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Biomedical subjects

Makoto Matsumoto

Publications and source records attributed to Makoto Matsumoto.

16 recordsLinked to original sources
PubMed2006

Synthesis and antituberculosis activity of a novel series of optically active 6-nitro-2,3-dihydroimidazo[2,1-b]oxazoles.

In an effort to develop potent new antituberculosis agents that would be effective against both drug-susceptible and drug-resistant strains of Mycobacterium tuberculosis, we prepared a novel series of optically active 6-nitro-2,3-dihydroimidazo[2,1-b]oxazoles substituted at the 2-position with various phenoxymethyl groups and a methyl group and investigated the in vitro and in vivo activity of these compounds. Several of these derivatives showed potent in vitro and in vivo activity, and compound 19 (OPC-67683) in particular displayed excellent in vitro activity against both drug-susceptible and drug-resistant strains of M. tuberculosis H37Rv (MIC = 0.006 microg/mL) and dose-dependent and significant in vivo efficacy at lower oral doses than rifampicin in mouse models infected with M. tuberculosis Kurono. The synthesis and structure-activity relationships of these new compounds are presented.

Antitubercular Agents↗
PubMed2006

TLR-dependent induction of IFN-beta mediates host defense against Trypanosoma cruzi.

Host resistance to the intracellular protozoan parasite Trypanosoma cruzi depends on IFN-gamma production by T cells and NK cells. However, the involvement of innate immunity in host resistance to T. cruzi remains unclear. In the present study, we investigated host defense against T. cruzi by focusing on innate immunity. Macrophages and dendritic cells (DCs) from MyD88(-/-)TRIF(-/-) mice, in which TLR-dependent activation of innate immunity was abolished, were defective in the clearance of T. cruzi and showed impaired induction of IFN-beta during T. cruzi infection. Neutralization of IFN-beta in MyD88(-/-) macrophages led to enhanced T. cruzi growth. Cells from MyD88(-/-)IFNAR1(-/-) mice also showed impaired T. cruzi clearance. Furthermore, both MyD88(-/-)TRIF(-/-) and MyD88(-/-)IFNAR1(-/-) mice were highly susceptible to in vivo T. cruzi infection, highlighting the involvement of innate immune responses in T. cruzi infection. We further analyzed the molecular mechanisms for the IFN-beta-mediated antitrypanosomal innate immune responses. MyD88(-/-)TRIF(-/-) and MyD88(-/-)IFNAR1(-/-) macrophages and DCs exhibited defective induction of the GTPase IFN-inducible p47 (IRG47) after T. cruzi infection. RNA interference-mediated reduction of IRG47 expression in MyD88(-/-) macrophages resulted in increased intracellular growth of T. cruzi. These findings suggest that TLR-dependent expression of IFN-beta is involved in resistance to T. cruzi infection through the induction of IRG47.

Adaptor Proteins, Vesicular Transport↗
PubMed2006

Exploration of a new type of antimalarial compounds based on febrifugine.

Febrifugine (1), a quinazoline alkaloid, isolated from Dichroa febrifuga roots, shows powerful antimalarial activity against Plasmodium falciparum. The use of 1 as an antimalarial drug has been precluded because of side effects, such as diarrhea, vomiting, and liver toxicity. However, the potent antimalarial activity of 1 has stimulated medicinal chemists to pursue compounds derived from 1, which may be valuable leads for novel drugs. In this study, we synthesized a new series of febrifugine derivatives formed by structural modifications at (i) the quinazoline ring, (ii) the linker, or (iii) the piperidine ring. Then, we evaluated their antimalarial activities. Thienopyrimidine analogue 15 exhibited a potent antimalarial activity and a high therapeutic selectivity both in vitro and in vivo, suggesting that 15 is a good antimalarial candidate.

Animals↗
PubMed2006

Impaired postnatal development in C/EBPbeta-deficient mice.

Tail DNA genotyping of fetal and neonatal mice from C/EBPbeta heterozygous parents was performed to determine whether the decreased number of surviving C/EBPbeta mutants was caused by prenatal or postnatal death. Eighty-four 3-week-old mice born of heterozygous parents had significantly lower numbers of C/EBPbeta-deficient offspring than the expected Mendelian ratio (29.8%+/+, 65.5%+/-, 4.76%-/-, P<0.05). The genotypes of 72 fetal mice from intercrossed heterozygotes showed approximately the expected 1:2:1 Mendelian ratio (18.1% +/+, 52.8% +/-, 29.2% -/-, P>0.05). No difference in the proportions by sex could be detected in these perinates. This data indicates that C/EBPbeta-deficient mice have unknown lethal problems between the embryonic stage and weaning.

