Influenza-associated Encephalopathy Research Articles

Diclofenac enhances proinflammatory cytokine-induced nitric oxide production through NF-κB signaling in cultured astrocytes

Recently, the number of reports of encephalitis/encephalopathy associated with influenza virus has increased. In addition, the use of a non-steroidal anti-inflammatory drug, diclofenac sodium (DCF), is associated with a significant increase in the mortality rate of influenza-associated encephalopathy. Activated astrocytes are a source of nitric oxide (NO), which is largely produced by inducible NO synthase (iNOS) in response to proinflammatory cytokines. Therefore, we investigated whether DCF enhances nitric oxide production in astrocytes stimulated with proinflammatory cytokines. We stimulated cultured rat astrocytes with three cytokines, interleukin-1β, tumor necrosis factor-α and interferon-γ, and then treated the astrocytes with DCF or acetaminophen (N-acetyl-p-aminophenol: APAP). iNOS and NO production in astrocyte cultures were induced by proinflammatory cytokines. The addition of DCF augmented NO production, but the addition of APAP did not. NF-κB inhibitors SN50 and MG132 inhibited iNOS gene expression in cytokine-stimulated astrocytes with or without DCF. Similarly, NF-κB p65 Stealth small interfering RNA suppressed iNOS gene expression in cytokine-stimulated astrocytes with or without DCF. LDH activity and DAPI staining showed that DCF induces cell damage in cytokine-stimulated astrocytes. An iNOS inhibitor, l-NMMA, inhibited the cytokine- and DCF-induced cell damage. In conclusion, this study demonstrates that iNOS and NO are induced in astrocyte cultures by proinflammatory cytokines. Addition of DCF further augments NO production. This effect is mediated via NF-κB signaling and leads to cell damage. The enhancement of DCF on NO production may explain the significant increase in the mortality rate of influenza-associated encephalopathy in patients treated with DCF.

Differences in clinical manifestations of influenza‐associated encephalopathy by age

Data from patients in Japan was analyzed to examine the age distribution and differences by age in the clinical manifestations of influenza-associated encephalopathy. Between 1998 and 2002, 472 cases of influenza-associated encephalopathy in patients aged 15 years or younger were reported to the Collaborative Study Group on Influenza-Associated Encephalopathy. These cases were divided into two groups by age: 0-5 and 6-15 years. The differences between the groups were estimated based on the data for those aged 0-5 years, and the odds ratios and 95% confidence intervals calculated. Distribution was inversely correlated with age, with a peak at 1-2 years old. In comparison with patients aged 0-5, those aged 6-15 years had a significantly greater incidence of type B infection, lower frequency of convulsions, higher frequency of loss of consciousness and altered consciousness as the initial neurological symptom, lower serum transaminase levels, lower frequency of low-density area for brain CT upon admission, and lower incidence of sequelae. Our analysis indicates that the clinical course, laboratory data, and brain imaging findings of influenza-associated encephalopathy exhibits patterns that vary with age.

Prognostic Factors in Influenza-associated Encephalopathy

Recently, reports of influenza-associated encephalopathy have increased worldwide. Given the high mortality and morbidity rates attributable to this severe neurologic complication of influenza, we conducted a nationwide study in Japan to identify the prognostic factors. We retrospectively evaluated 442 cases of influenza-associated encephalopathy that were reported to the Collaborative Study Group on Influenza-Associated Encephalopathy, which was organized by the Japanese Ministry of Health, Labor, and Welfare in collaboration with hospitals, clinics, and local pediatric practices in Japan between 1998 and 2002. The outcome for each patient was classified as either survival or death. Predictors of death were identified using logistic regression analysis. Four major prognostic factors for death were found to be significant by multivariate analysis (P < 0.05) in the 184 patients for whom we had complete data: elevation of aspartate aminotransferase, hyperglycemia, the presence of hematuria or proteinuria, and use of diclofenac sodium. We identified patients who had factors associated with a poor prognosis, and these findings might be clinically useful for the management of this illness.

