Kyoji Hioki

PD-1 and LAG-3 inhibitory co-receptors act synergistically to prevent autoimmunity in mice

A new mouse model of spontaneous autoimmune disease reveals an important role for the inhibitory co-receptor LAG-3 in suppressing autoimmunity.

Prostaglandin E2 Signaling and Bacterial Infection Recruit Tumor-Promoting Macrophages to Mouse Gastric Tumors

BACKGROUND & AIMS Helicobacter pylori infection induces an inflammatory response, which can contribute to gastric tumorigenesis. Induction of cyclooxygenase-2 (COX-2) results in production of prostaglandin E(2) (PGE(2)), which mediates inflammation. We investigated the roles of bacterial infection and PGE(2) signaling in gastric tumorigenesis in mice. METHODS We generated a germfree (GF) colony of K19-Wnt1/C2mE mice (Gan mice); these mice develop gastric cancer. We examined tumor phenotypes, expression of cytokines and chemokines, and recruitment of macrophages. We also investigated PGE(2) signaling through the PGE(2) receptor subtype 4 (EP4) in Gan mice given specific inhibitors. RESULTS Gan mice raised in a specific pathogen-free facility developed large gastric tumors, whereas gastric tumorigenesis was significantly suppressed in GF-Gan mice; reconstitution of commensal flora or infection with Helicobacter felis induced gastric tumor development in these mice. Macrophage infiltration was significantly suppressed in the stomachs of GF-Gan mice. Gan mice given an EP4 inhibitor had decreased expression of cytokines and chemokines. PGE(2) signaling and bacterial infection or stimulation with lipopolysaccharide induced expression of the chemokine C-C motif ligand 2 (CCL2) (which attracts macrophage) in tumor stromal cells or cultured macrophages, respectively. CCL2 inhibition suppressed macrophage infiltration in tumors, and depletion of macrophages from the tumors of Gan mice led to signs of tumor regression. Wnt signaling was suppressed in the tumors of GF-Gan and Gan mice given injections of tumor necrosis factor-α neutralizing antibody. CONCLUSIONS Bacterial infection and PGE(2) signaling are required for gastric tumorigenesis in mice; they cooperate to up-regulate CCL2, which recruits macrophage to gastric tumors. Macrophage-derived tumor necrosis factor-α promotes Wnt signaling in epithelial cells, which contributes to gastric tumorigenesis.

Prophylactic Effect of FK463, a Novel Antifungal Lipopeptide, against Pneumocystis carinii Infection in Mice

ABSTRACT The prophylactic effect of FK463, a new water-soluble echinocandin-like lipopeptide with inhibitory activity against 1,3-β-d-glucan synthase, against Pneumocystis carinii infection was investigated with the severe combined immunodeficient (SCID) mouse model. Treatment with FK463, pentamidine, and saline only was performed for 6 weeks from the day after the SCID mice were inoculated intranasally with infected lung homogenates. FK463 at 0.2 or 1.0 mg/kg of body weight, pentamidine at 4 mg/kg, or saline was subcutaneously administered daily into the backs of the SCID mice. The effects of the drugs were evaluated by detection of P. carinii cysts in mouse lung homogenates by toluidine blue O staining, lung histology, and PCR amplification of a P. carinii-specific DNA fragment from the lungs. P. carinii cysts were detected in the lungs of all mice administered saline. In contrast, no cysts were detected in mice administered both doses of FK463 and pentamidine. A specific DNA fragment was amplified from all mice administered saline and at least half or more of the mice administered FK463 and pentamidine. These results indicate that FK463 acts on cyst wall formation but not on trophozoite proliferation and is extremely effective in preventing P. carinii-associated pneumonia. These results suggest that FK463 is potentially useful as a prophylactic agent against P. carinii infection.

Involvement of Illegitimate V(D)J Recombination or Microhomology-Mediated Nonhomologous End-Joining in the Formation of Intragenic Deletions of the Notch1 Gene in Mouse Thymic Lymphomas

