Electroacupuncture inhibits IL-17/IL-17R and post-receptor MAPK signaling pathways in a rat model of chronic obstructive pulmonary disease.

OBJECTIVE: Interleukin (IL)-17, as a T-helper 17 cell (Th17) cytokine, plays a key role in chronic obstructive pulmonary disease (COPD) pathophysiology including chronic inflammation and airway obstruction, which lead to decreased pulmonary function. The aim of this study was to investigate the effect of acupuncture on IL-17, its receptor (IL-17R) and the mitogen-activated protein kinase (MAPK) signaling pathway, in a rat model of COPD. METHODS: The COPD model was induced in Sprague Dawley rats by exposure to cigarette smoke for 12 weeks. The model rats were treated with electroacupuncture (EA) at BL13 and ST36. The lung function and histology of the rats were observed. IL-17, tumor necrosis factor (TNF)-α, and IL-10 were detected by enzyme-linked immunosorbent assay (ELISA) in bronchoalveolar lavage fluid (BALF) and in plasma. The leukocytes and macrophages in the BALF were counted. The expression levels of IL-17R were assayed in lung tissue by real-time polymerase chain reaction (PCR), western blotting, and immunohistochemistry. MAPK signaling pathway molecules including c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK)1/2 and p38, and their phosphorylated forms, were observed in the lung by western blotting. RESULTS: Compared with the control group rats, lung function decreased and there was a severe inflammatory infiltration of the pulmonary parenchyma in the COPD rats. EA effectively improved lung function and alleviated the inflammatory infiltration in the lungs of COPD rats. EA also reversed the elevated total leukocyte and macrophage counts, the high levels of IL-17 and TNF-α, and the low IL-10 content in COPD rats. Meanwhile, EA downregulated the increased mRNA and protein expression of IL-17R, and significantly inhibited the elevated levels of phosphorylated JNK, ERK1/2, and p38 in the lungs of COPD rats. CONCLUSION: Our results suggest that the protective effects of acupuncture therapy on the lungs of COPD rats are likely related to inhibition of IL-17/IL-17R and the post-receptor MAPK signaling pathways.

Rapid linkage of innate immunological signals to adaptive immunity by the brain-fat axis.

Innate immunological signals induced by pathogen- and/or damage-associated molecular patterns are essential for adaptive immune responses, but it is unclear if the brain has a role in this process. Here we found that while the abundance of tumor-necrosis factor (TNF) quickly increased in the brain of mice following bacterial infection, intra-brain delivery of TNF mimicked bacterial infection to rapidly increase the number of peripheral lymphocytes, especially in the spleen and fat. Studies of various mouse models revealed that hypothalamic responses to TNF were accountable for this increase in peripheral lymphocytes in response to bacterial infection. Finally, we found that hypothalamic induction of lipolysis mediated the brain's action in promoting this increase in the peripheral adaptive immune response. Thus, the brain-fat axis is important for rapid linkage of innate immunity to adaptive immunity.

Pre-treatment with silymarin reduces brain myeloperoxidase activity and inflammatory cytokines in 6-OHDA hemi-parkinsonian rats.

Most chronic neurodegenerative diseases such as Parkinson's disease (PD) are accompanied by neuroinflammation which is associated with glial cells activation and production of different inflammatory cytokines. In the present study we evaluated the anti-cataleptic effect of silymarin pre-treatment in 6-hydroxydopamine (6-OHDA)-lesioned rats, striatum myeloperoxidase (MPO) activity and cerebrospinal fluid (CSF) levels of inflammatory cytokines. Male Wistar rats were pre-treated with intraperitoneal (i.p.) injections of silymarin (100, 200 and 300mg/kg) for 5 consecutive days. Then, catalepsy was induced by unilateral infusion of 6-OHDA (8µg/2µl/rat) into the central region of the SNc. The anti-cataleptic effect of silymarin was assessed by the bar test 3-weeks after neurotoxin injection. Striatal myeloperoxidase activity and CSF levels of TNF-α and IL-6 were assessed at the end of behavioral experiments. Our data demonstrated that silymarin pre-treatment decreased catalepsy. The most anti-cataleptic effect was observed at the dose of 300mg/kg of silymarin (p<0.001). There was a significant (p<0.001) increase in MPO activity of 6-OHDA-lesioned rats whereas; in silymarin (in all 3 doses, i.p. for 5 days) pre-treated hemi-parkinsonian rats' MPO activity was decreased markedly (p<0.001). Furthermore the CSF levels of TNF-α and IL-6 were decreased (p<0.001) in silymarin (100, 200 and 300mg/kg) pre-treated rats up to the range of normal non-parkinsonian animals. From these results, it may be concluded that pre-treatment with silymarin attenuates 6-OHDA-induced catalepsy by decreasing striatal MPO activity and restores CSF concentration of inflammatory cytokines, TNF-α and IL-6 to the levels of normal non-parkinsonian rats.

