Utililization of experimental animal model for correlative multispectral MRI and pathological analysis of brain tumors.

Magnetic resonance imaging is the method of choice for non-invasive detection and evaluation of tumors of the central nervous system. However, discrimination of tumor boundaries from normal tissue, and the evaluation of heterogeneous lesions have proven to be limitations in traditional magnetic resonance imaging. The use of post-image acquisition processing techniques, such as multispectral tissue segmentation analysis, may provide more accurate clinical information. In this report, we have employed an experimental animal model for brain tumors induced by glial cells transformed by the human neurotropic JC virus to examine the utility of multispectral tissue segmentation for tumor cell identification. Six individual tissue types were discriminated by segmentation analysis, including heterogeneous tumor tissue, a clear demarcation of the boundary between tumor and non-tumor tissue, deep and cortical gray matter, and cerebrospinal fluid. Furthermore, the segmentation analysis was confirmed by histopathological evaluation. The use of multispectral tissue segmentation analysis may optimize the non-invasive determination and volumetric analysis of CNS neoplasms, thus providing improved clinical evaluation of tumor growth and evaluation of the effectiveness of therapeutic treatments.

Ibuprofen treatment versus gradual introduction of interferon beta-1b in patients with MS.

Flu-like symptoms and injection site reactions are adverse effects of treatment with interferon beta-1b in patients with MS. We compared gradual dose escalation, ibuprofen treatment, or their combination in an open-label study. The combination reduced the incidence of flu-like symptoms to rates comparable with the placebo group in the pivotal trial but increased the frequency of injection site reactions, albeit modestly and transiently.

Improved intracranial lesion characterization by tissue segmentation based on a 3D feature map.

Our aim was to develop an accurate multispectral tissue segmentation method based on 3D feature maps. We utilized proton density (PD), T2-weighted fast spin-echo (FSE), and T1-weighted spin-echo images as inputs for segmentation. Phantom constructs, cadaver brains, an animal brain tumor model and both normal human brains and those from patients with either multiple sclerosis (MS) or primary brain tumors were analyzed with this technique. Initially, misregistration, RF inhomogeneity and image noise problems were addressed. Next, a qualified observer identified samples representing the tissues of interest. Finally, k-nearest neighbor algorithm (k-NN) was utilized to create a stack of color-coded segmented images. The inclusion of T1 based images, as a third input, produced significant improvement in the delineation of tissues. In MS, our 3D technique was found to be far superior to that based on any combination of 2D feature maps (P < 0.001). We identified at least two distinctly different classes of lesions within the same MS plaque, representing different stages of the disease process. Further, we obtained the regional distribution of MS lesion burden and followed its changes over time. Neuropsychological aberrations were the clinical counterpart of the structural changes detected in segmentation. We could also delineate the margins of benign brain tumors. In malignant tumors, up to four abnormal tissues were identified: 1) a solid tumor core, 2) a cystic component, 3) edema in the white matter, and 4) areas of necrosis and hemorrhage. Subsequent neurosurgical exploration confirmed the distribution of tissues as predicted by this analysis.

A multicenter, randomized, double-blind, placebo-controlled trial of influenza immunization in multiple sclerosis.

We determined the effect of influenza vaccine in patients with relapsing/remitting MS. Considerable controversy surrounds the question of whether to administer influenza vaccines to MS patients. Prevention of a febrile viral illness is clearly desirable in MS, and previous studies suggest that immunization is safe. Despite this, many clinicians avoid vaccination because they fear precipitating an MS exacerbation. We conducted a multicenter, prospective, randomized, double-blind trial of influenza immunization in patients with relapsing/remitting MS. In the autumn of 1993, 104 patients at five MS centers received either standard influenza vaccine or placebo. Patients were followed for 6 months for evaluation of neurologic status and the occurrence of influenza. Influenza was operationally defined as fever > or = 38 degrees C in the presence of coryza, cough, or sore throat at a time when the disease was present in the community. Attacks were defined in the standard manner, requiring objective change in the examination. Patients were examined at 4 weeks and 6 months after inoculation and were contacted by telephone at 1 week and 3 months. They were also examined at times of possible attacks but not when they were sick with flu-like illness. Three vaccine patients and two placebo patients experienced attacks within 28 days of vaccine (no significant difference). Exacerbation rates in the first month for both groups were equal to or less than expected from published series. The two groups showed no difference in attack rate or disease progression over 6 months. Influenza immunization in MS patients is neither associated with an increased exacerbation rate in the postvaccination period nor a change in disease course over the subsequent 6 months.

