IL6 trans-signaling promotes functional recovery of hypofunctional phagocytes through STAT3 activation during peritonitis.

OBJECTIVE: The role of high interleukin 6 (IL6) levels has not been clearly explained in severe sepsis. We show that the augmentation of the IL6 signal by recombinant IL6 receptors (rIL6R) delivery allows the functional recovery of phagocytes in a peritonitis mouse model. MATERIALS AND METHODS: Mice were challenged intraperitoneally (i.p.) with live Staphylococcus aureus for effect of IL6R delivery on the 24 h-survival, bacterial clearance and cellular responses. In additional experiments to assess the effect of IL6R delivery on phagocytosis, the model was i.p. inoculated with heat-killed S. aureus with or without rIL6R and the peritoneal lavage fluid and cells were collected at 1 h after the i.p. inoculation of S. aureus. RESULTS: The IL6R delivery tended to improve 24 h survival and increase bacteria clearance from the septic mice. The rIL6R treatment to heat-killed bacteria challenged mice augmented the uptake of bacteria and phagosome acidification, inducing the phosphorylation of STAT3 in peritoneal cells within 1 h after the IL6R delivery. Furthermore, the rIL6R delivery prevented the extracellular release of neutrophil elastase activity and myeloperoxidase (harmful factors). CONCLUSIONS: These results indicate that augmentation of IL6 signaling appears to be critical for the effective management of hypofunctional neutrophils during severe inflammation, such as sepsis.

Generation of monoclonal antibodies specific for ORF68 of koi herpesvirus.

Outbreaks of koi herpesvirus (KHV) infection in carp are still a serious problem worldwide. KHV is closely related to other two cyprinid herpesviruses, pox herpesvirus (CHV) and haematopoietic necrosis herpesvirus (CyHV-2) in goldfish. In this study, two major KHV antigenic proteins (ORF62 and ORF68) were identified by immunoscreening using a KHV-specific polyclonal antibody, and then monoclonal antibodies were generated for immunodiagnostic studies. After screening hybridoma cells, one mAb against ORF68 (mAb-7C6) was obtained but no mAbs against ORF62. mAb-7C6 specifically reacted with a lysate of KHV-infected koi fin cells (KF-1 cells) but not with lysates of CHV- or CyHV-2-infected KF-1 cells in an immuno-blotting analysis. Similar results were shown in the following tests: (1) a indirect fluorescent antibody test using infected KF-1 cells and (2) an immunohistochemical investigation by fast red stain (infected liver) or FITC detection (infected spleen). These results suggested that mAb-7C6 specifically reacts with KHV ORF68 protein.

Increased bacterial load in shrimp hemolymph in the absence of prophenoloxidase.

Invertebrates rely on their innate immune responses to protect themselves from pathogens, one of which is melanization of bacteria mediated by the activation of phenoloxidase (PO). Furthermore, invertebrate hemolymph, even that of healthy individuals, has been shown to contain bacterial species. The mechanisms that prevent these bacteria from proliferating and becoming deleterious to the host are, however, poorly understood. Here, we show that knocking down the activity of the inactive precursor of PO [prophenoloxidase (proPO)] by RNA interference resulted in a significant increase in the bacterial load of kuruma shrimp, Marsupenaeus japonicus, even in the absence of a bacterial or viral challenge. Silencing of proPO also led to a sharp increase in shrimp mortality. In addition, the hemolymph of proPO-depleted shrimp had significantly lower hemocyte counts and PO activity than control samples. Microarray analysis after proPO silencing also showed a decrease in the expression of a few antimicrobial peptides, but no effect on the expression of the genes involved in the clotting system. Treatment with antibiotics prior to and after proPO dsRNA injection, to counteract the loss of proPO, resulted in a significant increase in shrimp survival. Our results therefore show that the absence of proPO renders the shrimp incapable of controlling bacteria present in the hemolymph, and that proPO is therefore essential for its survival.

Characterization of polyclonal antibodies against Japanese flounder IgM derived from recombinant IgM constant region proteins.

