Leptomeningeal Cell Research Articles

B cell depletion with anti-CD20 promotes neuroprotection in a BAFF-dependent manner in mice and humans.

Anti-CD20 therapy to deplete B cells is highly efficacious in preventing new white matter lesions in patients with relapsing-remitting multiple sclerosis (RRMS), but its protective capacity against gray matter injury and axonal damage is unclear. In a passive experimental autoimmune encephalomyelitis (EAE) model whereby TH17 cells promote brain leptomeningeal immune cell aggregates, we found that anti-CD20 treatment effectively spared myelin content and prevented myeloid cell activation, oxidative damage, and mitochondrial stress in the subpial gray matter. Anti-CD20 treatment increased B cell survival factor (BAFF) in the serum, cerebrospinal fluid, and leptomeninges of mice with EAE. Although anti-CD20 prevented gray matter demyelination, axonal loss, and neuronal atrophy, co-treatment with anti-BAFF abrogated these benefits. Consistent with the murine studies, we observed that elevated BAFF concentrations after anti-CD20 treatment in patients with RRMS were associated with better clinical outcomes. Moreover, BAFF promoted survival of human neurons in vitro. Together, our data demonstrate that BAFF exerts beneficial functions in MS and EAE in the context of anti-CD20 treatment.

High Levels of Perivascular Inflammation and Active Demyelinating Lesions at Time of Death Associated with Rapidly Progressive Multiple Sclerosis Disease Course: A Retrospective Postmortem Cohort Study.

Analysis of postmortem multiple sclerosis (MS) tissues combined with in vivo disease milestones suggests that whereas perivascular white matter infiltrates are associated with demyelinating activity in the initial stages, leptomeningeal immune cell infiltration, enriched in B cells, and associated cortical lesions contribute to disease progression. We systematically examine the association of inflammatory features and white matter demyelination at postmortem with clinical milestones. In 269 MS brains, 20 sites were examined using immunohistochemistry for active lesions (ALs) and perivenular inflammation (PVI). In a subset of 22, a detailed count of CD20+ B cells and CD3+ T cells in PVIs was performed. ALs were detected in 22%, whereas high levels of PVI were detected in 52% of cases. ALs were present in 35% of cases with high levels of PVI. Shorter time from onset of progression to death was associated with increased prevalence and higher levels of PVI (both p < 0.0001). Shorter time from onset of progression to wheelchair use was associated with higher prevalence of ALs (odds ratio [OR] = 0.921, 95% confidence interval [CI] = 0.858-0.989, p = 0.0230) and higher level of PVI (OR = 0.932, 95% CI = 0.886-0.981, p = 0.0071). High levels of PVI were associated with meningeal inflammation and increased cortical demyelination and significantly higher levels of B lymphocytes within the PVI. ALs, a feature of early disease stage, persist up to death in a subgroup with high levels of PVI. These features link to a rapid progressive phase and higher levels of meningeal inflammation and B-cell infiltrates, supporting the hypothesis that chronic inflammation drives progression in MS. ANN NEUROL 2024;95:706-719.

Dissecting the human leptomeninges at single-cell resolution

Emerging evidence shows that the meninges conduct essential immune surveillance and immune defense at the brain border, and the dysfunction of meningeal immunity contributes to aging and neurodegeneration. However, no study exists on the molecular properties of cell types within human leptomeninges. Here, we provide single nuclei profiling of dissected postmortem leptomeninges from aged individuals. We detect diverse cell types, including unique meningeal endothelial, mural, and fibroblast subtypes. For immune cells, we show that most T cells express CD8 and bear characteristics of tissue-resident memory T cells. We also identify distinct subtypes of border-associated macrophages (BAMs) that display differential gene expressions from microglia and express risk genes for Alzheimer’s Disease (AD), as nominated by genome-wide association studies (GWAS). We discover cell-type-specific differentially expressed genes in individuals with Alzheimer’s dementia, particularly in fibroblasts and BAMs. Indeed, when cultured, leptomeningeal cells display the signature of ex vivo AD fibroblasts upon amyloid-β treatment. We further explore ligand-receptor interactions within the leptomeningeal niche and computationally infer intercellular communications in AD. Thus, our study establishes a molecular map of human leptomeningeal cell types, providing significant insight into the border immune and fibrotic responses in AD.

