Immunoglobulins in demyelinating lesions in canine distemper encephalitis

Summary The brains of 14 dogs with canine distemper encephalitis were examined with immunohistologic techniques to search for immunogobulin in demyelinating lesions. Four types of lesions presumably representing a temporal sequence of lesion development were distinguished. Immunohistologic findings included immunoglobulin bearing lymphoid cells, amorphous Ig containing material, immunoglobulin bound to the tissue and immunoglobulin containing macrophages and astrocytes. The humoral immune response was absent or very minimal in acute lesions and very intense in chronic lesions. It was concluded that early demyelination in canine distemper encephalitis occurs in the absence of a local humoral immune response but that this response may aggravate and accelerate myelin destruction in the later stages of the disease.Supported by the Schweizer Nationalfonds grant no 3.805.79 and the Schweizerische Multiple Sklerose Gesellschaft     

Primary demyelination in experimental canine distemper virus induced encephalomyelitis in gnotobiotic dogs

Summary Nine dogs with canine distemper encephalitis (CDE) were examined with immunological techniques including demonstration of antibodies against canine distemper virus (CDV) in the serum and against myelin basic protein (MBP) in serum and in CSF. Mitogen stimulation tests of lymphocytes were also done. The brains were examined pathologically and immunoglobulin and C₃ were demonstrated in lesions by means of immunohistological techniques.Six dogs with acute CDE had none or low antibody levels against CDV or MBP, and there was no immunoglobulin in demyelinating lesions. Some of these dogs had depressed lymphocyte mitogen responses. Two dogs with chronic CDE showed recovery of lymphocyte mitogen responses. One of these had a significant antibody response against CDV and MBP in the serum. Both dogs with chronic CDE had very high antibody titers against MBP in the CSF and demyelinating lesions contained immunoglobulin. These results suggest that acute demyelination in CDE is probably due to some direct viral activity and that the progression of demyelination in chronic CDE is associated with a local immune response.Supported by the Schweizer Nationalfonds grands nos. 3.805.79 (to M. V.) and 3.957-0.80 (to A. J. S.) and the Schweizerische Multiple Sklerose Gesellschaft     

Observations on the distribution of canine distemper virus in the central nervous system of dogs with demyelinating encephalitis

Summary The distribution of canine distemper virus in the central nervous system was examined in 11 dogs with demyelinating encephalitis by the direct fluorescent antibody technique on paraffin sections of brain and spinal cord. In the grey matter there was a good correlation between the presence and severity of lesions and presence and amount of viral antigen. Large concentrations of virus were found in neurons and their processes. In most demyelinating lesions only small amounts of viral antigen were found, mostly located in astrocytes. The potential importance of the role of the astrocyte in demyelination in canine distemper virus infection is stressed.     

Albumin leakage into cerebrospinal fluid of dogs lethally infected with R252 canine distemper virus

Cerebrospinal fluid (CSF) form nine lethally infected and three convalescent gnotobiotic dogs infected with the R252 strain of canine distemper virus (CDV) was evaluated prior to and following infection. Lethally infected dogs had a mean seven-fold increase in CSF albumin concentration compared to the preinoculation value, not present in dogs destined to survive. Immunochemical examination of tissue from these dogs revealed prominent perivascular localization of albumin. Examination of CSF cells demonstrated mild leukocytosis in both groups at the time when encephalopathic deaths occurred, with decreased lymphocyte percentages, particularly Thy-1-bearing lymphocytes, in lethally infected dogs. These dogs also had more extensive expression of viral antigens in CSF and peripheral blood leukocytes at the time of death than did surviving dogs, and failed to make antibody to viral antigens. The findings link terminal breakdown of the blood-brain barrier and extensive viral antigen expression in CSF leukocytes with experimental CDV infection resulting in death.     