Modulation of T cell-endothelial adhesion by astrocyte conditioned medium.

Astrocytes and derived factors maintain the morphologic, phenotypic, and physiological properties of the blood-brain barrier. Astroglial cells may also modulate endothelial cell properties associated with the entry of inflammatory cells into the brain. The study of mechanisms of lymphocyte migration through the blood-brain barrier is critical to understanding the pathophysiology of autoimmune (multiple sclerosis) and virus-induced central nervous system diseases (HIV-induced dementia). In this context the contribution of astrocyte derived factors in regulating the interactions between inflammatory cells and endothelial cells of the blood-brain barrier was studied. The treatment of endothelial cells derived from brain or peripheral sources (hepatic) with astrocyte conditioned medium resulted in a dose dependent enhancement of adhesion of T cells to endothelium. The antigen specificity of the T cells did not influence the findings. Identical results were obtained with fresh Concanavalin A activated T cells and T cell hybridomas generated using myelin basic protein or chicken ovalbumin as immunogens. Further studies are in progress to define the active components in astrocyte conditioned medium and endothelial cell adhesion molecules that are regulated in order to gain a better understanding of mechanisms of inflammatory cell entry into the central nervous system.

Formation of undifferentiated mesenteric tumors in transgenic mice expressing human neurotropic polymavirus early protein.

The human polyomavirus, JCV, is the established etiologic agent of the human demyelinating disease, progressive multifocal leukoencephalopathy (PML) seen in immunosuppressed individuals. In PML patients, the viral early protein, which is produced exclusively in glial cells is responsible for initiation of the viral lytic cycle. The JCV early protein, T-antigen, has greater than 70% homology to the well characterized SV40 early protein which has established oncogenic properties. To investigate the role of JCV T-antigen in tumorigenesis, transgenic mice containing the viral early genome were produced. Of the four positive transgenic animals, one developed severe neurological abnormalities and succumbed to death at 3 weeks of age. Another animal died with no visible gross pathology and the cause of death was not determined. The remaining two founders developed massive, undifferentiated, solid mesenteric tumors with no obvious neurological symptoms. Results from histologic analysis demonstrated the presence of highly cellular, poorly differentiated neoplastic cells in the tumor tissue. Electron microscopic evaluation of the tumor revealed the presence of a small blue cell-like tumor of epithelial/neuroectodermal origin. Results from RNA analysis by non-quantitative and highly sensitive RT-PCR indicated the presence of the JCV early transcript in various tissues, including kidney, liver, spleen, heart, lung, and brain, as well as in the tumors. However, analysis of the viral early protein by Western blot and immunohistochemistry indicated high level production of JCV early protein in the tumor tissue, but not in any other tissues. These observations present the first evidence for the development of inheritable neuroectodermal tumors induced by the human polyomavirus, JCV, early protein in a whole animal system.

Cutaneous ulcerations and pustular psoriasis flare caused by recombinant interferon beta injections in patients with multiple sclerosis.

Seven patients with multiple sclerosis who were receiving subcutaneous injections of recombinant interferon beta had pain followed by ulceration at the injection site. An eighth patient had a pustular flare of her usually mild psoriasis. No evidence of infection or contaminated medication was found.

Multiple sclerosis in the spinal cord: MR appearance and correlation with clinical parameters.

PURPOSE: To determine the characteristic magnetic resonance (MR) imaging features of multiple sclerosis (MS) that affect the spinal cord. MATERIALS AND METHODS: Sixty-eight patients underwent MR imaging of the cervical and/or thoracic spine. Plaques were analyzed for lesion length, cross-sectional area and location, signal intensity, and morphology. The clinical parameters of MS type, duration of disease, sex, and age were also correlated with these MR imaging findings. RESULTS: One hundred twenty-four demyelinating plaques were found in these 68 patients; 38 had more than one plaque. The majority of plaques were two body segments in length or less and peripherally located, and occupied less than 50% of the cross-sectional area of the cord. Plaques associated with cord atrophy were more likely to occur with the relapsing-progressive form of MS. Cord swelling was found only in the relapsing-remitting form of MS. CONCLUSION: Spinal cord MS plaques are characteristically peripherally located, are less than two vertebral segments in length, and occupy less than half the cross-sectional area of the cord.

