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encephalopathy by influenza virus
International Congress Series 1263 (2004) 468 – 471
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DNA microarray analysis of encephalitis/encephalopathy by influenza virus Tetsuya Toyoda a,b,*, Naoki Tsumura c, Toyojiro Matsuishi c, Muneaki Matsuo d, Sumio Miyazaki d, Hiroaki Saikusa e a Koyama Hospital, Fuji, Shizuoka 417-0801, Japan Aichi Medical University, Nagakute, Aichi 480-1195, Japan c Department of Pediatrics and Child Health, Kurume University School of Medicine, Kurume, Fukuoka 830-0011, Japan d Department of Pediatrics, Saga Medical School, Saga 849-8501, Japan e Saikusa Pediatric Clinic, Sasebo, Nagasaki 859-3214, Japan b
Abstract. Gene expression patterns between encephalitis/encephalopathy patients and respiratory infection patients by the influenza viral infection were compared by DNA microarray analysis. Expression of six genes (interferon (a, h, N) receptor, ephrin-B2, RNA binding motif protein 5 (RBM5), transcription factor EC (TFEC), Calsenilin/DREAM/KChIP3, and MEGF6 protein) was elevated and that of three genes (non-metastatic protein 23 (Nm23/Awd), neuropeptide Y/peptide YY receptor, and hippocalcin-like protein 4 (HPCAL4)) was decreased in encephalitis/ encephalopathy patients. D 2003 Elsevier B.V. All rights reserved. Keywords: Influenza virus; Encephalitis/encephalopathy; DNA microarray
1. Introduction The influenza virus sometimes causes encephalitis/encephalopathy in young children during epidemics, although virus isolation from the brain or cerebrospinal fluid (CSF) or detection of the viral antigens in the central nervous system (CNS) is infrequent [1]. The influenza virus generally causes only respiratory tract infections and viremia has been considered unusual in humans. However, some CNS diseases, including Reye’s syndrome, encephalitis/encephalopathy, myelitis, and acute necrotizing encephalitis, have been reported to be associated with the influenza viral infection [2]. The immunization policy of Japan changed in 1994, resulting in a dramatic decrease in influenza vaccine distribution [3]. Since then, sporadic outbreaks of influenza virus-related encephalitis/encephalopathy have been reported every winter. During 1997– 1999, when an epidemic of influenza A (H3N2) virus occurred, many cases of influenza-associated * Corresponding author. 2015-8 Ooka, Numazu, Shizuoka 410-0022, Japan. Tel./fax: +81-55-924-6950. E-mail address: [email protected] (T. Toyoda). 0531-5131/ D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.02.128
T. Toyoda et al. / International Congress Series 1263 (2004) 468–471
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encephalitis/encephalopathy were reported in Japan [1]. However, no factors responsible for the disease have been identified yet, and influenza virus types responsible for the encephalitis/encephalopathy remain unclear. We speculated that host factor(s) might be important for this disease because only a few patients suffered from encephalitis/ encephalopathy among the many people infected with the same influenza viruses. DNA microarray, in which mRNA transcription patterns can be determined for many thousands of genes simultaneously, is a new method for evaluating virus – host interactions. In the winter season of 2000 –2001, we analyzed the gene expression patterns by DNA microarray in two cases of influenza-associated encephalitis/encephalopathy and compared them with three cases of respiratory infection. We did not find specifically expressing genes up- or downregulated among the influenza respiratory infection cases. We found six elevating and three decreasing genes in two influenza encephalitis/ encephalopathy cases. 2. Materials and methods 2.1. Cases Three cases of influenza respiratory infections were chosen: case 1, 27-year-old male, influenza A (H1N1); case 2, 12-year-old male, influenza B; case 3, 1-year and 8-monthold male, influenza A (H3N2). Two cases of influenza encephalitis/encephalopathy were chosen: case 4, 2-year-old female, influenza A (H3N2); case 5, 2-month-old male, influenza A (H1N1). A new type of carbohydrate-deficient glycoprotein (CDG) syndrome type I with no neurologic symptoms nor mental retardation, characterized by a CDG syndrome type I transferring pattern was also diagnosed later in case 5 [4]. Influenza virus infection was determined by rapid diagnosis kit. The diagnosis of influenza encephalitis/ encephalopathy was made on the basis of all clinical signs. The first symptoms of both patients were convulsion and loss of consciousness. The brain CT scans and MRIs of these patients later indicated brain atrophy. Blood samples were taken on the second and the first day of disease onset in cases 4 and 5, respectively. Types of influenza viruses were determined by virus isolation and serum haemagglutinin inhibition test. These patients had not received immunization against influenza viruses. 