Animals↗
PubMed2006

Development of vaccines and passive immunotherapy against SARS coronavirus using mouse and SCID-PBL/hu mouse models.

We have investigated novel vaccines strategies against severe acute respiratory syndrome (SARS) CoV infection using cDNA constructs encoding the structural antigens; spike (S), membrane (M), envelope (E), or nucleocapsid (N) protein, derived from SARS CoV (strain HKU39849, TW1, or FFM-1). As SARS-CoV is thought to infect the alveolar epithelial cell of the lung,in the present study, a type II alveolar epithelial cell clone, T7, was used to analyze the mechanism of CTL against SARS CoV membrane antigens. Mice vaccinated with SARS CoV (N) DNA or (M) DNA using pcDNA 3.1 (+) plasmid vector showed T-cell immune responses (CTL induction and proliferation) against type II alveolar epithelial cells (T7) transfected with SARS (N) or (M) DNA, respectively. To determine whether these DNA vaccines could induce T-cell immune responses in humans as well as in mice, SCID-PBL/hu mice were immunized with these DNA vaccines. PBL from healthy human volunteers were administered i.p. into IL-2 receptor gamma-chain-disrupted NOD-SCID mice [IL-2R(-/-) NOD-SCID]. SCID-PBL/hu mice thus constructed can be used to analyze the human immune response in vivo. The SCID-PBL/hu mice were immunized with SARS (N) DNA or (M) DNA and analyzed for a human T-cell immune response. The M DNA vaccine enhanced CTL activity and proliferation in the presence of M peptide in SCID-PBL/hu mice. Furthermore, the SARS N DNA vaccine induced CTL activity (IFN-gamma production by recombinant N protein or N protein-pulsed autologous B blast cells) and proliferation of spleen cells in SCID-PBL/hu mice. These results, demonstrate that SARS M and N DNA vaccines induced human CTL and human T-cell proliferative responses. On the other hand, we have developed SARS DNA vaccines that induce human neutralizing antibodies and human monoclonal antibodies against SARS CoV. Transgenic mice expressing SARS-CoV receptor (angiotensin converting enzyme 2) are also under development. These vaccines are expected to induce immune responses specific for SARS CoV in human and should provide useful tool for development of protective vaccines.

Animals↗
PubMed2006

IkappaBNS inhibits induction of a subset of Toll-like receptor-dependent genes and limits inflammation.

Toll-like receptor (TLR)-mediated immune responses are downregulated by several mechanisms that affect signaling pathways. However, it remains elusive how TLR-mediated gene expression is differentially modulated. Here, we show that IkappaBNS, a TLR-inducible nuclear IkappaB protein, negatively regulates induction of a subset of TLR-dependent genes through inhibition of NF-kappaB activity. IkappaBNS-deficient macrophages and dendritic cells show increased TLR-mediated expression of genes such as IL-6 and IL-12p40, which are induced late after TLR stimulation. In contrast, IkappaBNS-deficient cells showed normal induction of genes that are induced early or induced via IRF-3 activation. LPS stimulation of IkappaBNS-deficient macrophages prolonged NF-kappaB activity at the specific promoters, indicating that IkappaBNS mediates termination of NF-kappaB activity at selective gene promoters. Moreover, IkappaBNS-deficient mice are highly susceptible to LPS-induced endotoxin shock and intestinal inflammation. Thus, IkappaBNS regulates inflammatory responses by inhibiting the induction of a subset of TLR-dependent genes through modulation of NF-kappaB activity.

Animals↗
PubMed2006

Differential modulatory effects of annexin 1 on nitric oxide synthase induction by lipopolysaccharide in macrophages.

