Toxicity Assessment of Mixed Exposure of Nine Perfluoroalkyl Substances at Concentrations Relevant to Daily Intake.

Per- and poly-fluoroalkyl substances (PFAS) exhibit high persistence in the environment and accumulate within the human body, warranting a thorough assessment of their toxicity. In this study, we exposed mice (male C57BL/6J mice aged 8 weeks) to a composite of nine PFAS, encompassing both long-chain PFAS (e.g., perfluorooctanoic acid and perfluorooctanesulfonic acid) and short-chain PFAS (e.g., perfluorobutanoic acid and perfluorobutanesulfonic acid). The exposure concentrations of PFAS were equivalent to the estimated daily human intake in the composition reported (1 µg/L (sum of the nine compounds), representing the maximum reported exposure concentration). Histological examination revealed hepatocyte vacuolization and irregular hepatocyte cord arrangement, indicating that exposure to low levels of the PFAS mixture causes morphological changes in liver tissues. Transcriptome analysis revealed that PFAS exposure mainly altered a group of genes related to metabolism and chemical carcinogenesis. Machine learning analysis of the liver metabolome showed a typical concentration-independent alteration upon PFAS exposure, with the annotation of substances such as glutathione and 5-aminovaleric acid. This study demonstrates that daily exposure to PFAS leads to morphological changes in liver tissues and alters the expression of metabolism- and cancer-related genes as well as phospholipid metabolism.

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.

[Newly developed MRSA medium].

We have developed a new selective medium, tentatively named MR(SA)2, for rapid identification of methicillin-resistant Staphylococcus aureus (MRSA) from clinical specimens. MR(SA)2 medium contained modified Müller-Hinton agar supplemented with 75 g of NaCl, 10 g of mannit, 20 mg of bromcresol purple, 20 g of egg-yolk, 4 mg of oxacillin, and 12.5 mg of ceftisoxime per 1,000 ml. There were no differences between the growth of MRSA strains on this medium at 30 C and that of 37 C. This medium can detect the egg-yolk reaction instead of the coagulase reaction. By single streaking of a test material on the surfaces of MR(SA)2 agar, MRSAs can easily be distinguished from methicillin-resistant coagulase-negative staphylococci (MR-CNS), other bacteria and fungi from their colony morphologies in a quantitative manner. A few MRSA strains would not form colonies on this medium because of their susceptibilities to ceftisoxime, but this may not inpede its use, since most MRSA strains isolated from clinical materials showed resistance to ceftisoxime. From the above results, the MR(SA)2 medium may be suitable for rapid detection of MRSA and MR-CNS.

[Proposal for the nosocomial infection control of methicillin-resistant Staphylococcus aureus (MRSA)].

To determine the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in hospital environments, quantitative isolation of MRSA from environmental and human specimens was performed. It was found that as many as 6 x 10(4) colony forming units (CFU)/100cm2 of MRSA and also methicillin-resistant coagulase-negative Staphylococcus (MRCNS) were isolated from 1) 25-100% of the bed-mattresses tested in 10 out of 11 (90%) hospitals, and also 2) bed sheets for patients, floors of patient wards, laundry, bath-room, toilet and laundry storage-room. And seven and eight palms of 20 patients were contaminated with MRCNS and MRSA, respectively, and MRCNS contamination was revealed in six of medical staffs. These results indicate that hospital environments, especially the mattresses and hospital floors are highly contaminated with MRCNS and MRSA, and sanitation and cleanliness of mattresses and floors are necessary to prevent the dissemination of both MRCNS and MRSA in hospital.

IgG2b-dependent down regulation of the LPS-induced PFC-response and its blockade by Fc gamma 2bR protein.

Fc gamma receptor (Fc gamma R)-dependent immunoregulation by murine heat-aggregated (HAgg) IgG subclasses on the bacterial lipopolysaccharide (LPS)-induced plaque forming cell (PFC) response to trinitrophenylated sheep red blood cell (TNP-SRBC) antigen and the competitive effect by Fc gamma 2bR-protein on the down regulation by HAgg-IgG2b were studied in murine T-cell-deprived spleen cell cultures. HAgg-IgG1 and HAgg-IgG3 enhanced the PFC response, but HAgg-IgG2b strongly suppressed the LPS-induced PFC response. HAgg-IgG1 could not compete with the suppressive effect of HAgg-IgG2b. The HAgg-IgG2b seemed to act on both macrophages (M phi) and B-cells, because the cell cultures that had been reconstituted with HAgg-IgG2b-pretreated M phi and untreated B-cells and vice versa showed poor PFC responses. The suppression induced by HAgg-IgG2b on the LPS-induced PFC response in the T-cell-deprived cultures was abolished by the addition of phospholipase C (PLC)-treated Fc gamma 2bR protein at the early stage of the culture. The mechanisms by which HAgg-IgG2b suppress the LPS-induced PFC response and PLC-treated Fc gamma 2bR protein restores this response were discussed.