2.2. DNA microarray analysis Blood samples of the patients with respiratory infection were taken on the first day of visit or disease onset and 1 month after they were healthy. For the encephalitis/ encephalopathy cases, blood samples were also taken approximately 2 months later when they had recovered from life-threatening illness and were in rehabilitation. Peripheral blood mononuclear cells (PBMC) were isolated in buffer EL (Qiagen, CA) according to the manufacturer’s protocol and immediately frozen in dry ice and stored at 80 jC until used. PBMC were processed for DNA microarray analysis using Atlas Glass Array Human 3.8I + 3.8II (7532 genes) at the Clontech Bioscience Laboratory (Clontech, Tokyo). Before DNA microarray analysis, the extracted RNA of PBMC was amplified by T7 RNA polymerase [5]. One sample of respiratory infection (case 1) was analyzed by UniGEMV Ver2.0 (9296 genes, Kurabo, Osaka).
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T. Toyoda et al. / International Congress Series 1263 (2004) 468–471
3. Results and discussion To identify gene(s) which showed unique expression pattern in patients with encephalitis/encephalopathy due to influenza virus infection, we performed a DNA microarray analysis using total RNA isolated from PBMC. No overlapping up- or downregulated genes were found among the influenza respiratory infection cases by DNA microarray analysis (cases 1– 3). Expression of six genes (interferon (a, h, N) receptor [6], ephrin-B2 [7], RNA binding motif protein 5 (RBM5) [8], transcription factor EC (TFEC) [9], Calsenilin/DREAM/KChIP3 [10], and MEGF6 protein [11]) was elevated more than threefold in both of the influenza encephalitis/encephalopathy patients (cases 4 and 5). No overlapping genes were found to be upregulated in one encephalitis/encephalopathy patient, but downregulated in the other. Expression of three genes (non-metastatic protein 23 (Nm23/Awd) [12], neuropeptide Y/peptide YY receptor [13], and hippocalcin-like protein 4 (HPCAL4) [14]) was decreased to less than one-third in these patients. Few overlapping genes were found between encephalitis/encephalopathy and respiratory infection patients. Expression of the interferon receptor (a, h, N) was elevated in cases 1, 4 and 5, and ribosomal protein L37a [15] was elevated in cases 3 and 4. The ras homolog gene family member H (ARHH) [16] and collagen type XVIIa1 [17] were elevated in cases 3 and 5. In the same cases, the myeloid cell nuclear differentiation antigen was decreased. The activation of macrophages by hypercytokinemia and resulting brain edema was proposed for the pathogenesis of influenza encephalitis/encephalopathy [1,18]. Elevation of interferon receptors (a, h, N) enhances the effect of hypercytokinemia. Ephrin-B2 may affect the endothelial cells of arterioles of the brain [7], which may result in their damage and brain edema. A decrease of neuropeptide Y/peptide YY receptor and HPCAL4 may affect brain damage through vasodilatation or calcium signaling in the brain [13]. Elevation of ephrin-B2, Calsenilin/DREAM/KChIP3, and decrease of Nm23/Awd may relate to apoptosis of endothelial cells of the brain vessel and neurons [7,10,19]. The response of these genes was analyzed in the PBMC, not in the brain. Although it is not clear that the response of these genes was also found in the brain, they may represent the response of the patients’ bodies. The gene(s) responsible for the encephalitis/encephalopathy associated with the influenza virus infection will be identified in these genes. Acknowledgements The protocol of this study was approved by the committee on medical ethics of Kurume University. The informed consent was obtained from the parents of the patients. This work was supported by Grants-in-Aid from the Ministry of Labor, Health and Welfare of Japan. References [1] T. Morishima, et al., Encephalitis and encephalopathy associated with an influenza epidemic in Japan, Clin. Infect. Dis. 35 (2002) 512 – 517. [2] F.A. Horner, Neurologic disorders after Asian influenza, N. Engl. J. Med. 258 (1958) 983 – 985. [3] Y. Hirota, D.S. Fedson, M. Kaji, Japan lagging in influenza jabs, Nature 380 (1996) 18. [4] J. Leonard, S. Gru¨newald, P. Clayton, Diversity of congenital disorders of glycosylation, Lancet 357 (2001) 1382 – 1383.
