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C F Brosnan

Publications and source records attributed to C F Brosnan.

At least 37 records · Page 2Linked to original sources
PubMed1999

Fetal human cortical neurons grown in culture: morphological differentiation, biochemical correlates and development of electrical activity.

Cultured fetal human cortical neurons derived from second trimester human fetal cortex were analyzed with regard to their morphological differentiation and expression of cell-specific markers. The culture method was adapted from standardized protocols originally developed for the isolation and culture of rodent oligodendrocytes and astrocytes. This technique takes advantage of the different adhesive properties and stratification of central nervous system cells in vitro. Under these culture conditions fetal human cortical neurons underwent morphological differentiation, expressed neuron-specific markers and voltage- and ligand-gated ion channels. Highly enriched cultures of microglia and astrocytes generated from the same starting material also expressed cell-type specific markers. These cultures serve as a valuable tool for the establishment of normative data and as experimental models for neurodevelopmental and neurodegenerative studies.

Astrocytes↗
PubMed1999

Differential chemokine induction by the mouse adenovirus type-1 in the central nervous system of susceptible and resistant strains of mice.

Mouse adenovirus-type 1 (MAV-1) has recently been shown to cause a fatal hemorrhagic encephalopathy in certain strains of mice whereas other strains are resistant. Morbidity is associated with a productive infection of cerebrovascular endothelial cells, resulting in necrosis of the vasculature, infarction, hemorrhage and death within 4 - 6 days. Previous studies were not able to define a role for the innate or acquired immune response. In the current study we have addressed the effect of MAV-1 on chemokine and chemokine receptor expression in the central nervous system (CNS) and spleen of susceptible (C57BL/6) and resistant (BALB/c) strains of mice. Intra-peritoneal infection with MAV-1 in C57BL/6 animals resulted in early and prominent induction of IP-10/crg-2 in the spleen and CNS. Increased expression of MCP-1, MIP-1alpha, MIP-1beta and RANTES was also noted in the CNS of MAV-1-infected C57BL/6 animals commencing around 72 h post-infection. In contrast, chemokine expression in BALB/c animals was more restricted with prominent upregulation only of MIP-2 in the CNS. In situ hybridization identified the vascular endothelium and CNS glia as the principal site of IP-10/crg-2 production in the C57BL/6 animals. The chemokine receptors CCR1-5 were upregulated in the CNS of both strains of mice. These data show that productive infection of the CNS with MAV-1 leads to the upregulation of a characteristic pattern of chemokines and their receptors, which may point to a role for these factors in disease pathogenesis.

Adenoviridae Infections↗
PubMed1998

IFNs are critical regulators of IL-1 receptor antagonist and IL-1 expression in human microglia.

Because IL-1 is implicated in the pathogenesis of multiple sclerosis, and IFNs are known to alter disease course, we sought to determine whether IFNs can regulate the expression of IL-1 and IL-1R antagonist (IL-1Ra) in primary cultures of human microglia and astrocytes. We found that IL-1 and IL-1Ra are products of microglia but not astrocytes, and IFN-beta and IFN-gamma differentially modulate LPS- and cytokine-induced IL-1 and IL-1Ra. IFN-beta induces IL-1Ra and augments LPS- and IL-4-induced IL-1Ra, but suppresses LPS- and IL-1-induced IL-1, shifting the balance toward the expression of the IL-1Ra. Like IFN-beta, IFN-gamma suppresses the expression of both LPS and IL-1-induced IL-1beta. However, IFN-gamma also suppresses the expression of IFN-beta- and IL-4-induced IL-1Ra so that IFN-gamma may enhance or suppress IL-1 activity depending on the other cytokines present. IL-4 has similar effects to IFN-beta; however, other anti-inflammatory cytokines, did not regulate IL-1 or IL-1Ra in human microglia. Our data demonstrate a novel suppressive effect of IFN-beta and IL-4 on IL-1 activity in human microglia, suggesting that IFN-beta, a therapeutic agent used for multiple sclerosis, could have wider applications in the treatment of other central nervous system disorders in which IL-1 activity has been implicated in the pathogenesis.