Annexin-1 (ANXA1) is a glucocorticoid-regulated protein that modulates the effects of bacterial lipopolysaccharide (LPS) on macrophages. Exogenous administration of peptides derived from the N-terminus of ANXA1 reduces LPS-stimulated inducible nitric oxide synthase (iNOS) expression, but the effects of altering the endogenous expression of this protein are unclear. We transfected RAW264.7 murine macrophage-like cell lines to over-express constitutively ANXA1 and investigated whether this protein modulates the induction of iNOS, cyclooxygenase-2 (COX-2) and tumour necrosis factor-alpha (TNF-alpha) in response to LPS. In contrast to exogenous administration of N-terminal peptides, endogenous over-expression of ANXA1 results in up-regulation of LPS-induced iNOS protein expression and activity. However, levels of iNOS mRNA are unchanged. ANXA1 has no effect on COX-2 or TNF-alpha production in response to LPS. In experiments to investigate the mechanisms underlying these phenomena we observed that activation of signalling proteins classically associated with iNOS transcription was unaffected. Over-expression of ANXA1 constitutively activates extracellular signal regulated kinase (ERK)-1 and ERK-2, components of a signalling pathway not previously recognized as regulating LPS-induced iNOS expression. Inhibition of ERK activity, by the inhibitor U0126, reduced LPS-induced iNOS expression in our cell lines. Over-expression of ANXA1 also modified LPS-induced phosphorylation of the ERK-regulated translational regulation factor eukaryotic initiation factor 4E. Our data suggest that ANXA1 may modify iNOS levels by post-transcriptional mechanisms. Thus differential effects on iNOS expression in macrophages are seen when comparing acute administration of ANXA1 peptides versus the chronic endogenous over-expression of ANXA1.

Animals↗
PubMed2006

OPC-67683, a nitro-dihydro-imidazooxazole derivative with promising action against tuberculosis in vitro and in mice.

BACKGROUND: Tuberculosis (TB) is still a leading cause of death worldwide. Almost a third of the world's population is infected with TB bacilli, and each year approximately 8 million people develop active TB and 2 million die as a result. Today's TB treatment, which dates back to the 1970s, is long and burdensome, requiring at least 6 mo of multidrug chemotherapy. The situation is further compounded by the emergence of multidrug-resistant TB (MDR-TB) and by the infection's lethal synergy with HIV/AIDS. Global health and philanthropic organizations are now pleading for new drug interventions that can address these unmet needs in TB treatment. METHODS AND FINDINGS: Here we report OPC-67683, a nitro-dihydro-imidazooxazole derivative that was screened to help combat the unmet needs in TB treatment. The compound is a mycolic acid biosynthesis inhibitor found to be free of mutagenicity and to possess highly potent activity against TB, including MDR-TB, as shown by its exceptionally low minimum inhibitory concentration (MIC) range of 0.006-0.024 microg/ml in vitro and highly effective therapeutic activity at low doses in vivo. Additionally, the results of the post-antibiotic effect of OPC-67683 on intracellular Mycobacterium tuberculosis showed the agent to be highly and dose-dependently active also against intracellular M. tuberculosis H37Rv after a 4-h pulsed exposure, and this activity at a concentration of 0.1 microg/ml was similar to that of the first-line drug rifampicin (RFP) at a concentration of 3 microg/ml. The combination of OPC-67683 with RFP and pyrazinamide (PZA) exhibited a remarkably quicker eradication (by at least 2 mo) of viable TB bacilli in the lung in comparison with the standard regimen consisting of RFP, isoniazid (INH), ethambutol (EB), and PZA. Furthermore, OPC-67683 was not affected by nor did it affect the activity of liver microsome enzymes, suggesting the possibility for OPC-67683 to be used in combination with drugs, including anti-retrovirals, that induce or are metabolized by cytochrome P450 enzymes. CONCLUSIONS: We concluded that based on these properties OPC-67683 has the potential to be used as a TB drug to help combat the unmet needs in TB treatment.

Animals↗
PubMed2005

DNA augments antigenicity of mycobacterial DNA-binding protein 1 and confers protection against Mycobacterium tuberculosis infection in mice.

Mycobacterium consists up to 7% of mycobacterial DNA-binding protein 1 (MDP1) in total cellular proteins. Host immune responses to MDP1 were studied in mice to explore the antigenic properties of this protein. Anti-MDP1 IgG was produced after infection with either bacillus Calmette-Guérin or Mycobacterium tuberculosis in C3H/HeJ mice. However, the level of Ab was remarkably low when purified MDP1 was injected. MDP1 is considered to be associated with DNA in nucleoid, which contains immunostimulatory CpG motif. Therefore, we examined coadministration of MDP1 and DNA derived from M. tuberculosis. Consequently, this procedure significantly enhanced the production of MDP1-specific IgG. Five nanograms of DNA was enough to enhance MDP1-specific IgG production in the administration of 5 microg of MDP1 into mice. Strong immune stimulation by such a small amount of DNA is noteworthy, because >1,000- to 100,000-fold doses of CpG DNAs are used for immune activation. A synthetic peptide-based study showed that B cell epitopes were different between mice administered MDP1 alone and those given a mixture of MDP1 and DNA, suggesting that DNA alters the three-dimensional structure of MDP1. Coadministration of DNA also enhanced MDP1-specific IFN-gamma production and reduced the bacterial burden of a following challenge of M. tuberculosis, showing that MDP1 is a novel vaccine target. Finally, we found that MDP1 remarkably enhanced TLR9-dependent immune stimulation by unmethylated CpG oligo DNA in vitro. To our knowledge, MDP1 is the first protein discovered that remarkably augments the CpG-mediated immune response and is a potential adjuvant for CpG DNA-based immune therapies.