The adjuvant effect of a muramyl dipeptide (MDP) analog on temperature-sensitive Salmonella mutant vaccine.

Adjuvant effect of a synthetic muramyl dipeptide analog, MDP-Lys (L18), MurNAc-L-Ala-D-glu[Lys(CO-(CH2)16-CH3)-OH]NH2 on a live Salmonella enteritidis vaccine, a temperature-sensitive mutant Ts-O strain that was obtained from the virulent S. enteritidis No.11 strain and could not grow at 37 degrees C, but could multiply at 25 degrees C as fast as the parent strain, was examined. Although the Ts-O organisms were avirulent and could hardly multiply in vivo when the organisms were injected into C57BL/6 mice and its mutant beige strain, which has a malfunction of phagocytic cells, injection of these mice with Ts-O endowed them with some protective immunity against infection by the virulent No.11 strain. When MDP-Lys(L18) was injected with Ts-O vaccine, the protection and the bactericidal capacity in the peritoneal cavities and spleens of these mice were augmented. MDP-Lys(L18) was still effective when it was injected at 48 h before or after the inoculation of Ts-O vaccine. Its effect was also observed against infection by the virulent S. cholerae-suis Hokkaido strain, a strain that does not share common O-antigenic determinants with the S. enteritidis No.11 or Ts-O strain. In addition, the mice that were inoculated simultaneously with Ts-O organisms and MDP-Lys(L18) were examined 10 days later for their footpad delayed type hypersensitivity reactions against Ts-O antigen. Mice inoculated with MDP-Lys(L18) and Ts-O showed augmented footpad swelling in comparison with the controls. These findings indicate that MDP-Lys(L18) is capable of augmenting the cellular immunity by live vaccine.

Phagocytosis and bactericidal action of mouse peritoneal macrophages treated with leukotriene B4.

The effects of exogenous leukotriene B4 (LTB4) on the resistance of mouse peritoneal macrophages against Salmonella (S.) typhimurium and Pseudomonas (P.) aeruginosa infections were studied. In vitro, LTB4 added to macrophage monolayers at final concentrations of 10(-12)-10(-8) M, enhanced their phagocytosis of S. typhimurium to 2.3 times the control level and that of P. aeruginosa to 1.8 times the control level. The intracellular killing rates were also elevated by the addition of LTB4: for S. typhimurium, 83.3% (LTB4) vs 59.1% (control) and for P. aeruginosa, 46.5% (LTB4) vs 9.2% (control). In vivo, intraperitoneally injected LTB4 (5 ng) enhanced the clearance at 24 h of intraperitoneally injected S. typhimurium from the mouse peritoneal cavity (2.38 x 10(3) +/- 0.94 x 10(3) cells [LTB4] vs 5.73 x 10(5) +/- 1.90 x 10(5) [control]) and spleen (5.00 x 10(2) +/- 0.94 x 10(2) [LTB4] vs 2.47 x 10(4) +/- 0.84 x 10(4) [control]), but this effect disappeared by 48 h. In contrast, in beige mice, an experimental model of the Chédiak-Higashi syndrome that is characterized by susceptibility to bacterial infection, there was no induction of the eliminating effect by intraperitoneal injection of LTB4. Activation of macrophages by exogenous LTB4 seemed to have contributed to such an augmented resistance of macrophages to bacterial infection. This study suggested a possible use of LTB4 in bacterial infectious diseases whereby phagocytes are able to play a key role in host defense.

Toxicity of Microcystis aeruginosa K-139 strain.

Toxicity of the cells of a newly established axenic Microcystis aeruginosa K-139 strain to mice was studied. LD50 of the cells harvested in the mid-log phase was 7.3 mg/kg. The organs of acute dead mice were examined histopathologically. The blood congestion and necrosis of the parenchymal cells around the central veins in the liver were observed, but other organs seemed to be normal. The liver damage was confirmed by the tests of glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) activities in the sera of the mice after the injection with the K-139 cells. Furthermore, the K-139 cells were capable of inducing interleukin 1 (IL-1) production by peritoneal macrophages in vitro.