Deregulated V(D)J recombination-mediated chromosomal rearrangements are implicated in the etiology of B- and T-cell lymphomagenesis. We describe three pathways for the formation of 5′-deletions of the Notch1 gene in thymic lymphomas of wild-type or V(D)J recombination-defective severe combined immune deficiency (scid) mice. A pair of recombination signal sequence-like sequences composed of heptamer- and nonamer-like motifs separated by 12- or 23-bp spacers (12- and 23-recombination signal sequence) were present in the vicinity of the deletion breakpoints in wild-type thymic lymphomas, accompanied by palindromic or nontemplated nucleotides at the junctions. In scid thymic lymphomas, the deletions at the recombination signal sequence-like sequences occurred at a significantly lower frequency than in wild-type mice, whereas the deletions did not occur in Rag2−/− thymocytes. These results show that the 5′-deletions are formed by Rag-mediated V(D)J recombination machinery at cryptic recombination signal sequences in the Notch1 locus. In contrast, one third of the deletions in radiation-induced scid thymic lymphomas had microhomology at both ends, indicating that in the absence of DNA-dependent protein kinase-dependent nonhomologous end-joining, the microhomology-mediated nonhomologous end-joining pathway functions as the main mechanism to produce deletions. Furthermore, the deletions were induced via a coupled pathway between Rag-mediated cleavage at a cryptic recombination signal sequence and microhomology-mediated end-joining in radiation-induced scid thymic lymphomas. As the deletions at cryptic recombination signal sequences occur spontaneously, microhomology-mediated pathways might participate mainly in radiation-induced lymphomagenesis. Recombination signal sequence-mediated deletions were present clonally in the thymocyte population, suggesting that thymocytes with a 5′-deletion of the Notch1 gene have a growth advantage and are involved in lymphomagenesis.

The role of leptin in the development of the cortical neuron in mouse embryos

Leptin is an obese gene product that decreases appetite and raises energy expenditure in adults. We previously reported that leptin was detected in the sera of mouse embryos and leptin receptors were expressed in the mouse embryonic cerebrum, suggesting that leptin plays a role in cerebral development. In this study, we injected leptin into the lateral ventricle of the cerebrum in leptin-deficient ob/ob mouse embryos to investigate the function of leptin in cerebral development. When leptin was injected on embryonic day (E) 14, the ratio of the number of cells in the cortical plate (CP) to that in the intermediate zone (IZ) was higher in leptin-injected than in vehicle-injected ob/ob embryos on E16, although there was no significant difference in the number of cells in the ventricular zone (VZ), IZ, or CP between these groups. The number of postmitotic BrdU-positive CP cells was larger in leptin-injected than in vehicle-injected ob/ob embryos on E16 and E17 when BrdU labeling and leptin injection were performed on E14. By in situ hybridization, NPY mRNA expression in CP neurons on E18 was weaker in leptin-injected than in vehicle-injected ob/ob embryos when leptin was injected on E16. These results suggest that leptin promotes the migration of neuronal lineage cells to CP and that the leptin-NPY axis in neurons works in the cerebral cortex on E16.

Importance of the interferon-α system in murine large intestine indicated by microarray analysis of commensal bacteria-induced immunological changes

BackgroundAlthough microbiota play a critical role in the normal development and function of host immune systems, the underlying mechanisms, especially those involved in the large intestine (LI), remain unknown. In the present study, we performed transcriptome analysis of the LI of germ-free (GF) and specific pathogen-free (SPF) mice of the IQI strain, an inbred strain established from ICR mice.ResultsGeneChip analysis, quantitative real-time RT-PCR, and reconfirmation using bacteria-inoculated GF mice revealed differences in the expression levels of several immune-related genes, such as cryptdin-related sequences (CRS), certain subsets of type 1 interferon (IFN)-related genes, class Ib MHC molecules, and certain complements. LI expressed no authentic cryptdins but predominantly expressed CRS2, 4, and 7. The mRNA levels of IFN-related genes, including Irf7, Isgf3g, Ifit1 and Stat1, were lower in SPF- and flora-reconstituted mice. When an oral IFN-α inducer tilorone analog, R11567DA, was administered to SPF mice, IFN-α was induced rapidly in the LI at 4 h, whereas no IFN-α protein was detected in the small intestine (SI) or blood. In situ hybridization and immunohistochemistry suggested that the IFN-α production originated from Paneth cells in the SI, and portions of lamina proprial CD11b- or mPDCA1-positive cells in the LI.ConclusionThe present study suggests that microbial colonization, while inducing the expression of anti-microbial peptides, results in the down-regulation of certain genes responsible for immune responses, especially for type I IFN synthesis. This may reflect the adaptation process of the immune system in the LI to prevent excessive inflammation with respect to continuous microbial exposure. Further, the repertoire of anti-microbial peptides and the extraordinary role of interferon producing cells in the LI have been found to be distinct from those in the SI.

