Tomokazu Indoh

Cytokine regulation in SARS coronavirus infection compared to other respiratory virus infections

The pathogenesis of severe acute respiratory syndrome (SARS) is poorly understood and cytokine dysregulation has been suggested as one relevant mechanism to be explored. We compared the cytokine profile in Caco2 cells after infection of SARS coronavirus (SARS‐CoV) with other respiratory viruses including respiratory syncytial virus (RSV), influenza A virus (FluAV), and human parainfluenza virus type 2 (hPIV2). Interferon (IFN) system (production and response) was not suppressed by SARS‐CoV infection. Therefore, SARS‐CoV replication was suppressed by pretreatment with IFN. SARS‐CoV and RSV induced high levels of IL‐6 and RANTES compared with FluAV and hPIV2. Induction level of suppressor of cytokine signaling‐3 (SOCS3) by SARS‐CoV was significantly lower than that by RSV in spite of the significant production of IL‐6. Toll‐like receptors 4 and 9, which correlate with the induction of inflammatory response, were upregulated by SARS‐CoV infection. Collectively, overinduction of inflammatory cytokine and dysregulation of cytokine signaling may contribute to the immunopathology associated with “severe” inflammation in SARS. J. Med. Virol. 78:417–424, 2006.

Cloning of a DNA fragment encoding the 5'-terminus of the botulinum type E toxin gene from Clostridium butyricum strain BL6340

Chromosomal DNA was extracted from toxigenic Clostridium butyricum strain BL6340 isolated from a case of infant botulism. After digestion by EcoRI, a DNA fragment of about 1 kbp was cloned into Escherichia coli using lambda gt11, and was subcloned into pUC118. The E. coli cells transformed with this cloned fragment produced a 33 kDa protein which reacted with monoclonal antibodies recognizing the light chain (Lc) component of botulinum type E toxin. The nucleotide sequence of the cloned fragment was determined. The sequence was similar to that from botulinum type E toxin gene fragments previously determined by our laboratory (strains Mashike, Otaru and Iwanai). Several highly homologous sequences among the botulinum type A, C, E, butyricum and tetanus toxin genes were found in both translated and untranslated regions. These results suggest that the toxin gene of C. butyricum may have evolved by transfer from C. botulinum.

The nucleotide and deduced amino acid sequences of EcoRI fragment containing the 5'-terminal region of Clostridium botulinum type E toxin gene cloned from Mashike, Iwanai and Otaru strains.

Chromosomal DNAs were extracted from toxigenic three Clostridium botulinum type E strains isolated from food‐borne botulism. After digestion by EcoRI, the fragments were cloned into Escherichia coli by using bacteriophage lambda gtl 1 and screened with monoclonal antibody recognizing the light chain component of botulinum type E toxin. The fragments (about 1 kbp size) cloned from each strain were recloned into a plasmid vector pUC118. The E. coli cells transformed with the recombinant plasmids produced 33 kDa protein with or without IPTG (isopropyl‐β‐D‐thiogalactopyranoside) which reacted with the monoclonal antibody. The nucleotide sequences of the cloned EcoRI fragments from the three type E strains were identical and contain the 5′‐terminal region of the type E toxin gene. It was also found that there exist several highly homologous nucleotide sequences among the botulinum types A, C and E, and tetanus toxin genes in both translated and untranslated regions.

Investigation of IFN Type-I Receptor and IFN Regulatory Factor Expression Relating to Induction of 2', 5'-Oligoadenylate Synthetase in Cells Persistently Infected with the Mumps Virus

Poor induction of interferon‐induced 2′, 5′‐oligoadenylate synthetase (2–5AS) activity has been demonstrated in cells persistently infected with the mumps virus or human T‐lymphotropic virus type‐I (HTLV‐I). The suppression of 2–5AS induction is the result of the repression of 2–5AS gene expression at the transcription level. In a general way, after the binding of interferon‐α (IFN‐α) to cell surface‐specific receptors, expression of 2–5AS gene is thought to be regulated by some transacting factors, IFN‐regulatory factors (IRF‐1 and IRF‐2) and the IFN‐stimulated gene factor (ISGF‐3, a complex consisting of STAT‐1α, STAT‐2 and p48). To clarify the cause of the suppression mechanism(s), fluctuation in the number of IFN receptors and the levels of mRNAs in both IRF‐1 and IRF‐2 were examined in cells persistently infected with the mumps virus (FLMT and KBMT). There were few defferences in the number of IFN receptors and the level of IRF‐2 mRNA between persistently infected cells and uninfected control cells. After the treatment of cells with IFN, a slight reduction of IRF‐1 mRNA was found in persistently infected cells as compared with that of the uninfected control cells.

Augmentation of Interferon Production after Cell‐Differentiation of U937 Cells by TPA

Persistent infections with mumps virus were established in several human lymphoid cells of T‐cell origin (Molt‐4, TALL‐1, and CCRF‐CEM) and human monocyte cells (U937 and THP‐1). 2′,5′‐Oligoadenylate synthetase (2–5AS) activity was demonstrated to be only slightly induced by interferon (IFN) or TPA (12‐O‐tetradecanoyl‐phorbol‐13‐acetate) treatment in these cells. Treatment of the persistently infected cells with IFN or TPA did not stimulate an increase in the amount of synthetase mRNA. Induction of cell differentiation and augmentation of IFN production by TPA were demonstrated in U937 cells persistently infected with mumps virus (U937‐MP). Similar results for IFN production were obtained from differentiated U937 cells. It is suggested that cell differentiation of U937 cells might be associated with the development of IFN inducibility.

Suppressed Induction of 2-5AS in Cells with HTLV-I Is Not Associated with the Fluctuation of IFN-Receptor, IFN-Regulatory Factors (IRF-1 and IRF-2) or STAT-1α

Some viruses seem to be capable of suppressing interferon (IFN)‐induced 2′, 5′‐oligoadenylate synthetase (2‐5AS) induction. Cells infected with human T‐lymphotropic virus type‐I (HTLV‐I) show different natures for the constitutive production of IFN‐γ or sensitivity to IFN. Poor induction of 2‐5AS was found in IFN‐γ producer cells carrying HTLV‐I (MT‐1, MT‐2 and SMT‐1). On the other hand, in non‐ or low‐producing cell lines of IFN‐γ such as HUT102 and OKM‐2, significant activity of 2‐5AS was induced by treatment with IFN‐α. A satisfactory level of IFN receptor was detectable in SMT‐1 cells in spite of the poor induction of 2‐5AS. There were no differences in either the interferon regulatory factor‐2 (IRF‐2) mRNA transcript or the level of STAT‐1α between SMT‐1 and HUT102 cells. However, the transcription of IRF‐1 mRNA was slightly reduced in SMT‐1 cells as compared with that of HUT102 cells.