Astrocytes↗
PubMed1998

The effect of gammadelta T cell depletion on cytokine gene expression in experimental allergic encephalomyelitis.

In experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis, we showed previously that depletion of gammadelta T cells using the mAb GL3 immediately before disease onset, or during the chronic phase, significantly ameliorated clinical severity. We now report on the effect of gammadelta T cell depletion on expression of five cytokine genes, IL-1, IL-6, TNF, lymphotoxin, and IFN-gamma in spinal cords of mice during the pre-onset, onset, height, and recovery phases of EAE, and on expression of type II nitric oxide synthase. In control animals, the mRNAs for IL-1 and IL-6 rose dramatically at disease onset and peaked before disease height, whereas the mRNAs for TNF, lymphotoxin, and IFN-gamma rose more slowly and peaked with peak of disease. In GL3-treated animals, a dramatic reduction in all five cytokines was noted at disease onset, but only IFN-gamma remained significantly reduced at a time point equivalent to height of disease in control animals. ELISA data confirmed the reduced levels of IL-1 and IL-6 at disease onset in GL3-treated animals, and pathologic analysis demonstrated a marked reduction in meningeal infiltrates at the same time point. Studies of type II NOS also demonstrated a significant reduction in both mRNA and protein expression at the height of disease in GL3-treated animals. These results suggest that gammadelta T cells contribute to the pathogenesis of EAE by regulating the influx of inflammatory cells into the spinal cord and by augmenting the proinflammatory cytokine profile of the inflammatory infiltrates.

Animals↗
PubMed1998

Mouse adenovirus type-1 replication is restricted to vascular endothelium in the CNS of susceptible strains of mice.

Previous studies have shown that mouse adenovirus type-1 (MAV-1) caused a fatal hemorrhagic encephalitis in certain strains of mice. C57BI/6 mice exhibited 100% mortality when given as little 10(3) plaque-forming units (PFU) of MAV, in contrast to BALB/c mice which were resistant to as many as 10(6) PFU. Susceptible animals died with a flaccid paralysis on the 3rd or 4th day after inoculation. The brains and spinal cords of these animals displayed numerous petechial hemorrhages that were found in virtually all areas of the brain, but were more numerous in white matter. In this paper, immunohistochemistry and electron microscopy were used to identify the viral target of replication within the CNS of susceptible mice. These studies showed that the CNS vascular endothelial cell was the primary site of viral replication within the CNS of mice infected with MAV-1. Characterization of cytokine mRNA levels and disease course in immunodeficient mice revealed that the host immune response played little, if any, role in the pathogenesis of MAV-1 disease in susceptible mice and was not responsible for the resistance of BALB/c mice. These results support the conclusion that disease course and outcome in susceptible and resistant strains of mice were determined primarily by the ability of the virus to replicate within the CNS vascular endothelium.

Animals↗
PubMed1998

MCP-1, MCP-2 and MCP-3 expression in multiple sclerosis lesions: an immunohistochemical and in situ hybridization study.

Chemokines are low molecular weight chemotactic cytokines that have been shown to play a central role in the perivascular transmigration and accumulation of specific subsets of leukocytes at sites of tissue damage. Two major families have been defined depending on the positioning of four conserved cysteines. The CXC chemokines predominantly attract neutrophils, whereas the CC chemokines predominantly attract monocytes and other leukocyte cell types. Members of the monocyte chemotactic protein (MCP)-1 family form a major component of the CC family of chemokines and are considered the principal chemokines involved in the recruitment of monocytes/macrophages and activated lymphocytes. In this study we addressed the expression and distribution of MCP-1, -2 and -3 in multiple sclerosis (MS) lesions of differing ages and levels of inflammatory activity using immunohistochemistry and in situ hybridization. In acute and chronic-active MS lesions immunoreactivity for MCP-1, -2 and -3 was prominent throughout the lesion center with reactivity diminishing abruptly at the lesion edge. Hypertrophic astrocytes were strongly reactive and inflammatory cells showed variable reactivity. Outside of the lesion only hypertrophic astrocytes were reactive. The results obtained by in situ hybridization for MCP-1 were in agreement with those obtained by immunostaining. In more chronic lesions immunoreactivity for MCP-1, -2 and -3 was considerably diminished, and in chronic-silent lesions immunoreactivity was restricted to a few scattered reactive astrocytes. Normal control brains showed no immunoreactivity for MCP-1, -2 and -3. Although the cellular distribution of all three members of this family was similar, antibodies to MCP-3 gave prominent staining of the extracellular matrix that was not noted for MCP-1 and -2. These results support the conclusion that members of the MCP family of chemokines are involved in the development of MS lesions in the central nervous system.