Cell‐type deconvolution analysis of RNAseq data reveals cell‐specific glycosylation changes in the brains of Alzheimer’s Disease patients

AbstractBackgroundIntriguing evidence that glycosylation is disrupted in Alzheimer’s Disease (AD) has emerged. However, our knowledge of cell‐specific alterations in the expression of genes involved in glycosylation in the brains of AD patients is limited.MethodIn this study, we conducted cell type deconvolution analysis of whole‐tissue RNAseq data to characterize cell type compositional differences and alterations in the expression of glycosylation related genes in the brains of AD patients and those with no cognitive impairment (NCI). Single‐cell human brain RNA‐seq data were obtained from the Allen Brain Map Atlas, consisting of 76,533 total nuclei derived from the cortical tissue of 2 humans. The identified cell types included GABAergic neurons, glutamatergic neurons, astrocytes, oligodendrocytes, oligodendrocyte precursor cells (OPC), microglia, endothelial cells, and vascular leptomeningeal cell (VLMC). The cell‐type deconvolution package CIBERSORT was applied in relative mode with S‐mode (single‐cell mode) batch correction to impute cell fractions and cell‐type‐specific gene expression profiles for NCI and AD samples in the ROSMAP dataset. The differences between cell type fractions of NCI and AD were analyzed by linear mixed regression model and t‐test.ResultIn the dorsolateral prefrontal cortex, neuronal cells represented the highest proportion of cells (∼30‐33%), followed by astrocytes (∼30%), and oligodendrocytes (∼20%), endothelial cells (∼7%), microglia (∼7%), VLMC (∼2‐3%), and OPC (&lt;1%). The overall cell type composition was not significantly different between NCI and AD patients, however, the proportion of microglia, VLMC, and OPC was significantly higher in AD patients (p&lt;0.05). The proportion of glutamatergic neuronal cells was significantly lower in female AD patients (p&lt;0.05) compared with NCI controls, but not in males after controlling for age. Microglia‐associated expression of the glycosyltransferases MGAT1, ST6GAL1, and ST6GALNAC3 was observed in AD patients but not in NCI controls. Glutamatergic neuronal cells and VLMC cells associated with the expression of GALNT10 was observed in AD patients but not in NCI controls. In contrast, GALNT11 expression associated with astrocytes was observed in NCI controls but not in AD patients.ConclusionOur findings indicate changes in cell‐type proportions and cell‐specific changes in glycosyltransferase gene expression in the brains of AD patients compared with NCI controls.

Characterization, isolation, and in vitro culture of leptomeningeal fibroblasts

Meninges, or the membranous coverings of the brain and spinal cord, play host to dozens of morbid pathologies. In this study we provide a method to isolate the leptomeningeal cell layer, identify leptomeninges in histologic slides, and maintain leptomeningeal fibroblasts in in vitro culture. Using an array of transcriptomic, histological, and cytometric analyses, we identified ICAM1 and SLC38A2 as two novel markers of leptomeningeal cells in vivo and in vitro. Our results confirm the fibroblastoid nature of leptomeningeal cells and their ability to form a sheet-like layer that covers the brain and spine parenchyma. These findings will enable researchers in central nervous system barriers to describe leptomeningeal cell functions in health and disease.

Leptomeninges-Derived Induced Pluripotent Stem Cells and Directly Converted Neurons From Autopsy Cases With Varying Neuropathologic Backgrounds.