Centrally administered lipopolysaccharide elicits sympathetic excitation via NAD(P)H oxidase-dependent mitogen-activated protein kinase signaling.

OBJECTIVE: The mechanisms by which inflammation activates sympathetic drive in heart failure and hypertension remain ill-defined. In this study, an intracerebroventricular injection of lipopolysaccharide (LPS) was used to induce the expression of cytokines and other inflammatory mediators in the brain, in the absence of other excitatory mediators, and the downstream signaling pathways leading to sympathetic activation were examined using intracerebroventricular injections of blocking or inhibiting agents. METHODS AND RESULTS: In anesthetized rats, intracerebroventricular injection of LPS (5 microg) increased (P < 0.05) renal sympathetic nerve activity, blood pressure and heart rate. LPS increased (P < 0.05) hypothalamic mRNA for NAD(P)H oxidase subunits p47 and gp91, NAD(P)H oxidase-dependent superoxide generation, hypothalamic mRNA for tumor necrosis factor-alpha, cyclooxygenase-2 and cerebrospinal fluid levels of tumor necrosis factor-alpha and prostaglandin E2. In the paraventricular nucleus of hypothalamus, dihydroethidium staining for superoxide expression and c-Fos activity (indicating neuronal excitation) increased. The superoxide scavenger tempol significantly (P < 0.05) diminished the expression of inflammatory mediators, as well as superoxide expression and neuronal excitation in paraventricular nucleus. SB203580 (p38 mitogen-activated protein kinase inhibitor) also reduced the expression of inflammatory mediators in hypothalamus and cerebrospinal fluid. Tempol, apocynin [NAD(P)H oxidase inhibitor], SB203580 and NS398 (cyclooxygenase-2 inhibitor) all reduced cerebrospinal fluid prostaglandin E2 and the sympathoexcitatory response to LPS. LPS also increased angiotensin II type 1 receptor mRNA, a response blocked by apocynin and tempol but not by SB203580. CONCLUSION: These findings suggest that central inflammation in pathophysiological conditions activates the sympathetic nervous system via NAD(P)H oxidase and p38 mitogen-activated protein kinase-dependent synthesis of prostaglandin E2.

Effects of MK-801 (dizocilpine) on brain cell membrane function and energy metabolism in experimental Escherichia coli meningitis in the newborn piglet.

We evaluated the efficacy of non-competitive N-methyl-D-aspartate receptor antagonist MK-801 (dizocilpine) as an adjuvant therapy in experimental neonal bacterial meningitis. Meningitis was induced by injecting 10(6) colony forming units of Escherichia coli into the cisterna magna. MK-801 3 mg/kg was given as a bolus intravenous injection, 30 min before the induction of meningitis. MK-801 did not down-modulate the inflammatory parameters, such as increased intracranial pressure, cerebrospinal fluid (CSF) leukocytosis, increased lactate and TNF-alpha levels in the CSF, and hypoglycorrhachia observed in the meningitis group. MK-801 did not significantly attenuate the elevated glutamate concentration in the CSF. However, MK-801 showed some neuroprotective effects as evidenced by significant attenuation of cerebral lipid peroxidation products (conjugated dienes) and increase of brain high-energy phosphate compounds (ATP and PCr). Improvement in cerebral cortical cell membrane Na+, K+ -ATPase activity did not reach a statistical significance. These results suggest that MK-801 was effective in ameliorating brain injury in neonatal bacterial meningitis, although it failed to attenuate the inflammatory responses.

Effects of MK-801 (dizocilpine) on Brain Cell Membrane Function and Energy Metabolism in Experimental Escherichia coli Meningitis in the Newborn Piglet

We evaluated the efficacy of non-competitive N-methyl-D-aspartate receptor antagonist MK-801 (dizocilpine) as an adjuvant therapy in experimental neonal bacterial meningitis. Meningitis was induced by injecting 10(6) colony forming units of Escherichia coli into the cisterna magna. MK-801 3 mg/kg was given as a bolus intravenous injection, 30 min before the induction of meningitis. MK-801 did not down-modulate the inflammatory parameters, such as increased intracranial pressure, cerebrospinal fluid (CSF) leukocytosis, increased lactate and TNF-alpha levels in the CSF, and hypoglycorrhachia observed in the meningitis group. MK-801 did not significantly attenuate the elevated glutamate concentration in the CSF. However, MK-801 showed some neuroprotective effects as evidenced by significant attenuation of cerebral lipid peroxidation products (conjugated dienes) and increase of brain high-energy phosphate compounds (ATP and PCr). Improvement in cerebral cortical cell membrane Na+, K+ -ATPase activity did not reach a statistical significance. These results suggest that MK-801 was effective in ameliorating brain injury in neonatal bacterial meningitis, although it failed to attenuate the inflammatory responses.