Adult↗
PubMed1998

Prevention and treatment of experimental autoimmune encephalomyelitis by CNI-1493, a macrophage-deactivating agent.

Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), are characterized by episodic neurologic dysfunction, perivascular mononuclear cell inflammation occurring mainly in white matter, and demyelination. Strong circumstantial evidence supports the conclusion that macrophage activation and local production of proinflammatory cytokines are necessary for disease induction and lesion formation. We now report that CNI-1493, a small m.w. compound, which inhibits macrophage activation and subsequent proinflammatory cytokine production, suppresses EAE induced in the genetically susceptible SJL/J mouse. Treatment with 5 mg/kg/day completely suppressed mild disease (clinical index of 1.6 +/- 0.5 in the untreated group as compared with 0.0 +/- 0.0 for the treated group) and significantly reduced acute disease (clinical index of 4.3 +/- 0.7 in the untreated group as compared with 0.5 +/- 0.3 for the treated group). Suppression of clinical manifestations of the disease correlated with a significant decrease in histopathology and proinflammatory cytokine expression at the lesion site. Moreover, drug treatment during the chronic phase resulted in amelioration of clinical signs. The data presented here should prove useful in developing novel chemotherapeutic approaches for the treatment of MS.

Animals↗
PubMed1998

A critical role for IL-4 in regulating disease severity in experimental allergic encephalomyelitis as demonstrated in IL-4-deficient C57BL/6 mice and BALB/c mice.

Experimental allergic encephalomyelitis (EAE) is a T cell-mediated autoimmune disease of the central nervous system (CNS) that has served as the principal experimental model for multiple sclerosis (MS). Susceptibility to disease is thought to correlate with the ability to generate a Th1-type cytokine profile in myelin-responsive T cells, whereas T cells producing a Th2 cytokine pattern, in particular IL-4, are thought to be nonencephalitogenic and also to confer protection against a Th1-type response. However, recent studies using a variety of genetically engineered animals in which the genes for Th1-type cytokines and/or their receptors have been inactivated have called into question the Th1-Th2 paradigm in experimental allergic encephalomyelitis. In this report we have addressed the contribution of IL-4 to disease expression by studying two strains of mice, C57BL/6 and BALB/c, in which the gene for IL-4 has been inactivated. The IL-4-deficient C57BL/6 mice, and to a lesser extent the IL-4-deficient BALB/c mice, developed a more severe form of clinical disease, a more extensive pathologic involvement of the spinal cord, and an increased expression of proinflammatory cytokines in the CNS than their wild-type littermates. BALB/c and C57BL/6 mice showed a slightly different cytokine profile in the CNS. Both groups of animals recovered from the acute clinical episode in a time frame that was essentially identical to that found in the wild-type controls. We conclude that IL-4 plays an important role in modulating the severity of the encephalitogenic process, but does not by itself contribute to spontaneous remission from the disease.

Animals↗
PubMed1998

Cytokine induction of MIP-1 alpha and MIP-1 beta in human fetal microglia.

