＳＰ２／０（Ｈ－２^d）中表达之后,注射ＢＡＬＢ／ｃ小鼠股四头肌。ＥＬＩＳＡ法检测血清中抗体水平;^3Ｈ－ＴｄＲ掺入法测定免疫小鼠脾细胞特异性增殖能力;^51Ｃｒ释放法检测免疫小鼠细胞毒性Ｔ淋巴细胞（ＣＴＬｓ）体外杀伤功能。结     

mpt64-卡介苗重组疫苗免疫原性研究

目的研究rapt64-卡介苗重组疫苗的免疫原性。方法以生理盐水、卡介苗、mpt64-卡介苗重组疫苗免疫小鼠，采用ELISA法检测小鼠血清中特异性抗体，MTS法分析小鼠脾淋巴细胞增殖指数。结果mpt64-卡介苗重组疫苗组小鼠体重变化与对照组相比，无显著性差异。应用不同抗原检测各组小鼠不同时间采集的血清，rapt64-卡介苗重组疫苗组有MPT64特异性抗体产生，而卡介苗组无MPT64特异性抗体产生；卡介苗、mpt64-卡介苗重组疫苗免疫后15d，抗体水平已有升高，45d达到最高水平。用不同抗原刺激实验组及对照组小鼠脾淋巴细胞，平均刺激指数均达2．0以上。结论结核分枝杆菌mpt64基因在卡介苗中能够表达特异的蛋白，刺激小鼠产生特异性抗体。     

DNA immunization encoding the secreted nonstructural protein 3 (NS3) of hepatitis C virus and enhancing the Th1 type immune response

To induce a sustained and specific cellular immune response to hepatitis C virus (HCV), DNA immunization of mice was performed using plasmids containing the HCV nonstructural gene 3 (HCV/NS3). Plasmids were constructed such that the NS3 gene was expressed in a secreted form, a nonsecreted form or as a membrane-bound antigen. The plasmid encoding the secreted antigen induced the strongest humoral and cellular immunity and favoured the T-helper type 1 (Th1) pathway as shown by cytokine profiles and switching of antibody subclasses. Our study indicates that DNA immunization with a secreted form of HCV/NS3 is an effective means of inducing primary Th1 immune responses in the murine model.     

Atopy and neural damage

The occurrence of myelitis with atopic diathesis (atopic myelitis) affecting young adults has recently been noted in Japan. The disease preferentially affects the posterior column of the cervical spinal cord, as shown clinically and by MRI. It is characterized by hyperIgEaemia and the presence of mite antigen-specific IgE. The spinal cord lesions have been shown to be eosinophilic inflammation on biopsy and thus an allergic mechanism is thought to be operative in this condition. In addition, we also found that Hirayama disease, juvenile muscular atrophy of the distal upper extremity, is also associated with airway allergy such as allergic rhinitis and atopic asthma. In children, poliomyelitislike illness after acute asthma attacks is well known as Hopkins syndrome. Moreover, by the prospective study of the history of allergic disorders in common neurologic diseases, an association between spinal progressive muscular atrophy (SPMA) and asthma as well as between myelitis and atopic dermatitis has been demonstrated. These observations strongly suggest a link between atopic diathesis and spinal cord damage. Central nervous system damage associated with atopic diathesis may be classified into two types; eosinophilic myelitis preferentially affecting the cervical spinal cord and lower motor neuron damage, such as Hopkins syndrome, Hirayama disease and SPMA. The former is typically associated with atopic dermatitis while the latter, with airway allergy.