Patient-specific stem cell technology from skin and other biopsy sources has transformed in vitro models of neurodegenerative disease, permitting interrogation of the effects of complex human genetics on neurotoxicity. However, the neuropathologic changes that underlie cognitive and behavioral phenotypes can only be determined at autopsy. To better correlate the biology of derived neurons with age-related and neurodegenerative changes, we generated leptomeningeal cell lines from well-characterized research subjects that have undergone comprehensive postmortem neuropathologic examinations. In a series of proof of principle experiments, we reprogrammed autopsy leptomeningeal cell lines to human-induced pluripotent stem cells (hiPSCs) and differentiated these into neurons. We show that leptomeningeal-derived hiPSC lines can be generated from fresh and frozen leptomeninges, are pluripotent, and retain the karyotype of the starting cell population. Additionally, neurons differentiated from these hiPSCs are functional and produce measurable Alzheimer disease-relevant analytes (Aβ and Tau). Finally, we used direct conversion protocols to transdifferentiate leptomeningeal cells to neurons. These resources allow the generation of in vitro models to test mechanistic hypotheses as well as diagnostic and therapeutic strategies in association with neuropathology, clinical and cognitive data, and biomarker studies, aiding in the study of late-onset Alzheimer disease and other age-related neurodegenerative diseases.

Abstract A08: IGF1R as a key target in high risk, metastatic medulloblastoma

Abstract The risk or presence of metastasis in childhood medulloblastoma represents a substantial cause of morbidity and mortality. Relatively little has been known about the molecular mechanisms of medulloblastoma dissemination throughout the cerebrospinal axis. To determine potential interventions that address both metastasis and tumor growth, we examined the expression of cytokines and growth factors in the CSF of metastatic medulloblastoma patients that were also present in the conditioned media of metastatic, MYC amplified medulloblastoma cell lines and/or cell culture models of the leptomeninges. The presence of IGF1, the IGF-sequestering protein Igfbp3, which can be cleaved by metal metalloproteases (MMP) and the serine protease tPA, and the presence of corresponding protease modulators TIMP1 and SerpinE1/PAI-1, led us to explore the bioactivity of IGF1 in medulloblastoma. IGF1 led not only to receptor phosphorylation but also increased migration and increased relative cell growth. A tumor cell viability screen of 60 clinically-related compounds prioritized IGF1R inhibitors. Our findings support a model whereby medulloblastoma tumor cells actively migrate, rather than arrive passively, to the leptomeningeal cell surface and provide a rationale for exploring IGF1R as a target for therapeutics in future clinical trials. Citation Format: Matthew N. Svalina, Ken Kikuchi, Jinu Abraham, Sangeet Lal, Monika A. Davare, Jennifer L. Peckham, Yoon-Jae Cho, Melanie Jackson, Daniel J. Guillaume, Nathan R. Selden, Darell D. Bigner, Kellie J. Nazemi, Sarah C. Green, Christopher L. Corless, Sakir Gultekin, Brian P. Rubin, Randall Woltjer, Charles Keller. IGF1R as a key target in high risk, metastatic medulloblastoma. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A08.

Bone morphogenetic protein-4 and 7 and receptors regulate vascular endothelial growth factor and receptors in human fetal leptomeninges

Bone morphogenetic proteins (BMP) 4 and 7 have important roles in neuronal differentiation and cortical development in the murine brain. However BMP4 and BMP7 expression and functions in the developing human brain are unknown. In this study, frozen tissue human fetal leptomeninges, formalin-fixed tissue and primary fetal leptomeningeal cell cultures were studied.By western blot, BMP4, BMP7 and BMPRIa were demonstrated in 15, 17 20, 23 week (wk) human leptomeninges. BMP receptor II was detected at 15 and 17wks. Immunohistochemically, BMP4 immunoreactivity was also found in 20 to 39wk human leptomeninges.BMP4 significantly reduced basal DNA synthesis at 22wks. BMP7 100 and 300ng/ml stimulated basal DNA synthesis in the 15, 17 and 22wk leptomeninges.BMP4 and BMP7 increased phosphorylation of SMAD-1, 5, 8 in most cells and had no effect on phosphorylation of p-38MAPK, or p44/42MAPK.BMP4 and BMP7 produced a decrease in VEGF RNA expression in 2 of 4 leptomeninges. BMP4 and BMP7 increased VEGFR1 RNA in 2 or 3 of 4 leptomeningeal cultures respectively. BMP4 produced a decrease in VEGFR2 RNA in 2 of 4 and BMP7 in 3 of 4 while BMP7 reduced VEGFR2 protein in the leptomeninges.The findings show, for the first time, BMP4, BMP7 and receptors are expressed and active in the human fetal leptomeninges. They suggest these BMPs influence vascular development in this tissue by regulating VEGF and its receptors.