Leukocyte infiltration into the central nervous system (CNS) is a key event in the inflammatory processes of neuroimmunologic diseases. Microglia, resident macrophages of the CNS, may contribute to this process by elaborating chemoattractants that are capable of recruiting leukocytes across the blood-brain barrier. Such factors have been detected in the CNS of animal models of multiple sclerosis and in the brains of human and nonhuman primates with AIDS encephalitis. As the expression of these chemoattractants may play an important role in the initiation and progression of neuroimmunologic diseases, we analyzed expression of the chemokines MIP-1 alpha, MIP-1 beta, MCP-1, and RANTES in human fetal microglial cultures. Unstimulated microglia expressed minimal levels of MIP-1 alpha, MIP-1 beta, and MCP-1, while RANTES was undetectable. In response to LPS, TNF-alpha, or IL-1 beta, both MIP-1 alpha and MIP-1 beta were induced at the mRNA and protein levels in a dose- and time-dependent manner. IFN-gamma did not significantly induce chemokine expression. MCP-1 was detectable in LPS- and cytokine-treated microglia. TGF-beta, a cytokine with down-modulatory effects on other cell types, had little effect on chemokine expression in microglia when used concomitantly before or during treatment with LPS. These results illustrate the ability of certain inflammatory stimuli to induce expression of MIP-1 alpha, MIP-1 beta, and MCP-1 by human fetal microglia. The expression of these chemoattractants may function to recruit inflammatory cells into the CNS during the course of neuroimmunologic diseases and may modulate the ability of HIV to infect the CNS.

Brain↗
PubMed1998

Selective inhibition of human glial inducible nitric oxide synthase by interferon-beta: implications for multiple sclerosis.

Nitric oxide generated from the inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of multiple sclerosis. Because significant species- and cell-specific differences exist in the expression of iNOS, we used primary human glial cell cultures to screen for an inhibitor of iNOS expression. Remarkably, among numerous soluble factors tested, interferon-beta (IFN-beta) alone showed a selective and potent inhibition of interleukin-1beta/interferon-gamma (IL-1beta/IFN-gamma)-induced iNOS expression in astrocytes. Inhibition of iNOS may provide a mechanism by which IFN-beta can ameliorate inflammation and cytotoxicity in the central nervous system of patients with multiple sclerosis.

Astrocytes↗
PubMed1998

MR microscopy of transgenic mice that spontaneously acquire experimental allergic encephalomyelitis.

Pathology of fixed spinal cords from transgenic mice with a myelin basic protein (MBP) specific T cell receptor was investigated. These mice spontaneously acquire the demyelinating disease experimental allergic encephalomyelitis (EAE). Several complementary imaging modalities, all on the same tissues, were used to visualize lesions; these included high-field (11.7-T) microscopic diffusion tensor imaging (DTI), T2*-weighted imaging, and optical microscopy on histological sections. Lesions were predominantly in white matter around meninges and vasculature and appeared hyperintense in anatomical images. DTIs showed reduced diffusion anisotropy in the same hyperintense regions, consistent with inflammation and edema. Histology in the same tissues exhibited the characteristic pathology of EAE. Two techniques for visualizing the effective diffusion tensor fields are presented, which display direction, organization, and integrity of neuronal fibers. It is shown that DTI offers intriguing possibilities for visualizing axonal organization and lesions within white matter.

Algorithms↗
PubMed1998

Cytokine-induced inflammation in the central nervous system revisited.

Cytokines play an essential role as mediators of the immune response. They usually function as part of a network of interactive signals that either activate, enhance, or inhibit the ensuing reaction. An important contribution of this cytokine cascade is the induction of an inflammatory response that recruits and activates subsets of leukocytes that function as effector cells in the response to the sensitizing antigen. Proinflammatory cytokines activate endothelial cells (EC) to express adhesion molecules and induce the release of members of the chemokine family, thus focusing and directing the inflammatory response to sites of antigen recognition. However, the vasculature of the central nervous system (CNS) is highly specialized and restricts the access of components of the immune system to the CNS compartment. In this review, we address the question as to whether endothelial cells in the CNS respond differently to specific cytokines known to induce either a proinflammatory effect or a regulatory effect in systemic vascular beds.