T. Toyoda et al. / International Congress Series 1263 (2004) 468–471
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[5] C. Pabon, et al., Optimized T7 amplification system for microarray analysis, BioTechniques 4 (2001) 874 – 879. [6] D. Novick, B. Cohen, M. Rubinstein, The human interferon alpha/beta receptor: characterization and molecular cloning, Cell 77 (1994) 391 – 400. [7] M.A. Takasu, et al., Modulation of MNDA receptor-dependent calcium influx and gene expression through EphB receptors, Science 295 (2002) 491 – 495. [8] L.C. Sutherland, et al., LUCA-15-encoded sequence variants regulate CD95-mediated apoptosis, Oncogene 19 (2000) 3774 – 3781. [9] M.C. Chung, H.K. Kim, S. Kawamoto, TFEC can function as a transcriptional activator of the nonmuscle myosin II heavy chain-A gene in transfected cells, Biochemistry 40 (2001) 8887 – 8897. [10] M.A. Leissring, et al., Calsenilin reversed presenilin-mediated enhancement of calcium signaling, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 8590 – 8593. [11] M. Nakayama, et al., Identification of high-molecular-weight proteins with multiple EGF-like motifs by motif-trap screening, Genomics 51 (1998) 27 – 34. [12] A.M. Rosengard, et al., Reduced Nm23/Awd protein in tumor metastasis and aberrant Drosophila development, Nature 342 (1989) 177 – 180. [13] C. Gerald, et al., Expression cloning and pharmacological characterization of a human hippocampal neuropeptide Y/peptide YY Y2 receptor subtype, J. Biol. Chem. 270 (1995) 26758 – 26761. [14] M. Kobayashi, et al., Isolation of two human cDNAs, HLP3 and HLP4, homologous to the neuron-specific calcium-binding protein genes, DNA Seq. 9 (1998) 171 – 176. [15] D.P. Saha, et al., Human ribosomal protein L37a: cloning of the cDNA and analysis of differential gene expression in tissues and cell lines, Gene 132 (1993) 285 – 289. [16] E. Dallery, et al., TTF, a gene encoding a novel small G protein, fuses to the lymphoma-associated LAZ3 gene by t(3;4) chromosomal translocation, Oncogene 10 (1995) 2171 – 2178. [17] C.W. Franzke, et al., Transmembrane collagen XVII, and epithelial adhesion protein, is shed from the cell surface by ADAMs, EMBO J. 21 (2002) 5026 – 5035. [18] T. Ichiyama, et al., Cerebrospinal fluid and serum levels of cytokines and soluble tumor necrosis factor receptor in influenza virus-associated encephalopathy, Scand. J. Infect. Dis. 35 (2003) 59 – 61. [19] J.D. Baxbaum, et al., Calsenillin: a calcium-binding protein that interacts with the presenilins and regulates the level of a presenilin fragment, Nat. Med. 4 (1998) 1177 – 1181.