Junctional region of the myelin basic protein-specific T cell receptor beta chain in mice.

Evidence for the existence of both conserved and diverse amino acid sequences in the junctional regions of the myelin basic protein (MBP)-specific T cell receptors (TCR) in mice is presented. The junctional region of the Nac1-11 MBP peptide-specific, H-2u-restricted TCR beta-chains is characterized by the utilization of similar amino acid sequences. In contrast, diverse junctional sequences within the TCR beta-chains of the p89-101 MBP peptide and H-2s-restricted T cell clones are reported. These findings demonstrate that a limited heterogeneity of the MBP-specific T cell clones does exist. However, it may not be universal even in inbred mouse strains.

Regulation of myelin basic protein gene transcription: identification of a distal cis-acting regulatory element.

The myelin basic protein (MBP) gene contains sequences located upstream of its transcription start site which play a key role in glial-specific transcription of the MBP promoter. Earlier analysis of the 320 bp upstream regulatory sequence of MBP has revealed multiple cis-acting regulatory motifs which differentially regulate transcription of a heterologous promoter fused to a reporter gene in glial and nonglial cells. In the present study, we have focused on a region designated MB3, which is located between -93 to -130 nucleotides with respect to the RNA start site, and contains a binding site for the NF1/CTF family of transcription activators. Results from DNase I footprint protection analysis of nuclear proteins prepared from mouse brain revealed a major region within the MB3 regulatory element that specifically interacts with the proteins derived from mouse brain at various stages of brain development. Using synthetic oligonucleotides spanning the protected region, we show that the double-stranded MB3 sequence interacts with nuclear proteins from mouse brain and forms specific major C1 and a minor C2 complex. Methylation interference experiments have allowed the identification of the G-residues within nucleotides -100 to -108, named MB3a, which are distinct from the NF1/CTF of MB3 that contact with nuclear proteins to form the major C1 complex. Results from band shift studies revealed assembly of the C1 complex upon incubation of MB3 DNA with the nuclear proteins from various cells of glial origin. Site-directed mutagenesis experiments revealed that the identified G-residues for DNA-protein interaction are important to confer transcriptional activity to this domain in transiently transfected glial cells.

Reflex sympathetic dystrophy revisited: MR imaging findings before and after infusion of contrast material.

PURPOSE: To determine the appearance of reflex sympathetic dystrophy (RSD) at magnetic resonance (MR) imaging. MATERIALS AND METHODS: Fifty-one patients with suspected RSD were prospectively evaluated at MR imaging with T1- and T2-weighted sequences and T1-weighted sequences with fat suppression before and after the intravenous administration of contrast material. RESULTS: RSD was confirmed in 45 patients. In 35 patients with stage 1 RSD, skin thickening (31 patients), tissue enhancement with contrast material (31 patients), and soft-tissue edema (six patients) were demonstrated. In five patients with stage 2 RSD, skin thickening (two patients), skin thinning (two patients), and infrequent contrast material enhancement (one patient) were demonstrated. There was no edema in this group of patients. In five patients with stage 3 RSD, inconsistent skin changes were also demonstrated; however, muscle atrophy (four patients) was demonstrated in this stage only. CONCLUSION: MR imaging was beneficial in the demonstration of soft-tissue abnormalities in patients with RSD. MR imaging may also help stage RSD, particularly stages 1 and 3.

Distributional patterns of multiple sclerosis brain lesions. Magnetic resonance imaging--clinical correlation.