Transgene stability and features of rasH2 mice as an animal model for short‐term carcinogenicity testing

The transgenic mouse rasH2 line, in which the mouse carries the human c‐Ha‐ras gene under the control of its own enhancer and promoter, has been proposed as one of the alternative short‐term models for carcinogenicity testing. To apply this purpose, we have produced a genetically homogeneous population as C57BL/6JJic‐TgN(RASH2) (Tg‐rasH2) by continuous backcrossing. In this study, we examined the transgene stability between different generations and the detailed transgene architecture of the integrated human c‐Ha‐ras gene. Fluorescence in situ hybridization analysis showed that the integrated human c‐Ha‐ras gene was stably located on chromosome 15E3 in Tg‐rasH2 mice at generation number (N) 15 and 20. Southern and Northern blot analysis did not show any differences in the hybridized band pattern in each generation. Southern blot analyses showed that the Tg‐rasH2 mouse contained three copies of the human c‐Ha‐ras gene arrayed in a head‐to‐tail configuration. We also determined the nucleotide sequence of the transgene in the Tg‐rasH2 mouse at N20 and confirmed that the sequence of the coding region was perfectly matched with human c‐Ha‐ras cDNA. Cloning and sequencing of genome/transgene junctions revealed that integration of the microinjected human c‐Ha‐ras gene into mouse host genome resulted in a 1820‐bp deletion in the rasH2 line. The deleted sequence did not have any sequence homologies with known functional genes. We assumed that either the deletion or the transgene insertion, or both, would not cause insertional mutation. In short‐term carcinogenicity testing with a genetically engineered mouse model, confirmation of the transgene or modified gene stability at each generation is one of the important factors that affect the sensitivity to carcinogenic compounds in the same way as the genetic background, age and route of administration.

Susceptibility to urethane carcinogenesis of transgenic mice carrying a human prototype c-Ha-ras gene (rasH2 mice) and its modification by butylhydroxytoluene

The susceptibility of rasH2 mice to urethane lung carcinogenesis and the modifying effects of butylhydroxytoluene (BHT) on development of pulmonary lesions were examined. Single i.p. injections of urethane at 250 mg/kg in males or 500 mg/kg in females induced alveolar/bronchiolar adenomas within 6 weeks. At 4 weeks after the injection with a dose of 1000 mg/kg, adenomas occurred in both sexes. BHT administration increased the multiplicity of hyperplasias observed 3 weeks after the urethane injection and additionally caused adenomas which did not occur in the urethane alone-treated animals. The overall data suggest the possibility of rapid assays for lung carcinogens using rasH2 mice.

Effect of leptin administration on myelination in ob/ob mouse cerebrum after birth.

Brain weight and size are known to be reduced in adult leptin-deficient Lepob/Lepob (OB) mice when compared with the wild-type (+/+) mice (C57BL/6: B6). We here analyzed leptin’s effects on myelination by examining morphometrically the myelin sheath (MS) in the cerebrum of postnatal day (P) 14 and P28 OB that had received leptin 1 nmol/capita/day from P7 to P14 or P28 (OB+lep), in comparison with OB and B6. We examined myelin basic protein (MBP) mRNA levels and the differentiation of oligodendrocytes by comparing the number of oligodendrocyte precursor cells (OPCs) and the mature oligodendrocytes in the cerebrum between OB, OB+lep, and B6 on P14 and P28. MBP-mRNA expression was lower in OB than in B6 on P14 and P28. On P14, it was higher in OB+lep than in OB but was still lower than in B6, whereas on P28 it was even higher in OB+lep than in B6. On P28, the radii of myelinated axons were larger in OB than in B6 and OB+lep. The MS on P28 was significantly thinner in OB than in B6, but there was no significant difference between OB and OB+lep. There were significantly fewer mature oligodendrocytes in OB and OB+lep than in B6 on P28, whereas on P14 there were significantly fewer OPCs in OB and OB+lep than in B6. Our results suggested that leptin regulates the myelination of oligodendrocytes and that the replenishment of leptin in OB recovered myelination but did not affect the differentiation of OPCs from P7 to P28.

Studies on beige–nude mice with low natural killer cell activity 1. Introduction of the bg gene into nude mice and the characteristics of beige–nude mice

To improve the take-rate of human tumours in nude mice, a nude mouse strain with the bg gene was established. The introduction of the bg gene was confirmed by examination of giant granules of blood neutrophils. The genetic profile of beige–nude mice was the same as that of the C57BL/6 strain according to genetic screening. The natural killer cell activity in the beige–nude mice was much lower than that in ordinary nude mice but slightly higher than that in beige mice. The reproductivity of beige–nude mice was as high as that of ordinary nude mice.