www.ics-elsevier.com
DNA microarray analysis of encephalitis/encephalopathy by influenza virus Tetsuya Toyoda a,b,*, Naoki Tsumura c, Toyojiro Matsuishi c, Muneaki Matsuo d, Sumio Miyazaki d, Hiroaki Saikusa e a Koyama Hospital, Fuji, Shizuoka 417-0801, Japan Aichi Medical University, Nagakute, Aichi 480-1195, Japan c Department of Pediatrics and Child Health, Kurume University School of Medicine, Kurume, Fukuoka 830-0011, Japan d Department of Pediatrics, Saga Medical School, Saga 849-8501, Japan e Saikusa Pediatric Clinic, Sasebo, Nagasaki 859-3214, Japan b
Abstract. Gene expression patterns between encephalitis/encephalopathy patients and respiratory infection patients by the influenza viral infection were compared by DNA microarray analysis. Expression of six genes (interferon (a, h, N) receptor, ephrin-B2, RNA binding motif protein 5 (RBM5), transcription factor EC (TFEC), Calsenilin/DREAM/KChIP3, and MEGF6 protein) was elevated and that of three genes (non-metastatic protein 23 (Nm23/Awd), neuropeptide Y/peptide YY receptor, and hippocalcin-like protein 4 (HPCAL4)) was decreased in encephalitis/ encephalopathy patients. D 2003 Elsevier B.V. All rights reserved. Keywords: Influenza virus; Encephalitis/encephalopathy; DNA microarray
1. Introduction The influenza virus sometimes causes encephalitis/encephalopathy in young children during epidemics, although virus isolation from the brain or cerebrospinal fluid (CSF) or detection of the viral antigens in the central nervous system (CNS) is infrequent [1]. The influenza virus generally causes only respiratory tract infections and viremia has been considered unusual in humans. However, some CNS diseases, including Reye’s syndrome, encephalitis/encephalopathy, myelitis, and acute necrotizing encephalitis, have been reported to be associated with the influenza viral infection [2]. The immunization policy of Japan changed in 1994, resulting in a dramatic decrease in influenza vaccine distribution [3]. Since then, sporadic outbreaks of influenza virus-related encephalitis/encephalopathy have been reported every winter. During 1997– 1999, when an epidemic of influenza A (H3N2) virus occurred, many cases of influenza-associated * Corresponding author. 2015-8 Ooka, Numazu, Shizuoka 410-0022, Japan. Tel./fax: +81-55-924-6950. E-mail address: [email protected] (T. Toyoda). 0531-5131/ D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.ics.2004.02.128
T. Toyoda et al. / International Congress Series 1263 (2004) 468–471
469
encephalitis/encephalopathy were reported in Japan [1]. However, no factors responsible for the disease have been identified yet, and influenza virus types responsible for the encephalitis/encephalopathy remain unclear. We speculated that host factor(s) might be important for this disease because only a few patients suffered from encephalitis/ encephalopathy among the many people infected with the same influenza viruses. DNA microarray, in which mRNA transcription patterns can be determined for many thousands of genes simultaneously, is a new method for evaluating virus – host interactions. In the winter season of 2000 –2001, we analyzed the gene expression patterns by DNA microarray in two cases of influenza-associated encephalitis/encephalopathy and compared them with three cases of respiratory infection. We did not find specifically expressing genes up- or downregulated among the influenza respiratory infection cases. We found six elevating and three decreasing genes in two influenza encephalitis/ encephalopathy cases. 2. Materials and methods 2.1. Cases Three cases of influenza respiratory infections were chosen: case 1, 27-year-old male, influenza A (H1N1); case 2, 12-year-old male, influenza B; case 3, 1-year and 8-monthold male, influenza A (H3N2). Two cases of influenza encephalitis/encephalopathy were chosen: case 4, 2-year-old female, influenza A (H3N2); case 5, 2-month-old male, influenza A (H1N1). A new type of carbohydrate-deficient glycoprotein (CDG) syndrome type I with no neurologic symptoms nor mental retardation, characterized by a CDG syndrome type I transferring pattern was also diagnosed later in case 5 [4]. Influenza virus infection was determined by rapid diagnosis kit. The diagnosis of influenza encephalitis/ encephalopathy was made on the basis of all clinical signs. The first symptoms of both patients were convulsion and loss of consciousness. The brain CT scans and MRIs of these patients later indicated brain atrophy. Blood samples were taken on the second and the first day of disease onset in cases 4 and 5, respectively. Types of influenza viruses were determined by virus isolation and serum haemagglutinin inhibition test. These patients had not received immunization against influenza viruses. 