Forty-one patients with multiple sclerosis were studied with magnetic resonance imaging (MRI) and a battery of neuropsychological tests to determine the pattern of lobar distribution of lesions in the relapsing-progressive and relapsing-remitting forms of the disease. Correlation of lesions with the patient's cognitive and emotional dysfunction was also obtained. The results of this study indicate that patients with relapsing-progressive multiple sclerosis are characterized by the large or coalescent lesions located predominantly in the periventricular area. These lesions were more frequently found in the parietooccipital areas and were accompanied by significant cognitive dysfunction and severe personality changes. The patients with relapsing-remitting disease, however, were characterized by punctiform or small discrete lesions predominantly located in the intermediate and subcortical areas. These lesions were more frequently seen in the parietooccipital regions and the patients had significantly less severe cognitive and emotional involvement than did the patients with relapsing-progressive disease. Recognition of these patterns was easily accomplished by MRI. In this investigation MRI studies were utilized as an index of the pathological changes occurring at one point in time in the evaluation of the disease. Only repeated MRI studies and continuous clinical observation can establish the final diagnosis.

Expression of the myelin basic protein gene in transgenic mice expressing human neurotropic virus, JCV, early protein.

Transgenic mice containing the early region of the JC virus encoding T-antigen developed neurological disease resulting from dysmyelination in the central nervous system. In this study, we investigate expression of the myelin basic protein (MBP) gene, a major constituent of the myelin sheath, at the RNA level by Northern blot and S1 nuclease assay and at the protein level by Western blot analysis using anti-MBP antibody in two distinct transgenic lines exhibiting different degrees of dysmyelination. Results from Western blot analysis of proteins from the brains of these mice revealed great reductions in MBP levels that parallel the severity of dysmyelination in the corresponding animals. Analysis of MBP RNA by Northern and quantitative S1 assays exhibited no alterations in the transcription initiation sites of the MBP gene in these animals; however, a significant decrease in the level of MBP mRNA was detected, suggesting that T-antigen may negatively influence transcription of the MBP gene. Results from Northern and Western blot analysis of proteolipid protein revealed low-level expression of this gene. Expression of JCV T-antigen is developmentally regulated in the transgenic mice; it appears at 8 days postnatal, peaks at 15 days, and substantially decreases in 18-day-old mice. The programmed expression of JCV T-antigen, which overlaps with MBP gene transcription at the early stage of myelination, suggests the involvement of a pathway which modulates stage-specific regulation of myelin genes and viral gene expression in transgenic mice during brain development.

Preferential but not exclusive T cell receptor V beta chain utilization of myelin basic protein and peptide-specific T cell clones in mice.

The repertoire of T cell receptor (TCR) V beta chain utilization was investigated in PL/J, CXJ-1, SJL/J and B10.S-->SJL/J chimeric mice in response to either myelin basic protein (MBP) or the strain-specific encephalitogenic peptide. Our analysis showed that there was an overlapping predominance in the TCR V beta gene utilization in the MBP-specific responses, which were independent of the major histocompatibility complex (MHC) class II haplotype present, and the immunodominant peptide region recognized in these different strains. In those mice having the TCR V beta b haplotype (PL/J, CXJ-1, and the B10.S-->SJL chimera) either the TCR V beta 4, 8, and 13 or the TCR V beta 4, 6, and 13 predominated. In contrast, in mice with TCR V beta a haplotype (SJL/J) V beta 4, 6, and 17a were found. However, the quantitative distribution of these preferentially utilized TCR V beta chains in each strain was defined by the MHC class II haplotype and the immunodominant peptide recognized. The expression of the V beta 8 gene product in the peripheral TCR repertoire did not always correlate with predominant V beta 8 utilization in the MBP-specific response.

Systemic recombinant human interferon-beta treatment of relapsing-remitting multiple sclerosis: pilot study analysis and six-year follow-up.

A pilot study was undertaken to test the safety and establish the side effect profile of recombinant human interferon-beta 1b (Betaseron, Berlex Laboratories, Richmond, CA), in patients with relapsing-remitting multiple sclerosis (RRMS). During the initial dose finding period (24 weeks), five groups of 6 patients each were treated by subcutaneous injection three times each week with either 0.8, 4, 8, or 16 million units (mU) of Betaseron or placebo (WHO Standard). Although some side effects were noted in all groups, a dose-related trend in reduction of exacerbation frequency and side-effect profile was noted. Patients given 16 mU had no exacerbations during the initial dosing period, but associated side effects led to dose reduction or dropout. An 8 mU dose was selected for further study after 24 weeks, and continuous dosing at 8 mU in 15 patients has now exceeded 6 years. Side effects abated over time. Neutralizing antibody developed in most patients, but titers were variable, fluctuated independently of clinical course, and tended to fall with prolonged treatment. A dose-dependent rise in neopterin levels was observed during the initial dosing period. This pilot study has demonstrated responsiveness to Betaseron, shown a stable safety profile over time, and established guidelines for a dosing regimen to evaluate and optimize further the efficacy of Betaseron in RRMS.