Radiation effects on human leptomeningeal cell response to cerebrospinal fluid and PDGF-BB

Purpose: The pathogenesis of meningiomas remains largely unknown, but there is evidence that exposure radiation increases the risk of meningiomas. We therefore evaluated whether radiation stimulates human leptomeningeal cell proliferation and activates growth regulatory pathways.Materials and methods: Cultures from six human primary leptomeningeal cells were established and irradiated with 0.5, 1 or 2 Gy. Cells were subsequently maintained for approximately 3–6 months and then treated with the mitogen, platlet-derived growth factor-BB (PDGF-BB), or human cerebrospinal fluid (CSF) obtained from patients with no neurological disease. Cell proliferation was evaluated by CyQUANT, and changes in the regulatory activation levels of signal transducer and activator of transcription (STAT3), mitogen-activated protein kinase kinase 1/2 (MEK1/2), mitogen activated protein kinase (MAPK 44/42), Akt, and mammalian target of rapamycin (mTOR) were assessed by Western blot.Results: At both 3 and 6 months post-irradiation, CSF stimulated cell proliferation in all six leptomeningeal cultures. At 3 months, radiation was associated with an increase in STAT3, MEK 1/2, p44/42 MAPK and variable Akt activation, and at 6 months, STAT3, p44/42 MAPK, variable Akt and mTOR activation was observed in response to PDGF-BB and CSF.Conclusions: Irradiation of fetal leptomeningeal cells was associated with loss of heterozygosity of 1p36 and persistent sensitization leading to upregulation in growth pathways in response to PDGF and CSF. These findings suggest a mechanistic basis for radiation's role in meningioma initiation.

Occult leptomeningeal large cell medulloblastoma in an adult

Large cell medulloblastoma is an uncommon malignancy of childhood that often pursues an aggressive clinical course. We report the first case of this entity in an adult that proved to be an unsuspected primary leptomeningeal tumor. A 30-year-old man complained of worsening neck pain over the course of 3 months. Neck pain increased a few days prior to admission and a cervical spine CT revealed tonsillar herniation. Cervical spine MRI performed the day prior to admission confirmed the diagnosis of Chiari I malformation and C3-4 disk herniation without spinal cord compression. On the day of admission, the patient became unresponsive and resuscitative measures were unsuccessful. Postmortem examination of the brain was notable for necrotic cerebellar tonsils, but demonstrated no evidence of an intraparenchymal mass lesion. Microscopic examination of the cerebellum revealed discohesive neoplastic cells, which showed characteristic dot-like immunoreactivity for synaptophysin, diagnostic of large cell medulloblastoma within the subarachnoid space. Our experience with this unique case illustrates the challenges of diagnosing a primary leptomeningeal neoplasm. This case also underscores the importance of maintaining a high degree of suspicion for leptomeningeal neoplasms in patients who present with imaging studies suspicious for Chiari I malformation.