Animals↗
PubMed1997

Phenotypic and cytokine analysis of human peripheral blood gamma delta T cells expressing NK cell receptors.

The presence of NK receptors (NKR) on populations of T cells has been proposed to play a regulatory role in T cell function, fine tuning the response to Ag, and influencing the nature of the immune response through rapid secretion of large amounts of cytokines. In this study, we assessed the nature and distribution of NKR on human peripheral blood gamma delta T cells and established clones to study cytokine release. In circulating gamma delta T cells, approximately 80% expressed CD94, approximately 25% expressed NKR-P1A, and approximately 20% expressed p58, values substantially higher than those found on alpha beta T cells from the same donors. When cloned for specific NKR expression, most cells in culture were NKR-P1A+ whereas p58 expression was variable, suggesting that the NKR-P1A phenotype can be acquired in culture whereas expression of p58 is more stable. Some clones were triple positive for CD94, NKR-P1A, and p58. V delta 2+ cells generally expressed a wider range of NKR than V delta 1+ cells. Following activation through CD3, all gamma delta T cell clones released large amounts of IFN-gamma, commencing as early as 4 h postactivation. Some clones also released TNF-alpha and IL-4, but no correlation with specific NKR expression was noted. Activation through NKR-P1A induced moderate levels of IFN-gamma without inducing IL-4. The results suggest that activation of most gamma delta T cells is regulated by signaling events occurring via both the TCR and the NKR. They further show that peripheral blood gamma delta T cells may function as a source of the proinflammatory cytokines IFN-gamma and TNF-alpha.

Cell Line↗
PubMed1997

Multiple sclerosis: altered expression of 70- and 27-kDa heat shock proteins in lesions and myelin.

Recent studies have implicated heat shock proteins (HSP) in the pathogenesis of the multiple sclerosis (MS) lesion. Expression of the 73 kDa constitutive HSP (HSC70), the 72 kDa stress-inducible HSP (HSP70), and the 27 kDa small HSP (HSP27) was analyzed in white matter and myelin from central nervous system (CNS) tissue of MS and normal subjects using a combination of immunocytochemistry and quantitative immunoblotting. Plaques of all types were sharply defined by reduced immunostaining for HSC70, and shown by immunoblotting to contain 30 to 50% less HSC70 than surrounding white matter or normal tissue. In contrast, HSP27 was markedly enhanced 2.5- to 4-fold in plaque regions, especially in fibrous astrocytes and in hyperplastic interfascicular oligodendrocytes at the lesion edge. HSP70 was less abundant than HSC70, and no significant differences in HSP70 levels were noted between MS and normal white matter. Myelin isolated from active plaques contained 3- to 4-fold more HSC70 than normal myelin. Pronounced expression of HSP70 and HSP27 was also found in MS myelin, although neither protein was detected in normal myelin. Thus, white matter undergoing immune-mediated destruction in MS was associated with altered distribution and expression of HSC70 and HSP27. These changes may initially serve to protect myelin from further destruction and facilitate repair; however, enhanced expression of HSC70, HSP70, and HSP27 in myelin may subsequently present as additional immune targets involved in the progression of disease.

Astrocytes↗
PubMed1996

Expression of type II nitric oxide synthase in primary human astrocytes and microglia: role of IL-1beta and IL-1 receptor antagonist.