2.2. DNA microarray analysis Blood samples of the patients with respiratory infection were taken on the first day of visit or disease onset and 1 month after they were healthy. For the encephalitis/ encephalopathy cases, blood samples were also taken approximately 2 months later when they had recovered from life-threatening illness and were in rehabilitation. Peripheral blood mononuclear cells (PBMC) were isolated in buffer EL (Qiagen, CA) according to the manufacturer’s protocol and immediately frozen in dry ice and stored at 80 jC until used. PBMC were processed for DNA microarray analysis using Atlas Glass Array Human 3.8I + 3.8II (7532 genes) at the Clontech Bioscience Laboratory (Clontech, Tokyo). Before DNA microarray analysis, the extracted RNA of PBMC was amplified by T7 RNA polymerase [5]. One sample of respiratory infection (case 1) was analyzed by UniGEMV Ver2.0 (9296 genes, Kurabo, Osaka).
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T. Toyoda et al. / International Congress Series 1263 (2004) 468–471
3. Results and discussion To identify gene(s) which showed unique expression pattern in patients with encephalitis/encephalopathy due to influenza virus infection, we performed a DNA microarray analysis using total RNA isolated from PBMC. No overlapping up- or downregulated genes were found among the influenza respiratory infection cases by DNA microarray analysis (cases 1– 3). Expression of six genes (interferon (a, h, N) receptor [6], ephrin-B2 [7], RNA binding motif protein 5 (RBM5) [8], transcription factor EC (TFEC) [9], Calsenilin/DREAM/KChIP3 [10], and MEGF6 protein [11]) was elevated more than threefold in both of the influenza encephalitis/encephalopathy patients (cases 4 and 5). No overlapping genes were found to be upregulated in one encephalitis/encephalopathy patient, but downregulated in the other. Expression of three genes (non-metastatic protein 23 (Nm23/Awd) [12], neuropeptide Y/peptide YY receptor [13], and hippocalcin-like protein 4 (HPCAL4) [14]) was decreased to less than one-third in these patients. Few overlapping genes were found between encephalitis/encephalopathy and respiratory infection patients. Expression of the interferon receptor (a, h, N) was elevated in cases 1, 4 and 5, and ribosomal protein L37a [15] was elevated in cases 3 and 4. The ras homolog gene family member H (ARHH) [16] and collagen type XVIIa1 [17] were elevated in cases 3 and 5. In the same cases, the myeloid cell nuclear differentiation antigen was decreased. The activation of macrophages by hypercytokinemia and resulting brain edema was proposed for the pathogenesis of influenza encephalitis/encephalopathy [1,18]. Elevation of interferon receptors (a, h, N) enhances the effect of hypercytokinemia. Ephrin-B2 may affect the endothelial cells of arterioles of the brain [7], which may result in their damage and brain edema. A decrease of neuropeptide Y/peptide YY receptor and HPCAL4 may affect brain damage through vasodilatation or calcium signaling in the brain [13]. Elevation of ephrin-B2, Calsenilin/DREAM/KChIP3, and decrease of Nm23/Awd may relate to apoptosis of endothelial cells of the brain vessel and neurons [7,10,19]. The response of these genes was analyzed in the PBMC, not in the brain. Although it is not clear that the response of these genes was also found in the brain, they may represent the response of the patients’ bodies. The gene(s) responsible for the encephalitis/encephalopathy associated with the influenza virus infection will be identified in these genes. Acknowledgements The protocol of this study was approved by the committee on medical ethics of Kurume University. The informed consent was obtained from the parents of the patients. This work was supported by Grants-in-Aid from the Ministry of Labor, Health and Welfare of Japan. References [1] T. Morishima, et al., Encephalitis and encephalopathy associated with an influenza epidemic in Japan, Clin. Infect. Dis. 35 (2002) 512 – 517. [2] F.A. Horner, Neurologic disorders after Asian influenza, N. Engl. J. Med. 258 (1958) 983 – 985. [3] Y. Hirota, D.S. Fedson, M. Kaji, Japan lagging in influenza jabs, Nature 380 (1996) 18. [4] J. Leonard, S. Gru¨newald, P. Clayton, Diversity of congenital disorders of glycosylation, Lancet 357 (2001) 1382 – 1383.