Hyperhomocysteinemia Increases β-Amyloid by Enhancing Expression of γ-Secretase and Phosphorylation of Amyloid Precursor Protein in Rat Brain

Hyperhomocysteinemia and beta-amyloid (Abeta) overproduction are critical etiological and pathological factors in Alzheimer disease, respectively; however, the intrinsic link between them is still missing. Here, we found that Abeta levels increased and amyloid precursor protein (APP) levels simultaneously decreased in hyperhomocysteinemic rats after a 2-week induction by vena caudalis injection of homocysteine. Concurrently, both the mRNA and protein levels of presenilin-1, a component of gamma-secretase, were elevated, whereas the expression levels of beta-secretase and presenilin-2 were not altered. We also observed that levels of phosphorylated APP at threonine-668, a crucial site facilitating the amyloidogenic cleavage of APP, increased in rats with hyperhomocysteinemia, although the phosphorylation per se did not increase the binding capacity of pT668-APP to the secretases. The enhanced phosphorylation of APP in these rats was not relevant to either c-Jun N-terminal kinase or cyclin-dependent kinase-5. A prominent spatial memory deficit was detected in rats with hyperhomocysteinemia. Simultaneous supplementation of folate and vitamin-B12 attenuated the hyperhomocysteinemia-induced abnormal processing of APP and improved memory. Our data revealed that hyperhomocysteinemia could increase Abeta production through the enhanced expression of gamma-secretase and APP phosphorylation, causing memory deficits that could be rescued by folate and vitamin-B12 treatment in these rats. It is suggested that hyperhomocysteinemia may serve as an upstream factor for increased Abeta production as seen in patients with Alzheimer disease.

Increased production of lipocalin-type prostaglandin D synthase in leptomeningeal cells through contact with astrocytes

Lipocalin-type prostaglandin D synthase (L-PGDS) is dominantly expressed in the leptomeninges surrounding the brain and secreted into the cerebrospinal fluid as beta-trace, a major cerebrospinal fluid protein. To examine the interaction between the leptomeninges and the brain parenchyma, we co-cultured rat leptomeningeal cells with cells dissociated from the neonatal rat cortex and found that the production of L-PGDS was remarkably increased after the co-cultivation. A similar increase in L-PGDS production was observed by the co-culturing of the leptomeningeal cells with cells dissociated from astrocyte-rich cultures or with 1321-N1 astrocytoma cells. When a crude membrane fraction prepared from 1321-N1 cells was added to leptomeningeal cell cultures, L-PGDS gene expression was slowly increased up to 48 h after the addition. These results indicate that leptomeningeal cells enhance their L-PGDS production by a slow activation of L-PGDS gene expression through their contact with astrocytes.

Constitutive integrin activation on tumor cells contributes to progression of leptomeningeal metastases1

Leptomeningeal metastases are a serious neurological complication in cancer patients and associated with a dismal prognosis. Tumor cells that enter the subarachnoid space adhere to the leptomeninges and form tumor deposits. It is largely unknown which adhesion molecules mediate tumor cell adhesion to leptomeninges. We studied the role of integrin expression and activation in the progression of leptomeningeal metastases. For this study, we used a mouse acute lymphocytic leukemic cell line that was grown in suspension (L1210-S cell line) to develop an adherent L1210 cell line (L1210-A) by selectively culturing the few adherent cells in the cell culture. beta1, beta2, and beta3 integrins were in a constitutively high active state on L1210-A cells and in a low, but inducible, active state on L1210-S cells. Expression levels of these integrins were comparable in the two cell lines. Static adhesion levels of L1210-A cells on a leptomeningeal cell layer were significantly higher than those of L1210-S cells. All mice that were injected intrathecally with L1210-A cells died rapidly of leptomeningeal leukemia. In contrast, 45% long-term survival was seen after intrathecal injection of mice with L1210-S cells. Our data indicate that constitutive integrin activation on leukemic cells promotes progression of leptomeningeal leukemia by increased tumor cell adhesion to the leptomeninges. We argue that an aberrantly regulated inside-out signaling pathway underlies constitutive integrin activation on the adherent leukemic cell population.

Effects of interleukin-1beta and prostaglandin E2 on prostaglandin D synthase production in cultivated rat leptomeningeal cells.