Cell and determinant markers are important in fish immunology and have vast applications in aquaculture but the availability of such markers are quite limited. Hence, there is a need to identify and also further improve existing markers in fish. Here, we developed effective polyclonal antibodies (pAbs) targeting specific parts of the Japanese flounder (Paralichthys olivaceus) IgM constant (C) region. Recombinant proteins from the CHmu2 and CHmu3 termed IgM fragment 1 (rIgM1) and from CHmu4 termed IgM fragment 2 (rIgM2) were expressed and used to construct mouse pAb-IgM1 and pAb-IgM2, respectively. pAb-IgM1 detected both the approximately 77 kDa and the approximately 72 kDa heavy chains detected while pAb-IgM2 marked only the approximately 77 kDa heavy chain of Japanese flounder. Both pAbs detected IgM heavy chain in immune-related tissues, heart and serum. pAb-IgM2, but not pAb-IgM1, revealed cross reactions with other fish species detecting pronounced multiple IgM bands suggesting that the CHmu4 is an important functional region in the teleost IgM molecules. Finally, the pAb-IgMs detected surface IgM+ (sIgM+) and cytoplasmic IgM+ (cIgM+) B cells in Japanese flounder kidney in vivo.

[Educational program in the Medical Science Course, Kitasato University School of Allied Health Sciences].

The aim of education in the Medical Laboratory Science course, Kitasato University School of Allied Health Sciences, is to bring up train students who have Kitasato spirit, for careers in laboratory medicine of hospital or scientific staff of medical companies or as researchers. General and enlightening education concerning "Kitasato spirit" and professional education composed of major subjects was carried out in the first and during the 2nd and two third of 3rd grade, respectively. Medical practice and research training were alternatively carried out for 6 months between November of the 3rd year and November of the 4th year, in order to gain practical experience. Two problem-based learning (PBL) tutorial courses, "Infectious Diseases Course" and "Team Medical Care--Interprofessional Collaborations" were also carried out at the end of the 3rd and beginning of the 4th years, respectively, in order to convert a memory to knowledge. Team medical care course enrolls 1000 students at the School of Allied Health Sciences, Medicine, Nursing, Pharmacy and Kitasato College Applied Clinical Dietetics Course, is now one of special courses available at our university. This attempt is thought to result in a way of thinking that recognizes the importance of co-operation as a team member and personal contributions to actual team medical care.

Essential function of transglutaminase and clotting protein in shrimp immunity.

Blood coagulation is a conserved defense mechanism among invertebrates and it has been well studied in horseshoe crab and freshwater crayfish but is ill defined in shrimp. Transglutaminase (TGase) and clotting protein (CP) are molecules involved in the blood clotting system of shrimp. Here, we demonstrate in vivo the functional involvement of TGase and CP in the shrimp blood coagulation system using RNA interference. Expression of TGase mRNA was inhibited in gills, heart, hemocyte, hepatopancreas, intestine and lymphoid organ while the CP gene was suppressed only in gills and heart tissues on day-1 post-injection with 1 microg and 10 microg of TGase- and CP-dsRNA, respectively. However, at day-7 post-injection, systemic gene silencing was observed for both genes and dosages as shown by mRNA expression. We also show the efficiency of dsRNA silencing the protein expression as well as inhibited blood coagulation. Such silencing at the transcription, translation and phenotypic level is the first to be documented in the shrimp system. Challenge test with white spot virus and Vibrio penaecida revealed that TGase and CP are critical molecules for the immune function of shrimp against bacterial and viral infection.

Molecular cloning, characterization, expression and functional analysis of Japanese flounder Paralichthys olivaceus Fas ligand.

We isolated and sequenced Fas ligand cDNA and its gene from Japanese flounder (JF), Paralichthys olivaceus. The JF-Fas ligand cDNA consisted of 1016 bp and encoded 230 amino acid residues. The identities of the deduced amino acid sequence of the JF-Fas ligand to human Fas ligand, Tumor necrosis factor-alpha and Lymphotoxin-alpha were 26.1%, 24.5% and 23.0%, respectively. A proline-rich domain (PRD) that is important for localization of the protein was found in the N-terminal region, and two cysteine residues, which form a disulfide bond, were conserved. The JF-Fas ligand gene has a length of 1.8 kb and consists of four exons and three introns. The length of the JF-Fas ligand second intron is shorter than that in the human and pig Fas ligand genes. However, the organization of the exons and introns is similar to that of mammals. RT-PCR was conducted for 12 tissues, and expression of JF-Fas ligand mRNA was detected in the kidney, thymus, gills, stomach and spleen. The recombinant JF-Fas ligand prepared in an Escherichia coli protein expression system showed cytotoxic activity against Japanese flounder cell line HINAE and caused the fragmentation of genomic DNA. The cytotoxic activity was measured by MTT assay. These results indicate that fish possess a Fas ligand system.