Restricted expression of viral surface proteins in canine distemper encephalitis

Summary Sixteen dogs with naturally occurring acute and chronic canine distemper virus (CDV) encephalitis were examined immunohistochemically for the presence of the five major CDV-specific proteins in the central nervous system. Monoclonal antibodies (mAbs) directed against two, three, four and five epitopes of the nucleo-(N), phospho- (P), fusion (F), and hemagglutinin (H) protein, respectively, and a polyclonal monospecific antibody recognizing the matrix (M) protein were used. Both core proteins and their epitopes, three F protein epitopes and the M protein were demonstrated in all animals examined. A fourth F protein epitope was found only in 13 animals. The H-2 and H-3 epitope of the H protein were detected in 15, the H-1 and H-5 epitope in 14, and the H-4 epitope in 3 animals. All viral proteins were observed in the same types of brain cells including neurons and astrocytes. The N and P protein were demonstrated in nucleus, cytoplasm and cell processes, whereas M, H and F protein were observed in the cytoplasm only and rarely in cell processes. In addition, the M protein was detected occasionally in the nucleus of neurons and reactive astrocytes. Intralesional distribution of CDV-specific proteins varied between core and surface proteins. In acute and subacute lesions without associated inflammation, expression of the M, H and F protein was only slightly diminished compared to the N and P protein. However, plaques with severe inflammation were either devoid of viral antigen or exhibited N-and P protein-specific immunoreactivity exclusively at the periphery, whereas expression of surface proteins was severely reduced or absent. These results are suggestive of restricted synthesis of CDV envelope proteins in acute, and more prominent in chronic, distemper encephalitis.Supported by the Gemeinnützige Hertie-Stiftung (grant no. GHS 203/91)     

Demyelination in experimental canine distemper virus infection: Immunological,pathologic, and immunohistological studies

Summary White matter lesions were induced in the brains of eight of nine dogs by means of experimental canine distemper virus (CDV) infection. Dogs were killed at 21, 24, 31, and 42 days after infection. Lymphocyte responsiveness to Con A and PHA stimulation in vitro was severely suppressed up to 31 days post infection (p.i.), followed by partial recovery as tested at 42 days p.i. Anti-CDV neutralizing antibody response was very weak in most dogs. There was a weak increase in antimyelin and antimyelin basic protein antibodies in most dogs during the course of the experiment. Dogs killed up to 31 days p.i. developed non-inflammatory demyclinating lesions in which no immunoglobulin could be detected. One of the three dogs that were killed at 42 days p.i. developed severe inflammatory demyelination. This was the only dog with a strong anti-MBP antibody response in the CSF and immunoglobulin demonstrated in demyelinating lesions. The present study supports previous observations that demyelination in acute CDV infection is not an immune mediated lesion but that these lesions may progress as a result of the local immune response. It is uncertain at this stage whether the local immune reaction specifically causes myelin destruction or whether bystander demyelination occurs in chronic CDE.Supported by Schweizer Nationalfonds grant no. 3.809.81 (to M.V.) and grant no. 3.957.0.80 (to A.J.S.)     

Demyelinating canine distemper encephalomyelitis: measurement of myelin basic protein in cerebrospinal fluid

Beagle dogs were experimentally infected with the Cornell A75-17 strain of canine distemper virus. At three time points post-infection (PI), immunoreactive myelin basic protein (MBP) was measured in cerebrospinal fluid (CSF). Levels were correlated with neuropathological findings, interferon in CSF and virus isolation from the brain. CSF from animals inoculated with Cornell A75-17 strain often showed detectable immunoreactive MBP late in the disease course. As anticipated from earlier morphological studies, CSF drawn around day 20 PI lacked MBP while subsequent samples were positive. Dogs with severe demyelination had elevated values of immunoreactive MBP while dogs with only mild inflammation had little or none. Release of MBP or MBP peptides into CSF of dogs with canine distemper may be a valuable laboratory test in studies of the natural history of this disease and in assessing the response to treatment. Whether an immune response to MBP plays an immunopathogenic role in the chronic, demyelinating phase of canine distemper encephalitis remains to be determined.     

Spread and distribution of viral antigen in nervous canine distemper

Summary Canine distemper virus (CDV) antigen was demonstrated immunocytochemically in the central nervous system (CNS) of 19 dogs killed from 16 to 170 days after infection. In the earliest lesions, infection of glial cells preceded demyelination, and the degree of myelin destruction correlated with the amount of viral antigen in the tissue. It was concluded that initial demyelination in distemper is directly viral-induced, but the nature of the infected glial cells remains uncertain. Ependymal infection and spread of virus in the subependymal white matter was often seen, suggesting invasion of CDV into the CNS along the CSF pathways. Inflammation during the latter stages of the infection appeared to be associated with viral clearance from the CNS in most dogs. In two dogs with chronic progressive neurologic distemper, viral antigen was still present in the brain suggesting that viral persistence and associated immunologic reactions may contribute to further myelin damage. With the exception of one dog that survived for 6 months after infection, viral antigen was no longer detected in the dogs that had reeovered.Supported by the Swiss National Science Foundation (Grant no. 3.809.81) and the Swiss M. S. Society     

Antibody-induced generation of reactive oxygen radicals by brain macrophages in canine distemper encephalitis: a mechanism for bystander demyelination