Although the interleukin (IL)-1 receptor is densely distributed in the leptomeninges constituting the blood/cerebrospinal fluid barrier, its physiologic significance has remained unclear. In the present study, we show that in cultured leptomeningeal cells, IL-1beta, tumor necrosis factors, or lipopolysaccharide causes a prominent increase in the synthesis and release of prostaglandin (PG) D synthase, which catalyzes the final step in the biosynthesis of PGD2. Although significant increases in the amount of PGD synthase were also observed with cells exposed to somatostatin, thrombin, or ciliary neurotrophic factor, these were much smaller than were those induced by the proinflammatory cytokines. Other agents tested including IGF-I had no effect upon the enzyme levels in the culture media. Furthermore, we found that the increased secretion of PGD synthase by IL-1beta was completely inhibited by 10(-7) M PGE2. The same dose of PGD2 or 15-deoxy-Delta(12-14)PGJ2 had no effect upon the IL-1beta action. In addition, PGE2 increased the level of fibronectin and eliminated the expression of zonula occludentes-1, a tight junction-associated protein from cultured cells, effects likely reflecting a loss of barrier integrity. These results demonstrate the importance of inflammatory stimuli as a physiologic regulator of the leptomeningeal cell function.

Neurofibromatosis 2.

Recent clinical and molecular research on neurofibromatosis 2 (NF2) is reviewed, and the implications for clinical practice and research are discussed. NF2 patients who are treated in specialty centers have a significantly lower risk of mortality than those who are treated in non-specialty centers. Vestibular schwannoma growth rates in NF2 are generally higher in younger people but are highly variable, even among multiple NF2 patients of similar ages in the same family. Radiation therapy is best reserved for NF2 patients who have particularly aggressive tumors, those who are poor surgical risks, those who refuse surgery, or those who are elderly. In-vivo studies have demonstrated that leptomeningeal cell activation of in mice results in leptomeningeal hyperplasia and meningioma formation. In-vitro studies have identified molecules that interact with the product (merlin or schwannomin), some of which (e.g., CD44 and paxillin) may play critical roles in merlin growth regulation. NF2 patients should be referred to specialty treatment centers for optimal care. Clinical management of multiple patients in NF2 families cannot be based on the expectation of similar vestibular schwannoma growth rates, even when other clinical aspects of disease severity are similar. The availability of accurate mouse models of human NF2-associated tumors and the identification of molecules involved in merlin growth regulation now provide an opportunity to design targeted treatments for schwannomas and meningiomas.

Vascular Cell Adhesion Molecule-1 Is a Key Adhesion Molecule in Melanoma Cell Adhesion to the Leptomeninges

Leptomeningeal metastases occur in up to 8% of patients with systemic malignancies and have a poor prognosis. A better understanding of the pathophysiologic processes underlying leptomeningeal metastases is needed for more effective treatment strategies. We hypothesized that tumor cells will have to adhere to the well-vascularized leptomeninges, because the cerebrospinal fluid lacks nutrients and growth factors for efficient tumor cell proliferation. Specific receptor-ligand interactions, which are unknown until now, will mediate this adhesion process. We determined the growth characteristics of B16F-10 melanoma cells in cerebrospinal fluid. The expression levels of specific adhesion molecules on both mouse leptomeningeal cells (MLMC) and murine B16F-10 melanoma cells were measured by immunofluorescence flow cytometry. We used mAbs to determine the function of these specific adhesion molecules on B16F-10 melanoma cell adhesion to a leptomeningeal cell layer under static and (cerebrospinal fluid-like) flow conditions. B16F-10 melanoma cells did not proliferate in cerebrospinal fluid because of a lack of nutrients and growth factors. MLMC expressed low levels of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), β1- and β3-integrin subunits, and CD44. VCAM-1 expression on MLMC was shown to be up-regulated by TNF-α. Blocking VCAM-1 on the MLMC with a mAb resulted in a 60% inhibition of melanoma cell adhesion to a leptomeningeal cell layer under flow but not under static conditions. No additive inhibitory effect on melanoma cell adhesion was found by concomitant blocking of the β1- and β3-integrin subunits and CD44 with mAbs. Our experiments indicate that cerebrospinal fluid does not support B16F-10 melanoma cell proliferation, suggesting the need for melanoma cell adhesion to the well-vascularized leptomeninges. VCAM-1, expressed on MLMC, is an important mediator of in vitro melanoma cell adhesion under (cerebrospinal fluid-like) flow conditions.