Thermal instability of compound variants of carnitine palmitoyltransferase II and impaired mitochondrial fuel utilization in influenza-associated encephalopathy

Influenza-associated encephalopathy (IAE) is characterized by persistent high fever, febrile convulsions, severe brain edema, and high mortality in otherwise apparently healthy individuals. We have reported that a large proportion of patients suffering from disabling or fatal IAE, with transiently elevated serum acylcarnitine during high fever, exhibit a thermolabile phenotype of compound homo-/heterozygous variants of carnitine palmitoyltransferase II (CPT II, gene symbol CPT2). We characterized the enzymatic properties of five single and three compound CPT II variants in patients with IAE. The kinetic characteristics of WT and variant CPT IIs, expressed in COS-7 cells, indicated that the variants exert a dominant-negative effect on the homotetrameric protein of the enzyme. Among the variants, three compound variations found in patients with severe encephalopathy; [c.1055T>G (p.Phe352Cys); c.1102G>A (p.Val368Ile)], [c.1511C>T (p.Pro504Leu); c.1813G>C (p.Val605Leu)], and [c.1055T>G (p.Phe352Cys); c.1102G>A (p.Val368Ile); c.1813G>C (p.Val605Leu)], showed reduced activities, thermal instability, and short half-lives compared with the WT. Like other disease-causing mutant proteins, these variant proteins were poly-ubiquitinated and rapidly degraded by a lactacystin-sensitive proteasome pathway. COS-7 cells transfected with the compound variants had their fatty acid beta-oxidation decreased to 30-59% and intracellular ATP levels to 48-79%, and a marked reduction of mitochondrial membrane potential at 41 degrees C, compared with control cells transfected with WT at 37 degrees C. The unstable CPT II variants with decreased enzymatic activities may bring mitochondrial fuel utilization below the phenotypic threshold during high fever, and thus may play an important etiopathological role in the development of brain edema of IAE.

Urinary organic metabolite screening of children with influenza-associated encephalopathy for inborn errors of metabolism using GC/MS

Background: Influenza-associated encephalopathy (IAE) occurs in childhood often with a serious clinical course and fatal outcomes. We screened children with IAE using GC/MS to determine whether they have metabolic disorders such as organic acidemias. Methods: Urine samples from 70 Japanese children with IAE were analyzed between 2001 and 2005 using GC/MS with solvent extraction and direct drying methods. Results: Apparent metabolic disorders in 6 of 70 tested children included vitamin B12-responsive methylmalonic acidemia (MMA; n = 1), fructose-1,6-diphosphatase (FDPase) deficiency ( n = 1) and non-ketotic dicarboxylic aciduria ( n = 4) suggesting disorders of fatty acid oxidation. One child had an FDPase deficiency, for which glycerol infusion was contraindicated. Valproic acid metabolites were detected in 10 children and urinary glycerol excretion was increased in 22 of them after glycerol treatment. Discussion: Our results showed that inborn errors of metabolism (IEM), such as organic acidemias, are detectable among children with IAE. Patients with metabolic disorders such as an FDPase deficiency and MMA often have hypoglycemia, hyperammonemia and acute lactic acidemia. Importantly, a detection of FDPase deficiency in which glycerol infusion is contraindicated suggests careful selection of a treatment strategy for “acute encephalopathy”. We detected valproic acid and its metabolites at a rate of 14%, which was considerably higher than that in a control population. Thus, convulsive disorders might be a risk factor associated with IAE.

高齢者インフルエンザ脳症の１剖検例

Influenza-associated encephalopathy (IAE) usually occurs in children aged <5 years. Adult cases of IAE are very rare and, thus far, no definite adult autopsy case has been reported. Here, we present the first definite adult autopsy case of IAE. A 76-year-old man presented with sudden coma a day after the onset of fever caused by infection with influenza type A virus. Soon after admission, his condition was complicated by DIC, shock, and multiple organ failure, and he was diagnosed with IAE. Oseltamivir administration and steroid pulse therapy were performed but these proved to be ineffective. The patient died about 24 hours after the onset of encephalopathy. The autopsy revealed massive brain edema and diffuse increase of amoeboid glias without inflammatory cell infiltration. Influenza type A/Hong Kong virus (H3) was isolated from his lungs. Serum IL-6 level was extremely high (35,800 pg/ml; normal, 0.221-4.62 pg/ml). The clinical course, and the laboratory and pathological findings of this adult case resembled those of a typical childhood-onset IAE, suggesting the same pathogenesis. During the influenza season, IAE should be taken into account for differential diagnosis in adult patients with altered mental status and fever.

Reversible Splenial Lesion in Influenza Virus Encephalopathy

We describe a rare case of clinically mild, influenza-associated encephalopathy with a reversible splenial lesion. A 12-year-old Japanese girl presented with fever and headache, followed by muscle weakness and somnolence. Magnetic resonance imaging on day 4 of her illness showed a solitary lesion of the splenium of the corpus callosum that was most prominently visualized on diffusion-weighted images. The patient was diagnosed with influenza B-associated encephalopathy. Her neurologic signs had completely recovered by day 6, and the splenial abnormalities disappeared on day 11. A review of the literature identified four additional pediatric cases of this type of influenza-associated encephalopathy: three and one were caused by influenza A and B viruses, respectively. Common features include prompt and complete recovery from clinical and radiologic abnormalities, a relatively older age (> or = 5 years), and a higher incidence among the Japanese. To better understand the pathophysiology of this encephalopathy, we examined interleukin-6, tumor necrosis factor-alpha, and soluble tumor necrosis factor receptor 1 levels in serum and cerebrospinal fluid from this patient. The results did not reveal any elevations of these cytokines in the sera or cerebrospinal fluid, suggesting that this condition is not mediated by augmented cytokine responses.