Summary The mechanism of inflammatory demyelination in canine distemper encephalitis (CDE) is uncertain but macrophages are thought to play an important effector role in this lesion. Serum and cerebrospinal fluid (CSF), containing anti-canine distemper virus and anti-myelin antibodies from dogs with CDE were tested for their ability to generate reactive oxygen species (ROS) in macrophages in primary dog brain cell cultures using a chemiluminescence (CL) assay. The majority of serum samples and several CSF samples from animals with inflammatory demyelination elicited a CL signal in infected dog brain cell cultures. In contrast, none of these samples induced a positive response in uninfected cultures which contained large numbers of myelin antigen-presenting cells, although defined anti-myelin antibodies lead to a marked secretion of ROS in this system. It was concluded that antiviral antibody-induced secretion of ROS, known to be highly toxic for brain tissue, may play an important role in white matter damage in inflammatory lesions supporting a previous hypothesis of bystander demyelination in CDE. No evidence was found for a similar antibody-dependent cellular cytotoxicity-like mechanism mediated by anti-myelin antibodies in CDE, which does not support the concept of autoimmunity in this disease.Supported by the Swiss National Foundation Grants nos. 3.956.87 and 3.636.87 and the Swiss Multiple Sclerosis Society     

84例病毒性脑炎的脑电图、脑脊液、头颅CT及临床分析

目的　回顾分析脑电图对病毒性脑炎的早期协助诊断价值以及脑电图结合脑脊液、头颅CT及临床资料对病毒性脑炎的早期诊断价值。方法　分析治愈的 84例病毒性脑炎患者住院 2 4h内的脑电图、腰穿结果 (脑压、脑脊液外观、常规、生化检查 )、头颅CT以及复查的腰穿结果及临床资料。结果　 84例病毒性脑炎患者住院 2 4h内的脑电图异常 77例 ,其中 9例腰穿正常 ;同期腰穿异常结果中以脑压增高例数最多 ( 68例 ) ,其EEG均不正常。EEG和腰穿的检出结果比较有非常显著性差异 ( χ2 =7 11,P <0 0 1) ;头颅CT异常 3 2例。 84例CSF的糖和氯化物初查及复查均正常。临床症状以头痛 ( 82例 )、发热 ( 76例 )最常见。结论　脑电图对病毒性脑炎的早期协助诊断有重要价值。以发热头痛为主症而且脑电图异常 ,结合CSF的压力、细胞数、蛋白的增高、氯化物正常、糖正常或偶有轻度增高及典型病史 ,病毒性脑炎的诊断基本可以确定     

Naturally occurring canine distemper virus encephalitis: distribution and expression of viral polypeptides in nervous tissues

Summary Formalin-fixed brain tissues from 16 dogs with naturally occurring canine distemper virus (CDV) infection were investigated immunohistochemically by a panel of eight monoclonal antibodies (mAb) directed against four structural CDV proteins. Three mAb recognizing different epitopes of the polymerase (P-1, P-2, P-3) protein, two clones identifying different epitopes on each, the nucleocapsid (NP-1, NP-2) and fusion (F-2, F-3) protein, and one mAb directed against the hemagglutinin (H-2) protein were used. The immunoreactivity of the clones was tested on formalin-fixed, paraffin-embedded Vero cells, which were lytically infected with the neurotropic R252 (R252-CDV) or the Onderstepoort (CDV/Ond) strain of CDV. Clones directed against the H-2 and F-3 epitope recognized CDV/Ond but not R252-CDV. The remaining six clones showed positive immunoreaction with both CDV strains. In vivo expression and distribution of the individual proteins and their epitopes varied substantially between animals and within lesions from the same animal. The NP-2 epitope showed positive immunostaining in all 16 cases. The P-2 epitope was demonstrated in 13, the NP-1 epitope in 12, the P-3 epitope in 9, and the P-1 epitope in 3 brains, but staining was severely reduced compared with the NP-2 epitope and restricted to areas with strong NP-2 expression. Immunostaining was prominent in early and subacute and reduced in chronic demyelinating lesions. mAb directed against the H and F protein showed no immunoreaction in diseased brains.Supported by a grant from the Deutsche Forschungsgemeinschaft     

Phase-dependent expression of matrix metalloproteinases and their inhibitors in demyelinating canine distemper encephalitis