The expression of tumour necrosis factor in the hypothalamus after treatment with lipopolysaccharide.

To investigate the effects of tumour necrosis factor (TNF) in the hypothalamus, Wistar rats received an intravenous administration of lipopolysaccharide (LPS) at a dose of 3.0 mg/100 g. Concentrations of TNF-alpha in the cerebral liquor and blood sera rapidly increased at 30 minutes after administration of LPS, rose to the maximum level at 1 hour, and then gradually decreased. Using horse-radish peroxidase as a tracer, a transient increase in paracellular permeability throughout the tight junctions of the ependymal cell layer covering the third ventricle was observed by electron microscopy at 30 minutes and in that of the capillary endothelium at 1 hour after administration, respectively. Following LPS administration, TNF was preferentially localized by immunoelectron microscopy in the tight junctional area of the ependymal cell layer and the capillary. These data indicate that TNF, synthesized in the ependymal cell layer, induces a deterioration in the cerebrospinal fluid-brain barrier and subsequently in the blood-brain barrier. The present study suggests that oedematous changes in the hypothalamic areas determined by ultrastructural and magnetic resonance analyses were mainly due to TNF conveyed from the ependymal cell layer to the hypothalamus after administration of LPS.

Production of systemic and hypothalamic cytokines during the early phase of endotoxin fever.

Changes in concentrations of cytokines in plasma and in hypothalamic push-pull perfusates of guinea pigs were measured within the 1st hour after intramuscular injections of bacterial lipopolysaccharide (LPS; Escherichia coli, 20 micrograms/kg) or solvent (0.9% saline). In control animals injected with solvent, interleukin (IL)-1 and tumor necrosis factor alpha (TNF-alpha) were not detectable in plasma. Only IL-6 was present in picogram quantities. Within 45 min after injection of LPS, the concentrations of IL-1, TNF-alpha, and IL-6 increased in the plasma: by several orders of magnitude for TNF-alpha and about tenfold for IL-G. Picogram amounts of biologically active IL-1 were detected in plasma after injection of LPS. No steady state levels of systemic cytokines were reached during the experimental period. In hypothalamic perfusates of animals injected with the solvent, no IL-1 was detectable. TNF-alpha could be detected at higher concentrations than IL-6. IL-6 was detectable at tenfold lower concentrations than in the plasma. In animals injected with LPS, the hypothalamic concentration of IL-6 started to increase during the period 15-30 min and the concentrations of TNF-alpha during the period 30-45 min after LPS injection. The concentrations of IL-6 increased by 300-400% and did not exceed picogram values. No progressive increase of hypothalamic levels of these cytokines was observed during the time course of the experiment. The method used did not detect any changes in the amount of biologically active IL-1 in hypothalamic perfusates of LPS-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunological fluctuations during intrathecal immunotherapy in three patients affected by CNS tumours disseminating via CSF.

The immunological therapy of cancer has been proposed in a number of neoplasms (Borden, Sondel, 1989; Foon, 1989; Rosenberg, 1992) and has recently been adopted in the treatment of Central Nervous System (CNS) tumors in combination with conventional surgical and radiotherapeutical approach. In this context, loco-regional administration of immunomodulating agents (for instance in post-surgical cavity) allows to achieve much higher in situ concentrations than by systemic route. Since these treatments have potential adverse effects, careful assessment of clinical and immunological parameters in phase I trials is needed. CNS tumors disseminating via Cerebrospinal Fluid (CSF) pathways offer a stimulating opportunity for intrathecal immunotherapy. In this context, alpha-IFN and IL2 (alone or in combination with LAK cells) have been employed either loco-regionally or intrathecally (Merchant, Mc Vicar, Merchant & Young, 1992; Schiller, Hank, Storer, Borchert, Moore, Albertini, Bechhofer, Wesley, Brown, Bastin & Sondel, 1993). The rationale for the use of both these substances includes the known anti-tumor action of alpha-IFN (Mahaley, Urso, Whaley, Blue, Williams, Guaspari & Selker, 1985; Nagai, 1988) and the ability of r-IL2 to generate activated cells effective in lysing tumor cell targets (Hayes, Moore, Pierz, Chen, Da Rosso, Nirenberg & Allen, 1993). We treated 3 patients (2 affected by disseminating cerebellar medulloblastoma, 1 by disseminating thalamic glioblastoma) by intrathecal r-IL2 via recervoir. In the first 2 patients, this treatment was preceded by alpha-IFN (also intrathecally). Monitoring of immunological effects of the treatment schedule involved kinetics of CSF and serum TNF-alpha, IL2s and IL2R during the first day of r-IL2 treatment, as well as on day +2 and +4 of both r-IL2 cycles, and assessment of CSF cells, protein and CSF and PB NK cell activity and CD3-CD56+ cells during the course of all treatment cycles. We also assessed clinical and neuroradiological effects of immunotherapy.