Animals↗
PubMed2005

The development of vaccines against SARS corona virus in mice and SCID-PBL/hu mice.

We have investigated to develop novel vaccines against SARS CoV using cDNA constructs encoding the structural antigen; spike protein (S), membrane protein (M), envelope protein (E), or nucleocapsid (N) protein, derived from SARS CoV. Mice vaccinated with SARS-N or -M DNA using pcDNA 3.1(+) plasmid vector showed T cell immune responses (CTL induction and proliferation) against N or M protein, respectively. CTL responses were also detected to SARS DNA-transfected type II alveolar epithelial cells (T7 cell clone), which are thought to be initial target cells for SARS virus infection in human. To determine whether these DNA vaccines could induce T cell immune responses in humans as well as in mice, SCID-PBL/hu mice was immunized with these DNA vaccines. As expected, virus-specific CTL responses and T cell proliferation were induced from human T cells. SARS-N and SARS-M DNA vaccines and SCID-PBL/hu mouse model will be important in the development of protective vaccines.

Animals↗
PubMed2005

Novel recombinant BCG and DNA-vaccination against tuberculosis in a cynomolgus monkey model.

We have developed two novel tuberculosis (TB) vaccines: a DNA vaccine combination expressing mycobacterial heat shock protein 65 (Hsp65) and interleukin-12 (IL-12) by using the hemagglutinating virus of Japan (HVJ)-liposome (HSP65+IL-12/HVJ) and a recombinant BCG harboring the 72f fusion gene (72f rBCG). These vaccines provide remarkable protective efficacy in mouse and guinea pig models, as compared to the current by available BCG vaccine. In the present study, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis, to evaluate the HSP65+IL-12/HVJ and 72f rBCG vaccines. Vaccination with HSP65+IL-12/HVJ as well as 72f rBCG vaccines provided better protective efficacy as assessed by the Erythrocyte Sedimentation Rate, chest X-ray findings and immune responses than BCG. Most importantly, HSP65+IL-12/HVJ resulted in an increased survival for over a year. This is the first report of successful DNA vaccination and recombinant BCG vaccination against M. tuberculosis in the monkey model.

Animals↗
PubMed2005

The nuclear IkappaB protein IkappaBNS selectively inhibits lipopolysaccharide-induced IL-6 production in macrophages of the colonic lamina propria.

Macrophages play an important role in the pathogenesis of chronic colitis. However, it remains unknown how macrophages residing in the colonic lamina propria are regulated. We characterized colonic lamina proprial CD11b-positive cells (CLPMphi). CLPMphi of wild-type mice, but not IL-10-deficient mice, displayed hyporesponsiveness to TLR stimulation in terms of cytokine production and costimulatory molecule expression. We compared CLPMphi gene expression profiles of wild-type mice with IL-10-deficient mice, and identified genes that are selectively expressed in wild-type CLPMphi. These genes included nuclear IkappaB proteins such as Bcl-3 and IkappaBNS. Because Bcl-3 has been shown to specifically inhibit LPS-induced TNF-alpha production, we analyzed the role of IkappaBNS in macrophages. Lentiviral introduction of IkappaBNS resulted in impaired LPS-induced IL-6 production, but not TNF-alpha production in the murine macrophage cell line RAW264.7. IkappaBNS expression led to constitutive and intense DNA binding of NF-kappaB p50/p50 homodimers. IkappaBNS was recruited to the IL-6 promoter, but not to the TNF-alpha promoter, together with p50. Furthermore, small interference RNA-mediated reduction in IkappaBNS expression in RAW264.7 cells resulted in increased LPS-induced production of IL-6, but not TNF-alpha. Thus, IkappaBNS selectively suppresses LPS-induced IL-6 production in macrophages. This study established that nuclear IkappaB proteins differentially regulate LPS-induced inflammatory cytokine production in macrophages.

Animals↗
PubMed2004

Extracellular mycobacterial DNA-binding protein 1 participates in mycobacterium-lung epithelial cell interaction through hyaluronic acid.