Matrix metalloproteinases (MMPs) are zinc- and calcium-dependent enzymes that cleave molecules of the extracellular matrix, and thus are able to open the blood-brain-barrier and affect myelin. Their inhibitors (TIMPs) are important candidates for the therapy of demyelinating diseases. To establish an immunohistochemical profile of MMP and TIMP expression in plaque variants in dogs with spontaneous demyelinating distemper encephalitis, paraffin-embedded cerebella were studied employing the avidin-biotin-peroxidase complex method with a panel of nine polyclonal (anti-MMP-1, -3, -7, -9, -12, -13, -14, -TIMP-1, and -2) and two monoclonal antibodies (anti-latent MMP-2, and -MMP-11). All MMPs and TIMPs were prominently up-regulated in acute and subacute non-inflammatory lesions, and double-labeling techniques showed that they were mainly expressed by astrocytes and brain macrophages/microglia. In subacute inflammatory and chronic plaques, a moderate to strong decrease of MMP and TIMP expression compared to acute lesions was observed. In these phases MMP-11, -12, and -13 were still moderately present. In addition to astro- and microglia, invading perivascular mononuclear cells were positive for MMPs and TIMPs. In summary, there seems to be a phase-dependent expression of MMPs and TIMPs in demyelinating canine distemper encephalitis, and an MMP-TIMP imbalance might account for the lesion progression in this disease.     

Up-regulation of major histocompatibility complex class II antigen expression in the central nervous system of dogs with spontaneous canine distemper virus encephalitis

Major histocompatibility complex class II (MHC II) and canine distemper virus (CDV) antigen expression were compared by immunohistochemistry in the cerebellar white matter of ten dogs with naturally occurring canine distemper encephalitis. In addition, infiltrating mononuclear cells were characterized by employing poly- and monoclonal antibodies directed against human CD3, canine MHC II, CD5, B cell antigen and CDV-specific nucleoprotein. Positive antigen-antibody reaction was visualized by the avidin-biotin-peroxidase complex method on frozen sections. Histologically, neuropathological changes were categorized into acute, subacute, and chronic. In control brains, MHC II expression was weak and predominantly detected on resident microglia of the white matter and on endothelial, perivascular and intravascular cells. In CDV antigen-positive brains, MHC II was mainly found on microglia and to a lesser extent on endothelial, meningeal, choroid plexus epithelial, ependymal and intravascular cells. In addition, virtually all of the perivascular cells expressed MHC II antigen. CDV antigen was demonstrated most frequently in astrocytes. Of the perivascular lymphocytes, the majority were CD3-positive cells, followed by B cells. Only a small proportion of perivascular cells expressed the CD5 antigen. In addition, B cells and CD3 and CD5 antigen-positive cells were found occasionally in subacute and frequently in chronic demyelinating plaques. In acute encephalitis, CDV antigen exhibited a multifocal or diffuse distribution, and MHC II was moderately up-regulated throughout the white matter and accentuated in CDV antigen-positive plaques. In subacute encephalitis, moderate multifocal CDV antigen and moderate to strong diffuse MHC II-specific staining, especially prominent in CDV antigen-positive lesions, were observed. In chronic encephalitis, CDV antigen expression was restricted to single astrocytes at the edge of the lesions or was absent, while MHC II expression, especially prominent on microglia, was strongly up-regulated throughout the white matter, most pronounced in demyelinated plaques. In summary, in acute and subacute lesions without perivascular cuffs, MHC II expression correlated with the presence of CDV antigen. In contrast, in chronic lesions, MHC II expression on microglial cells was the most prominent despite a few CDV antigen-positive astrocytes, indicating that nonviral antigens may play an important role as triggering molecules for the process of demyelination. Received: 13 September 1995 / Revised: 26 February 1996 / Accepted: 1 April 1996     

Prognostic value of intrathecal antibody production and DNA viral load in cerebrospinal fluid of patients with herpes simplex encephalitis

Herpes simplex encephalitis is a devastating disease. In the early 1980s our group conducted a nationwide clinical trial of acyclovir versus vidarabine in patients with herpes simplex encephalitis in whom intrathecal herpes simplex virus (HSV) antibodies were assayed. The purpose of this study was to investigate if antibody levels and viral load correlate with outcome in herpes simplex encephalitis. We have analysed the prognostic value of HSV antibody levels in serum and cerebrospinal fluid (CSF) at the start of antiviral treatment in the 53 included patients. Frozen samples from a subset of patients were analysed with quantitative polymerase chain reaction (PCR) to assess the prognostic value of the viral load in CSF. IgG-levels in CSF at presentation were significantly higher in vidarabine-treated patients with a favourable outcome than in those treated with vidarabine but with an unfavourable outcome. The intrathecal viral load at presentation showed no correlation with outcome. However, the duration of positive HSV-PCR in CSF was longer in vidarabine-treated than in acyclovir-treated patients. These findings indicate that the B-cell response is important in the pathogenetic process of herpes simplex encephalitis. However, neither antibody levels nor viral load at presentation are useful as prognostic markers for the individual patient in this study.     