Severe influenza resembling hemorrhagic shock and encephalopathy syndrome

Influenza-associated encephalopathy is a clinically diverse syndrome and severe cases are not well documented outside Japan. Clinical, pathological and molecular aspects are described of two fatal cases presenting during 2004 and 2005 winter seasons in The Netherlands. Results showed that severe influenza can resemble hemorrhagic shock and encephalopathy syndrome, and proper testing for influenza virus should be considered in similar cases. The failure to detect viral replication in non-pulmonary organs including the brain would support the pathogenesis of this syndrome is based on proinflammatory cytokine responses.

Acute encephalopathy associated with influenza and other viral infections

Acute encephalopathy is the most serious complication of pediatric viral infections, such as influenza and exanthem subitum. It occurs worldwide, but is most prevalent in East Asia, and every year several hundreds of Japanese children are affected by influenza-associated encephalopathy. Mortality has recently declined, but is still high. Many survivors are left with motor and intellectual disabilities, and some with epilepsy. This article reviews various syndromes of acute encephalopathy by classifying them into three major categories. The first group caused by metabolic derangement consists of various inherited metabolic disorders and the classical Reye syndrome. Salicylate is a risk factor of the latter condition. The second group, characterized by a systemic cytokine storm and vasogenic brain edema, includes Reye-like syndrome, hemorrhagic shock and encephalopathy syndrome, and acute necrotizing encephalopathy. Non-steroidal anti-inflammatory drugs, such as diclofenac sodium and mephenamic acid, may aggravate these syndromes. Severe cases are complicated by multiple organ failure and disseminated intravascular coagulation. Mortality is high, although methylprednisolone pulse therapy may be beneficial in some cases. The third group, characterized by localized edema of the cerebral cortex, has recently been termed acute encephalopathy with febrile convulsive status epilepticus, and includes hemiconvulsion-hemiplegia syndrome and acute infantile encephalopathy predominantly affecting the frontal lobes. Theophylline is a risk factor of these syndromes. The pathogenesis is yet to be clarified, but an increasing body of evidence points to excitotoxicity and delayed neuronal death.

重症インフルエンザ脳症発症５年後のＣＴ所見と臨床症状が乖離した１例

症例は発症時3歳の男児。インフルエンザ脳症に罹患し, バルビタール療法と低体温療法を併用することにより頭蓋内圧をコントロールできた。初診時のCTでは大脳両半球における低吸収, 脳腫脹, 皮髄境界の消失が顕著であった。第17病日には不明瞭であった脳槽の輪郭が描出され始め, やがて広範な低吸収域のなかに脳回とみられる相対的な高吸収域がわずかに認められるに至った。ただし, 両側大脳皮質の広範な低吸収の状態は変わらなかった。第39病日, 大脳皮質の相対的な高吸収域がよりコントラストを増し, その数も増加した。第52病日の転院までには四肢ともに運動が活発化するなど, 神経機能は徐々に回復した。その後, さらに認識能及び運動能は向上し, 5年後の現在, 介助なしで階段の昇降が可能となり公立小学校の養護学級に通学している。したがって画像においても相応な改善が期待された。ところが実際には5年後のCTでは全く逆の所見を呈している。転院前にはすでに脳回が明瞭化してきていたにもかかわらず, 発症5年後には再び不明瞭化へと逆行した。運動野を中心として脳回が残存している部分もみられるが, 両側中大脳動脈領域を主体に全体的に神経細胞は脱落しており, 脳萎縮は顕著になっていた。このような画像の変化と相反する脳機能の回復は, 頭部外傷のごく一部に見出されるdelayed neuronal lossと呼ばれる病態に類似している。以上の点から, 少なくともCT画像が大脳皮質における器質的障害の程度を直接反映するとは限らないため, 小児インフルエンザ脳症については, 急性期の集学的治療のみならず理学療法を含めた長期にわたる積極的な治療が重要となろう。

Impaired long-chain fatty acid metabolism in mitochondria causes brain vascular invasion by a non-neurotropic epidemic influenza A virus in the newborn/suckling period: implications for influenza-associated encephalopathy