Effects of antifungal therapy on inflammation, sterilization, and histology in experimental Candida albicans meningitis.

To assess the effects of antifungal therapy on the course of Candida albicans central nervous system infection and inflammation, we inoculated intracisternally 10(5) CFU of C. albicans into rabbits. Fluconazole (10 mg/kg of body weight) or amphotericin B (1 mg/kg) was infused intravenously daily for 14 days. Treatment was initiated 24 h or 5 days after infection. Cerebrospinal fluid (CSF) was repeatedly obtained to culture the organisms, assess the level of inflammation, and measure drug concentrations. Brain tissue was obtained at the end of therapy for culture, drug concentration determinations, and histopathology. The median number of days of treatment required to sterilize CSF cultures was 4 days for fluconazole therapy and 1 day for amphotericin B therapy (P = 0.037). There was a significant reduction in tumor necrosis factor alpha and leukocyte concentrations in the CSF of animals treated early versus those in untreated control animals (P < 0.05 and P < 0.001, respectively; analysis of variance). Compared with treated animals, a higher proportion of cultured CSF samples from untreated animals were positive for Candida (P < 0.001). A cultured brain sample from 1 of the 12 animals treated early with amphotericin B was positive for C. albicans (P < 0.01 versus controls); cultures of brain samples from 3 of 12 animals treated early with fluconazole were positive, whereas cultures of brain samples from 10 of 12 controls were positive (P < 0.05). The mean density of C. albicans was lower in the single culture-positive amphotericin B recipient (1 x 10(1) CFU/g of brain tissue) than in those treated with fluconazole (1 x 10(3) CFU/g) and in controls (8 x 10(4) CFU/g). In animals treated late, the density of C. albicans in the brain in relation to the number of days of therapy was significantly lower in amphotericin B recipients than in those treated with fluconazole (P < 0.01) and untreated controls (P < 0.01; analysis of covariance). By histopathology, a larger proportion of untreated animals compared with those treated early demonstrated features of severe infection such as perivasculitis, ventriculitis, and evidence of fungal organisms. Compared with amphotericin B-treated rabbits, those given fluconazole had a trend toward more severe pathologic lesions. Reduced susceptibility to both fluconazole and amphotericin B was observed in the C. albicans organisms isolated from the brain of one fluconazole-treated animal. These data suggest that amphotericin B is the preferred treatment for C. albicans infections of the central nervous system.

[In vitro and in vivo immunobiological responses of glioblastoma to human natural tumor necrosis factor-alpha].

Biological effects of human natural tumor necrosis factor-alpha (TNF) on glioblastoma cells in vitro and on glioma patients were investigated. TNF treatment on glioblastoma cells, even at a high dose (256 U/ml), exhibited no remarkable cytocidal activity in MTT assay, but at lower doses significantly inhibited colony forming and DNA synthesis. TNF at a low dose (10 U/ml) stimulated production of prostaglandin E2, Mn-superoxide dismutase, interleukin (IL)-6 and IL-8 by glioblastoma cells. These results indicated that the direct effect of TNF on human glioblastoma cells is rather antiproliferative than cytotoxic and is to modulate their metabolic pathways. In an early Phase I clinical trial, TNF was administered intracranially to six patients bearing glioblastoma. In this trial, the author studied in vivo immunological responses in the cerebrospinal fluid and regional fluid after the regional TNF injections. TNF in these body fluids were detected with a half life of several hours. There occurred a substantial number of leukocyte migration after the TNF administration. Neutrophils appeared first peaking at 8 to 12 hours, and then CD4+CD8-T cells and CD11b+CD13+CD14+ monocytes followed. IL-8 activity in the cerebrospinal fluid simultaneously corresponded to peak of the neutrophil migration. Increases in IL-6, IL-1 beta and prostaglandin E2 levels in the cerebrospinal fluid, regional fluid or both occurred peaking at 8 to 12 hours after TNA infection. Neither IL-2 nor interferons was detected. In conclusion, TNF may act as an antineoplastic agent by its direct cytostatic effects and indirectly through immune modulatory effects.