磁共振成像及脑脊液细胞学对病毒性脑(膜)炎的诊断性研究

目的探讨MRI和脑脊液细胞学检测阳性率、异常程度和发病时间对病毒性脑(膜)炎诊断和制定治疗方案的指导价值。方法回顾189例病毒性脑(膜)炎患者的头部MRI及脑脊液检查资料,分析MRI正常或异常患者在疾病早期的脑脊液细胞学改变。结果 189例患者中96例(50.79%)呈现MRI异常影像、129例(68.25%)脑脊液细胞学检测异常。MRI异常患者中脑脊液细胞学检测异常率为72.92%(70/96),与MRI正常患者(63.44%,59/93)比较差异具有统计学意义(P=0.000)。结论病毒性脑(膜)炎患者在疾病早期MRI改变晚于脑脊液细胞学改变,但二者对提示诊断和制定治疗方案均具有重要临床意义。     

Modifications of the cerebrospinal fluid IgG concentrations in patients with multiple sclerosis treated with intrathecal steroids

Summary Forty-one patients with multiple sclerosis (MS), in the acute exacerbation phase of the disease, were treated with three or four intrathecal injections of triamcinolone retard, 40 mg. Cerebrospinal fluid (CSF) specimens were collected at the first and at the last lumbar puncture, and analyzed for IgG and albumin. There was a decrease of CSF IgG concentration in the specimens of 85% of the cases which were collected at the last lumbar puncture, compared to the initial concentration. Similarly, the CSF IgG/albumin ratio in the last specimens collected were reduced in 78% of the cases. Both these results are statistically significant.Presented to the Meeting of Swiss Neuropathologists with International Participation, St. Moritz, 6 to 9 March 1978     

病毒性脑炎患者血液和脑脊液博尔纳病病毒p24的检测

目的探讨博尔纳病病毒（BDV）与病毒性脑炎（VE）的关系。方法采用巢式逆转录聚合酶链反应（RT-PCR）结合荧光定量（FQ）PCR检测了52例VE患者和32例外科手术患者外周血液（PBMC）和脑脊液有核细胞（CSFMC）中的BDV p24基因片段，对阳性产物进行了基因序列测定、同源性和氨基酸顺序分析。结果VE患者PBMC和CSFMC中BDV p24基因片段阳性率（分别为9．6％和11．5％）明显高于对照组（0，P〈0．05）；PBMC和CSFMC中BDV p24基因片段拷贝数的对数值呈显著正相关（r=0．653，P〈0．05）；CSFMC所含目的基因片段阳性产物测序后，与BDV标准病毒株V核苷酸序列比较同源性为96．5％，在3个位点出现突变（nt1649T→C，nt1670C→T，nt1673C→T），突变率为3．5％。从系统发生树图形可以看出，该目的基因片段与澳大利亚马脑组织分离的S88和德国绵羊血液分离的H544病毒株亲缘关系最近。结论宁夏及其周边地区部分VE患者中可能存在BDV感染。     

单纯疱疹病毒性脑炎患者脑脊液和血清S-100 B蛋白及NSE的测定

目的　测定单纯疱疹病毒性脑炎 (HSE)患者脑脊液和血清中的S 1 0 0B蛋白和神经元特异性烯醇化酶(NSE)含量 ,并探讨S 1 0 0B蛋白和NSE含量对HSE患者脑损害的评估价值。方法　 4 2例HSE患者 (昏迷组 1 6例 ,无昏迷组 2 6例 )和 2 2例正常人对照组脑脊液和血清的S 1 0 0B蛋白和NSE含量 ,均采用酶联免疫吸附试验双抗体夹心法检测 ,并进行动态观察。结果　①HSE昏迷组的脑脊液和血清S 1 0 0B蛋白和NSE含量显著高于无昏迷组和对照组 (P均小于 0 0 1 ) ,无昏迷组脑脊液S 1 0 0B蛋白和NSE含量与对照组也有差异 (P <0 0 1和 <0 0 1 ) ;②脑脊液S 1 0 0B蛋白和NSE含量随HSE患者病情变化而发生相应的改变 ;③脑脊液S 1 0 0B蛋白和NSE含量分别与其各自的血清含量呈正相关。结论　脑脊液和血清的S 1 0 0B蛋白和NSE可以作为HSE患者脑损害的标志物 ,其含量的高低与胶质细胞和神经元的损害程度有关