The neuropathogenesis of influenza-associated encephalopathy in children and Reye's syndrome remains unclear. A surveillance effort conducted during 2000-2003 in South-West Japan reveals that almost all fatal and handicapped influenza-associated encephalopathy patients exhibit a disorder of mitochondrial beta-oxidation with elevated serum acylcarnitine ratios (C(16:0)+C(18:1))/C(2). Here we show invasion by a non-neurotropic epidemic influenza A H3N2 virus in cerebral capillaries with progressive brain edema after intranasal infection of mice having impaired mitochondrial beta-oxidation congenitally or posteriorly in the newborn/ suckling periods. Mice genetically lacking of carnitine transporter OCTN2, resulting in carnitine deficiency and impaired beta-oxidation, exhibited significant higher virus-genome numbers in the brain, accumulation of virus antigen exclusively in the cerebral capillaries and increased brain vascular permeability compared to in wild type mice. Mini-plasmin, which proteolytically potentiates influenza virus multiplication in vivo and destroys the blood-brain barrier, accumulated with virus antigen in the brain capillaries of OCTN2-deficient mice but only a little in wild-type mice. These results suggest that the impaired mitochondrial beta-oxidation changes the susceptibility to a non-neurotropic influenza A virus as to multiplication in the brain capillaries and to cause brain edema. These pathological findings in the brain of mice having impaired mitochondrial beta-oxidation after influenza virus infection may have implications for human influenza-associated encephalopathy.

14-3-3 Protein Detection in the Cerebrospinal Fluid of Patients With Influenza-Associated Encephalopathy

Influenza-associated encephalopathy is characterized by high fever, convulsions, and loss of consciousness associated with influenza infection in children, but its pathophysiology remains to be clarified. We examined 14-3-3 proteins, which are acidic brain proteins, in cerebrospinal fluid by immunoblotting in four patients with influenza-associated encephalopathy, four patients with influenza without encephalopathy, and four patients with another encephalopathy. Interestingly, we detected 14-3-3 proteins in all four patients with influenza-associated encephalopathy (100%) but not in any of the other patients. 14-3-3 isoforms, including beta, gamma, epsilon, xi, and theta, were found in the cerebrospinal fluid of the patients with influenza-associated encephalopathy, suggesting extensive damage to the brain. We conclude that 14-3-3 proteins in cerebrospinal fluid are highly detectable in influenza-associated encephalopathy and thus can be used as a rapid diagnostic marker.

A missense mutation of the Toll-like receptor 3 gene in a patient with influenza-associated encephalopathy

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns and mediate the activation of NF-κB and the production of proinflammatory cytokines, which is critical for the innate immune system. TLR3 recognizes both double-stranded RNA and the influenza A virus. Since influenza-associated encephalopathy is frequent in Japan and East Asia and its pathological mechanism remains unknown, we analyzed several genes including TLRs and the retinoic acid inducible gene I, which could be involved in the recognition of the RNA virus. In one of three patients with influenza-associated encephalopathy, we detected a novel missense mutation (F303S) in just the TLR3 gene. This was confirmed as a loss-of-function mutant in a dose-dependent manner by NF-κB and IFN-β reporter assays using wild-type and mutant TLR3-transfected HEK293 cells. Our results imply that a mutation of the TLR3 gene could be one of the factors responsible for influenza-associated encephalopathy.

Investigation of Equine Influenza Cases Exhibiting Neurological Disease: Coincidence or Association?

Equine influenza is usually a transient and self-limiting disease. However, during an outbreak of equine influenza in the UK in 2003 there were reports of unusually severe clinical signs among unvaccinated animals. Two influenza-infected horses developed neurological signs, and one was subjected to euthanasia. Post-mortem examination of the brain revealed viral-type non-suppurative encephalitis, and influenza virus antigen was demonstrated by immunolabelling of sections of nasal mucosa. A syndrome known as influenza-associated encephalopathy has been described in man. Although not proved, the data suggest that similar disease mechanisms may operate in horses, and that equine influenza virus infection can result in encephalitis in the natural host, perhaps due to an aberrant host immune response.

PRIMARY BIOMARKERS IN CEREBRAL SPINAL FLUID OBTAINED FROM PATIENTS WITH INFLUENZA-ASSOCIATED ENCEPHALOPATHY ANALYZED BY METABOLOMICS

In order to search for the specific biomarkers of patients with influenza-associated encephalopathy this article analyzed all metabolites in cerebrospinal fluid (CSF) by using metabolome analysis. In all metabolites, the peaks of two molecular weights, 246.0092 and 204.0611, were significantly higher than those in other diseases including influenza without convulsion (p < .05). The peak of a molecular weight 228.0247 in all of the patients except one was less than that in other patients. These results indicate that the new metabolites detected in CSF would be primary markers for the diagnosis of influenza-associated encephalopathy.