Molecular cloning and characterization of Toll-like receptor 9 in Japanese flounder, Paralichthys olivaceus.

Toll-like receptor (TLR) 9 cDNA and gene were cloned from Japanese flounder, Paralichthys olivaceus. The Japanese flounder TLR9 cDNA encodes 1065 amino acids. The leucine-rich domain (LRD) and the Toll/interleukin-1 receptor (TIR) domain found in other vertebrate TLR9s were conserved in Japanese flounder TLR9. The gene is composed of three exons and two introns. The Japanese flounder tumor necrosis factor (TNF) gene promoter was activated in Japanese flounder TLR9-transformed hirame natural embryo (HINAE) cells upon stimulation with synthesized CpG oligodeoxynucleotide (ODN), but not by stimulation with GpC ODN. The Japanese flounder TLR9 gene was highly expressed in epithelial and lymphoid organs, such as the gills, intestines, kidney, spleen and stomach in an apparently healthy fish. The mRNA copy numbers of Japanese flounder TLR9 and its adapter protein, the myeloid differentiation factor 88 (MYD88) were increased in some organs including blood, gill, kidney and spleen after Edwardsiella tarda challenge. Immunohistochemical analysis revealed that TLR9 and MYD88 were expressed in the same cells of kidney. Few TLR9-expressing cells were found in gill, kidney and spleen in healthy Japanese flounder, but many were found in these organs after E. tarda challenge and were coincident with lesions that had been colonized by the bacteria.

Identification and characterization of a myeloid differentiation factor 88 (MyD88) cDNA and gene in Japanese flounder, Paralichthys olivaceus.

The interleukin-1 receptor/toll-like receptor (IL-1R/TLR) superfamily signaling involves myeloid differentiation factor 88 (MyD88) that acts as an important adapter protein. A Japanese flounder (Paralichthys olivaceus) MyD88 (jfMyD88) cDNA and gene were cloned, and found to have lengths of 1.5 and 3.01 kb, respectively. The ORF encodes 285 amino acids that contain a death domain and a Toll/IL-1 receptor domain. The gene is composed of 5 exons and 4 introns. The jfMyD88 gene is highly expressed in organs involved in immune functions, including the gills, intestines, kidney, skin and spleen. Three days after a fish was infected with Edwardsiella tarda, staining with anti-jfMyD88 polyclonal antibody revealed an increased population of MyD88-positive cells in the kidney and spleen. These results imply that MyD88 has an important role in the innate immune system in Japanese flounder.

Immunoanalysis of antiviral Mx protein expression in Japanese flounder (Paralichthys olivaceus) cells.

Mx proteins are interferon-inducible GTPases that possess antiviral properties in vertebrates. Japanese flounder (Paralichthys olivaceus) Mx protein has previously been shown to possess some antiviral activity against rhabdoviruses. A polyclonal antibody was generated against a purified peptide fragment of Japanese flounder Mx protein that had been produced in an Escherichia coli expression system. The PAb detected the approximately 71 kDa Mx protein from Japanese flounder (hirame) natural embryo (HINAE) cells that had been cultured with poly I:C, an interferon inducer, but not in unstimulated cells. The polyclonal antibody did not cross react with Mx protein from carp epithelial, grouper fin and zebrafish embryo cell lines that had been similarly induced or transfected with poly I:C. By immunofluorescence cytochemistry, Japanese flounder Mx protein was localized to the cell cytoplasm. Hirame rhabdovirus stimulated expression of Mx protein in the infected and surrounding HINAE cells. Within virus-infected cells, there was some indication of Mx protein colocalizing with viral proteins. Poly I:C stimulation of HINAE cells induced an early increase in Mx protein mRNA transcripts, but maximum Mx mRNA transcript and protein expression was reached after 48 h. Both Mx mRNA transcripts and protein levels were maintained till at least 72 h.