Transient appearance of amyloid precursor protein plaques in the brain of thymectomized rats after human leptomeningeal cell grafts

Cells cultured from Alzheimer disease leptomeninges or skin were grafted into the cortex of adult thymectomized rats. At 3 days post-implant, plaque-like aggregates were found in the cortex, corpus callosum, septum and caudate nucleus. These structures were immunopositive for human amyloid precursor protein (APP), human amyloid beta peptide (Aβ), cathepsin D, apolipoprotein E and ubiquitin. Aberrant tau + neurites, reactive astrocytes and microglia were associated with many aggregates. Although birefringent amyloid occupied the central area of most aggregates, these structures had disappeared by l month post-implant. Aβ and APP produced by grafted non-neural human cells can penetrate rat brain and form plaque-like structures, which can be effectively cleared by the rat.

Differential Expression of TGF-β, IL-2, and Other Cytokines in the CNS of Theiler's Murine Encephalomyelitis Virus-Infected Susceptible and Resistant Strains of Mice

Intracranial inoculation of susceptible SJL mice with Theiler's murine encephalomyelitis virus (TMEV) results in biphasic disease consisting of early acute disease, followed by late chronic demyelinating disease, associated with mononuclear infiltrates and demyelinating lesions. In contrast, resistant C57BL/6 (B6) mice develop only early acute disease. We employed cytokine-specific RT-PCR to determine the expression of cytokine transcripts in the CNS of TMEV-infected SJL and B6 mice. During early acute disease, we have found a strong proinflammatory (Th1) cytokine response in the CNS of both TMEV-infected SJL and B6 mice, demonstrated by the expression of transcripts for IFN-γ, IL-1, IL-6, IL-12p40, and TNF-α. At 8 days postinfection (p.i.), TGF-β1 and TNF-α transcripts were present at significantly higher levels (P < 0.01) in the CNS of SJL susceptible mice in comparison to those found in the CNS of B6 mice. Immunohistochemical staining revealed that TGF-β protein was expressed in leptomeningeal mononuclear inflammatory cell infiltrates in the brain of SJL mice but not in B6 mice, at 8 days p.i. TGF-β may be responsible for the failure of SJL mice to develop an effective anti-TMEV CTL response. During late chronic demyelinating disease, high levels of proinflammatory Th1 cytokines were found in the CNS of SJL mice, but not B6 mice. Significantly higher levels (P < 0.01) of anti-inflammatory cytokine transcripts (IL-4, IL-5, and IL-10 (Th2 cytokines) and TGF-β) were found in the spinal cord of TMEV-infected SJL mice with chronic demyelinating disease than in the spinal cord of B6 mice during the same time period (39 or 60 days p.i.). These anti-inflammatory cytokines may contribute to the downregulation of the proinflammatory response in SJL mice. High levels of IL-2 transcripts and protein appeared transiently in the spinal cord of TMEV-infected SJL mice before the onset of demyelinating disease and coincided with an influx of new T cells into the CNS and/or expansion of remaining T cells that have not been eliminated after viral clearance.

Central nervous system delivery of interleukin 4 by a nonreplicative herpes simplex type 1 viral vector ameliorates autoimmune demyelination.