T. Toyoda et al. / International Congress Series 1263 (2004) 468–471
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[5] C. Pabon, et al., Optimized T7 amplification system for microarray analysis, BioTechniques 4 (2001) 874 – 879. [6] D. Novick, B. Cohen, M. Rubinstein, The human interferon alpha/beta receptor: characterization and molecular cloning, Cell 77 (1994) 391 – 400. [7] M.A. Takasu, et al., Modulation of MNDA receptor-dependent calcium influx and gene expression through EphB receptors, Science 295 (2002) 491 – 495. [8] L.C. Sutherland, et al., LUCA-15-encoded sequence variants regulate CD95-mediated apoptosis, Oncogene 19 (2000) 3774 – 3781. [9] M.C. Chung, H.K. Kim, S. Kawamoto, TFEC can function as a transcriptional activator of the nonmuscle myosin II heavy chain-A gene in transfected cells, Biochemistry 40 (2001) 8887 – 8897. [10] M.A. Leissring, et al., Calsenilin reversed presenilin-mediated enhancement of calcium signaling, Proc. Natl. Acad. Sci. U. S. A. 97 (2000) 8590 – 8593. [11] M. Nakayama, et al., Identification of high-molecular-weight proteins with multiple EGF-like motifs by motif-trap screening, Genomics 51 (1998) 27 – 34. [12] A.M. Rosengard, et al., Reduced Nm23/Awd protein in tumor metastasis and aberrant Drosophila development, Nature 342 (1989) 177 – 180. [13] C. Gerald, et al., Expression cloning and pharmacological characterization of a human hippocampal neuropeptide Y/peptide YY Y2 receptor subtype, J. Biol. Chem. 270 (1995) 26758 – 26761. [14] M. Kobayashi, et al., Isolation of two human cDNAs, HLP3 and HLP4, homologous to the neuron-specific calcium-binding protein genes, DNA Seq. 9 (1998) 171 – 176. [15] D.P. Saha, et al., Human ribosomal protein L37a: cloning of the cDNA and analysis of differential gene expression in tissues and cell lines, Gene 132 (1993) 285 – 289. [16] E. Dallery, et al., TTF, a gene encoding a novel small G protein, fuses to the lymphoma-associated LAZ3 gene by t(3;4) chromosomal translocation, Oncogene 10 (1995) 2171 – 2178. [17] C.W. Franzke, et al., Transmembrane collagen XVII, and epithelial adhesion protein, is shed from the cell surface by ADAMs, EMBO J. 21 (2002) 5026 – 5035. [18] T. Ichiyama, et al., Cerebrospinal fluid and serum levels of cytokines and soluble tumor necrosis factor receptor in influenza virus-associated encephalopathy, Scand. J. Infect. Dis. 35 (2003) 59 – 61. [19] J.D. Baxbaum, et al., Calsenillin: a calcium-binding protein that interacts with the presenilins and regulates the level of a presenilin fragment, Nat. Med. 4 (1998) 1177 – 1181.