Mycobacterium tuberculosis infects not only host macrophages but also nonprofessional phagocytes, such as alveolar epithelial cells. Glycosaminoglycans (GAGs) are considered as the component of mycobacterial adherence to epithelial cells. Here we show that extracellularly occurring mycobacterial DNA-binding protein 1 (MDP1) promotes mycobacterial infection to A549 human lung epithelial cells through hyaluronic acid (HA). Both surface plasmon resonance analysis and enzyme-linked immunosorbent assay revealed that MDP1 bound to HA, heparin, and chondroitin sulfate. Utilizing synthetic peptides, we next defined heparin-binding site of 20 amino acids from 31 to 50 of MDP1, which is responsible for the specific DNA-binding site of MDP1. MDP1 bound to A549 cells, and exogenous DNA and HA interfered with the interaction. The binding was also abolished by treatment of A549 cells with hyaluronidase, suggesting that HA participates in the MDP1-A549 cell interaction. Adherence of bacillus Calmette-Guérin (BCG) and M. tuberculosis to A549 cells was inhibited by addition of HA, DNA, and anti-MDP1 antibody, showing that MDP1 participates in the interaction between mycobacteria-alveolar epithelial cells. Simultaneous treatment of intratracheal BCG-infected mice with HA reduced the growth of BCG in vivo. Taken together, theses results suggest that HA participates in Mycobacterium-lung epithelium interaction and has potential for therapeutic and prophylactic interventions in mycobacterial infection.

Amino Acid Sequence↗
PubMed2004

Simvastatin augments lipopolysaccharide-induced proinflammatory responses in macrophages by differential regulation of the c-Fos and c-Jun transcription factors.

The 3-hydroxyl-3-methylglutaryl-coenzyme A reductase inhibitors, or statins, are a widely used class of drugs for cholesterol reduction. The reduction in mortality and morbidity in statin-treated patients is incompletely explained by their effects on cholesterol, and an anti-inflammatory role for the drug has been proposed. We report in this work that, unexpectedly, simvastatin enhances LPS-induced IL-12p40 production by murine macrophages, and that it does so by activating the IL-12p40 promoter. Mutational analysis and dominant-negative expression studies indicate that both C/EBP and AP-1 transcription factors have a crucial role in promoter activation. This occurs via a c-Fos- and c-Jun-based mechanism; we demonstrate that ectopic expression of c-Jun activates the IL-12p40 promoter, whereas expression of c-Fos inhibits IL-12p40 promoter activity. Simvastatin prevents LPS-induced c-Fos expression, thereby relieving the inhibitory effect of c-Fos on the IL-12p40 promoter. Concomitantly, simvastatin induces the phosphorylation of c-Jun by the c-Jun N-terminal kinase, resulting in c-Jun-dependent activation of the IL-12p40 promoter. This appears to be a general mechanism because simvastatin also augments LPS-dependent activation of the TNF-alpha promoter, perhaps because the TNF-alpha promoter has C/EBP and AP-1 binding sites in a similar configuration to the IL-12p40 promoter. The fact that simvastatin potently augments LPS-induced IL-12p40 and TNF-alpha production has implications for the treatment of bacterial infections in statin-treated patients.

Adjuvants, Immunologic↗
PubMed2002

Lipopolysaccharide-dependent prostaglandin E(2) production is regulated by the glutathione-dependent prostaglandin E(2) synthase gene induced by the Toll-like receptor 4/MyD88/NF-IL6 pathway.

Macrophages produce a large amount of PGE(2) during inflammation. This lipid mediator modulates various immune responses. PGE(2) acts on macrophages and inhibits production of cytokines such as TNF-alpha and IL-12. Membrane-bound glutathione-dependent PGE(2) synthase (mPGES) has been shown to be a terminal enzyme of the cyclooxygenase-2-mediated PGE(2) biosynthesis. Here we identified mPGES as a molecule that is induced by LPS in macrophages. The expression of mPGES was not induced by LPS in mice lacking Toll-like receptor 4 or MyD88. Furthermore, mice deficient in NF-IL6 showed neither induction of mPGES nor biosynthesis of PGE(2) in response to LPS, indicating that mPGES expression in response to LPS is regulated by a Toll-like receptor 4/MyD88/NF-IL6-dependent signaling pathway. We generated mPGES-deficient mice and investigated the role of mPGES in vivo. The mice showed no augmentation of the PGE(2) production in response to LPS. However, they were not impaired in the LPS-induced production of inflammatory cytokines and showed normal response to the LPS-induced shock. Thus, mPGES is critically involved in the biosynthesis of PGE(2) induced by LPS, but is dispensable for the modulation of inflammatory responses.

Adaptor Proteins, Signal Transducing↗