Multiple sclerosis (MS) is a T cell-mediated organ-specific inflammatory disease leading to central nervous system (CNS) demyelination. On the basis of results obtained in experimental autoimmune encephalomyelitis (EAE) models, MS treatment by administration of antiinflammatory cytokines such as interleukin 4 (IL-4) is promising but is hampered by the limited access of the cytokines to the CNS and by the pleiotropic effects of systemically administered cytokines. We established a cytokine delivery system within the CNS using non-replicative herpes simplex type 1 (HSV-1) viral vectors engineered with cytokine genes. These vectors injected into the cisterna magna (i.c.) of mice diffuse in all ventricular and subarachnoid spaces and infect with high efficiency the ependymal and leptomeningeal cell layers surrounding these areas, without obvious toxic effects. Heterologous genes contained in the vectors are efficiently transcribed in infected ependymal cells, leading to the production of high amounts of the coded proteins. For example, 4.5 ng of interferon gamma (IFN-gamma) per milliliter is secreted into the cerebrospinal fluid (CSF) up to day 28 postinjection (p.i.) and reaches the CNS parenchyma in bioactive form, as demonstrated by upregulation of MHC class I expression on CNS-resident cells. We then exploited the therapeutic potential of the vectors in EAE mice. An HSV-1-derived vector containing the IL-4 gene was injected i.c. in Biozzi AB/H mice at the time of EAE induction. We found the following in treated mice: (1) delayed EAE onset, (2) a significant decrease in clinical score, (3) a significant decrease in perivascular inflammatory infiltrates and in the number of macrophages infiltrating the CNS parenchyma and the submeningeal spaces, and (4) a reduction in demyelinated areas and axonal loss. Peripheral T cells from IL-4-treated mice were not affected either in their antigen-specific proliferative response or in cytokine secretion pattern. Our results indicate that CNS cytokine delivery with HSV-1 vectors is feasible and might represent an approach for the treatment of demyelinating diseases. Advantages of this approach over systemic cytokine administration are the high cytokine level reached in the CNS, the absence of effects on the peripheral immune system, and the long-lasting cytokine production in the CNS after a single vector administration.

Central Nervous System Delivery of Interleukin 4 by a Nonreplicative Herpes Simplex Type 1 Viral Vector Ameliorates Autoimmune Demyelination

Multiple sclerosis (MS) is a T cell-mediated organ-specific inflammatory disease leading to central nervous system (CNS) demyelination. On the basis of results obtained in experimental autoimmune encephalomyelitis (EAE) models, MS treatment by administration of antiinflammatory cytokines such as interleukin 4 (IL-4) is promising but is hampered by the limited access of the cytokines to the CNS and by the pleiotropic effects of systemically administered cytokines. We established a cytokine delivery system within the CNS using nonreplicative herpes simplex type 1 (HSV-1) viral vectors engineered with cytokine genes. These vectors injected into the cisterna magna (i.c.) of mice diffuse in all ventricular and subarachnoid spaces and infect with high efficiency the ependymal and leptomeningeal cell layers surrounding these areas, without obvious toxic effects. Heterologous genes contained in the vectors are efficiently transcribed in infected ependymal cells, leading to the production of high amounts of the coded proteins. For example, 4.5 ng of interferon gamma (IFN-gamma) per milliliter is secreted into the cerebrospinal fluid (CSF) up to day 28 postinjection (p.i.) and reaches the CNS parenchyma in bioactive form, as demonstrated by upregulation of MHC class I expression on CNS-resident cells. We then exploited the therapeutic potential of the vectors in EAE mice. An HSV-1-derived vector containing the IL-4 gene was injected i.c. in Biozzi AB/H mice at the time of EAE induction. We found the following in treated mice: (1) delayed EAE onset, (2) a significant decrease in clinical score, (3) a significant decrease in perivascular inflammatory infiltrates and in the number of macrophages infiltrating the CNS parenchyma and the submeningeal spaces, and (4) a reduction in demyelinated areas and axonal loss. Peripheral T cells from IL-4-treated mice were not affected either in their antigen-specific proliferative response or in cytokine secretion pattern. Our results indicate that CNS cytokine delivery with HSV-1 vectors is feasible and might represent an approach for the treatment of demyelinating diseases. Advantages of this approach over systemic cytokine administration are the high cytokine level reached in the CNS, the absence of effects on the peripheral immune system, and the long-lasting cytokine production in the CNS after a single vector administration.