In this work, we studied the expression of type II nitric oxide synthase (NOS) in primary cultures of human astrocytes and microglia. Cytokine-activated human fetal astrocytes expressed a 4.5-kb type II NOS mRNA that was first evident at 8 h, steadily increased through 48 h, and persisted through 72 h. The inducing signals for astrocyte NOS II mRNA expression were in the order IL-1beta + IFN-gamma > IL-1beta + TNF-alpha > IL-1beta. SDS-PAGE analysis of cytokine-stimulated astrocyte cultures revealed an approximately 130-kDa single NOS II band that was expressed strongly at 48 and 72 h (72 h > 48 h). Specific NOS II immunoreactivity was detected in cytokine-treated astrocytes, both in the cytosol and in a discrete paranuclear region, which corresponded to Golgi-like membranes on immunoelectron microscopy. In human microglia, cytokines and LPS failed to induce NOS II expression, while the same stimuli readily induced TNF-alpha expression. In cytokine-treated human astrocytes, neither NOS II mRNA/protein expression nor nitrite production was inhibited by TGF-beta, IL-4, or IL-10. In contrast, IL-1 receptor antagonist exerted near complete inhibition of NOS II mRNA and nitrite induction. Monocyte chemoattractant peptide-1 mRNA was induced in TGF-beta-treated astrocytes, demonstrating the presence of receptors for TGF-beta in astrocytes. These results confirm that in humans, cytokines stimulate astrocytes, but not microglia, to express NOS II belonging to the high output nitric oxide system similar to that found in rodent macrophages. They also show that the regulation of type II NOS expression in human glia differs significantly from that in rodent glia. A crucial role for the IL-1 pathway in the regulation of human astrocyte NOS II is shown, suggesting a potential role for IL-1 as a regulator of astrocyte activation in vivo.

Astrocytes↗
PubMed1996

A pathogenic role for gamma delta T cells in relapsing-remitting experimental allergic encephalomyelitis in the SJL mouse.

Previous studies have detected gamma delta T cells in multiple sclerosis and experimental allergic encephalomyelitis (EAE) lesions but their role remains obscure. In the present study, we assessed gamma delta T cell dynamics and distribution in spleen and central nervous system (CNS) from mice with relapsing-remitting EAE, and studied the effect of depleting these cells on clinical and pathologic expression of disease using the mAb GL3. By immunohistochemistry and FACS analysis, striking disease-related changes were observed in the gamma delta T cell population in the CNS. FACS analysis showed that while gamma delta T cells remained low in the spleen (approximately 2% total CD3+ T cells) at all stages, in the CNS they increased to approximately 12% at the height of the acute attack, fell to approximately 5% during the recovery phase, but rose again to approximately 12% during the chronic phase. In animals in which gamma delta T cells were depleted immediately before the onset of acute disease, or during the chronic stage, a striking and significant reduction in the severity of the clinical signs was observed that was associated with a decrease in the percentage of CD3+/gamma delta T cells in the CNS. In depleted animals a statistically significant reduction in inflammation and demyelination was noted during the acute stage, but only marginal effects on these disease parameters were found in the chronic phase. Taken together, the data support the conclusion that gamma delta T cells play an important role in the pathogenesis of EAE in mice during both acute and chronic/progressive phases of the disease process.

Acute Disease↗
PubMed1996

Localization of monocyte chemoattractant peptide-1 expression in the central nervous system in experimental autoimmune encephalomyelitis and trauma in the rat.

Monocyte chemoattractant protein-1 (MCP-1) is a member of the chemokine beta family of chemoattractants that has been shown to play a major role in the initiation of monocyte and T cell inflammation to sites of tissue injury. In this study, we have examined the distribution of MCP-1 expression in inflammation in the central nervous system (CNS) associated with the autoimmune disease experimental autoimmune encephalomyelitis (EAE) and compared the results with those detected in inflammation associated with trauma. In EAE, MCP-1 expression was detected at the onset of inflammation, prior to clinical expression of disease, in lymphocytes and endothelial cells in subarachnoid locations. Monocyte infiltration into these areas appeared 24 h later. After the onset of clinical signs, MCP-1 expression was widely distributed in the spinal cord with levels increasing and decreasing in association with disease activity. Lymphocytes, macrophages, astrocytes, and endothelial cells could be identified as sources of MCP-1 by immunoreactivity and in situ hybridization. A similar close correlation between macrophage infiltration and the levels of mRNA for MCP-1 was found in the CNS of rats subjected to trauma, and in these animals MCP-1 was detected by immunohistochemistry in macrophages and endothelial cells. The results support the conclusion that MCP-1 is an important mediator of inflammation in the CNS.

Animals↗