Interferon-gamma-inducible endothelial cell class II major histocompatibility complex expression correlates with strain- and site-specific susceptibility to experimental allergic encephalomyelitis.

The interaction between encephalitogenic lymphocytes and the cerebral microvascular lining is considered to be an important initial step in the recruitment of immune cells into the central nervous system (CNS) under pathological conditions such as multiple sclerosis (MS) and its investigative analog, experimental allergic encephalomyelitis (EAE). This study was conducted in order to examine whether differences in microvascular endothelial cell expression of several molecules involved in lymphovascular interactions correlate with the strain and organ-specific development of EAE. Cerebral and epididymal microvascular endothelial cells (EC) were isolated from SJL and B10.S mice, which, despite MHC-compatibility (H-2S), differ in their ability to develop EAE. The subcultured cells were then analyzed by flow cytometry for their ability to express class I MHC, class II MHC and ICAM-1 molecules in response to treatment with murine recombinant interferon-gamma (IFN-gamma). Over a range of doses and times, cerebral EC cultures derived from EAE-susceptible SJL mice expressed two-fold higher levels and higher cell surface densities of class II molecules than cerebral EC cultures derived from EAE-resistant B10.S mice, whereas class I and ICAM-1 molecules were comparably upregulated on both SJL and B10.S cerebral EC. In contrast, both SJL and B10.S epididymal EC cultures expressed lower but comparable levels of class II molecules in response to IFN-gamma. Class I and ICAM-1 molecules, however, were upregulated to at least the same degree as that observed on cerebral EC derived from both strains.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of staphylococcal enterotoxin B on T cell receptor V beta utilization and clinical manifestations of experimental allergic encephalomyelitis.

Staphylococcal enterotoxin B (SEB) is a superantigen (SA) that up-regulates and then subsequently down-regulates and deletes T cells expressing V beta 8 T cell receptor (TcR) chains (Marrack and Kappler, 1990; Johnson et al., 1991). We have investigated the effect of SEB on experimental allergic encephalomyelitis (EAE) in PL/J mice, where the predominant encephalitogenic T cells are V beta 8+ (Acha Orbea et al., 1988; Zamvil et al., 1988). SEB did not enhance induction of EAE when administered prior to or after immunization for EAE. PL/J mice pretreated with SEB developed anergy and deletion of V beta 8 bearing cells and concomitant reduction in the incidence of EAE. Following SEB pretreatment, a redistribution in the TcR utilization of MBP-specific lymphocytes occurred. As a result, there was a low frequency of V beta 8 and expansion of other, normally less frequent, myelin basic protein (MBP)-specific clones. These observations indicate that systemic exposure to superantigen can influence organ-specific autoimmune diseases. We observed V beta-specific elimination, rather than activation, of autoimmune clones, a finding of potential therapeutic value. Modification of the TcR repertoire by systemic exposure to this SA indicates plasticity of immune reactivity and demonstrates a mechanism by which an environmental exposure (SEB) can influence a genetically determined, T cell mediated autoimmune disease.

T cell receptor V beta gene utilization in myelin basic protein specific clones from CXJ1 recombinant inbred mice.

CXJ1 mice are a recombinant inbred strain generated from experimental allergic encephalomyelitis (EAE) resistant BALB/c and EAE susceptible SJL/J progenitors. CXJ1 derive their major histocompatibility complex (MHC) class II and TCR genes from the BALB/c progenitor. However, their susceptibility to EAE is similar to SJL/J. Utilizing myelin basic protein (MBP)-specific CD4+ hybridoma clones and a MBP-specific T cell line (TCL) from CXJ1, we found the predominant T cell receptor (TCR) V beta chain expression to be V beta 8 and V beta 13. Our data support the concept of preferential, but not exclusive, TCR V beta usage in the MBP-specific response which is independent of MHC class II haplotype or immunodominant peptide.