Influenza-Associated Encephalopathy with Onset of Prolonged Convulsion: a Case Report

Influenza-Associated Encephalopathy with Onset of Prolonged Convulsion: a Case Report

Identification of trypsin I as a candidate for influenza A virus and Sendai virus envelope glycoprotein processing protease in rat brain

Extracellular cleavage of virus envelope fusion glycoprotein hemagglutinin (HA0) by host trypsin-like proteases is a prerequisite for the infectivity and pathogenicity of human influenza A viruses and Sendai virus. The common epidemic influenza A viruses are pneumotropic, but occasionally cause encephalopathy or encephalitis, although the HA0 processing enzyme in the brain has not been identified. In searching for the brain processing proteases, we identified a processing enzyme in rat brain that was inducible by infection with these viruses. The purified enzyme exhibited an apparent molecular mass of approximately 22 kDa on SDS-PAGE and the N-terminal amino acid sequence was consistent with that of rat pancreatic trypsin I. Its substrate specificities and inhibition profiles were the same as those of pancreatic trypsin I. In situ hybridization and immunohistochemical studies on trypsin I distribution revealed heavy deposits in the brain capillaries, particularly in the allocortex, as well as in clustered neuronal cells of the hippocampus. The purified enzyme efficiently processed the HA0 of human influenza A virus and the fusion glycoprotein precursor of Sendai virus. Our results suggest that trypsin I in the brain potentiates virus multiplication in the pathogenesis and progression of influenza-associated encephalopathy or encephalitis.

Cytochrome c and Tumor Necrosis Factor-α Values in Serum and Cerebrospinal Fluid of Patients With Influenza-Associated Encephalopathy

Cytochrome c and tumor necrosis factor-alpha concentrations were measured in serum and cerebrospinal fluid samples from 10 patients with influenza-associated encephalopathy. In the acute exacerbation phase, serum tumor necrosis factor-alpha and cytochrome c values were high in patients with a poor prognosis. In the convalescent phase, cerebrospinal fluid cytochrome c values increased remarkably in patients with subsequent brain atrophy.

Apoptosis under hypercytokinemia is a possible pathogenesis in influenza‐associated encephalopathy

Influenza-associated encephalopathy is reported to be frequent in Japan and East Asia. No evaluating markers except interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha and no likely pathological mechanism for the disease have yet been elucidated. In this study, influenza-associated encephalopathy was defined by clinical symptoms, and the use of an anti-influenza antibody test and/or influenza antigen detection kits, as well as computed tomography and/or magnetic resonance imaging. The levels of proinflammatory cytokines, acute phase proteins, endothelial markers and cytochrome c were compared in sera from 11 patients with and 42 without encephalopathy. Cytochrome c concentration in sera from patients with encephalopathy was markedly increased compared with that from patients without encephalopathy and normal controls. Although levels of several other proinflammatory cytokines and acute phase proteins such as TNF-alpha and IL-8 were also elevated in patients with influenza virus infection, the difference between those with and without encephalopathy, though significant, was less dramatic. The mean serum concentration of cytochrome c in 11 patients with encephalopathy, consisting of four deceased, four with and three without residual central nervous system sequelae, was 26.7 +/- 19.5 ng/mL on admission. In contrast, cytochrome c levels in 42 patients without encephalopathy were 0.3 +/- 0.7 ng/mL. The present results indicate that cytochrome c is a useful marker to follow patients with influenza-associated encephalopathy and suggest that an apoptosis of cells in several organs including the cerebrum and liver under the influence of hypercytokinemia is a possible mechanism of the disease.

Thermolabile phenotype of carnitine palmitoyltransferase II variations as a predisposing factor for influenza-associated encephalopathy

To assess the etiology of influenza-associated encephalopathy (IAE), a surveillance effort was conducted during 2000–2003 in South-West Japan. All fatal and handicapped patients except one (4/34 patients) exhibited a disorder of mitochondrial β-oxidation evoked by the inactivated carnitine palmitoyltransferase II (CPT II) with transiently elevated serum acylcarnitine ratios (C 16:0 + C 18:1)/C 2 > 0.09 during high-grade fever. Analyses of genotypes and allele compositions of CPT II revealed a thermolabile phenotype of compound heterozygotes for [1055T > G/F352C] and [1102G > A/V368I], which shows a higher frequency in IAE patients than healthy volunteers ( P < 0.025). The thermolabile phenotype of CPT II variations may be a principal genetic background of IAE in Japanese.