Hypascyrins A-E, Prenylated Acylphloroglucinols from Hypericum ascyron.

Six new prenylated acylphloroglucinols with menthane moieties, hypascyrins A-E (1-5) and ent-hyphenrone J (6), together with four known analogues, were isolated from Hypericum ascyron roots. Detailed spectroscopic data analyses resulted in the assignment of their structures. The absolute configuration of 1 was deduced by experimental and calculated ECD data, while those of 2-6 were assigned by ECD data analyses as well as chemical conversions. Hypascyrins A (1), C (3), and E (5) and ent-hyphenrone J (6) exhibited antimicrobial activity against MRSA (MIC50 values of 4.0, 8.0, 2.0, and 4.0 µM, respectively) and Bacillus subtilis (MIC values of 4.0, 4.0, 2.0, and 4.0 µM, respectively).

Inhibition of tumor formation and metastasis by a monoclonal antibody against lymphatic vessel endothelial hyaluronan receptor 1.

Although cancer metastasis is associated with poor prognosis, the mechanisms of this event, especially via lymphatic vessels, remain unclear. Lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1) is expressed on lymphatic vessel endothelium and is considered to be a specific marker of lymphatic vessels, but it is unknown how LYVE-1 is involved in the growth and metastasis of cancer cells. We produced rat monoclonal antibodies (mAb) recognizing the extracellular domain of mouse LYVE-1, and investigated the roles of LYVE-1 in tumor formation and metastasis. The mAb 38M and 64R were selected from hybridoma clones created by cell fusion between spleen cells of rats immunized with RH7777 rat hepatoma cells expressing green fluorescent protein (GFP)-fused mouse LYVE-1 proteins and mouse myeloma cells. Two mAb reacted with RH7777 and HEK293F human embryonic kidney cells expressing GFP-fused mouse LYVE-1 proteins in a GFP expression-dependent manner, and each recognized a distinct epitope. On immunohistology, the 38M mAb specifically stained lymphatic vessels in several mouse tissues. In the wound healing assay, the 64R mAb inhibited cell migration of HEK293F cells expressing LYVE-1 and mouse lymphatic endothelial cells (LEC), as well as tube formation by LEC. Furthermore, this mAb inhibited primary tumor formation and metastasis to lymph nodes in metastatic MDA-MB-231 xenograft models. This shows that LYVE-1 is involved in primary tumor formation and metastasis, and it may be a promising molecular target for cancer therapy.

[M1 AND M2 MACROPHAGE POPULATIONS: THOSE INDUCED AND ACTIVATED BY MYCOBACTERIAL INFECTIONS].

In the advanced stages of mycobacterial infections, host immune systems tend to change from a Th1-type to Th2-type immune response, resulting in the abrogation of Th1 cell- and macrophage-mediated antimicrobial host protective immunity. Notably, this type of immune conversion is occasionally associated with the generation of. certain types of suppressor macrophage populations. During the course of infections due to pathogenic mycobacteria, the generation of macrophages which possess strong suppressor activity against host T- and B-cell functions is frequently encountered. This review describes the immunological properties of M1- and M2-type macrophages generated in hosts with certain microbial infections including mycobacteriosis and those generated in tumor-bearing animals. Particularly, this paper highlights the immunological and molecular biological characteristics of M1 and M2 macrophage populations, which are induced by mycobacterial infections

Roles of autophagy in killing of mycobacterial pathogens by host macrophages - Effects of some medicinal plants.

Autophagy is a cellular stress-induced intracellular process, through which damaged cellular components are decomposed via lysosomal degradation. This process plays important roles in host innate immunity, particularly the elimination of intracellular pathogens inside host macrophages. A more detailed understanding of the roles of autophagic events in the effective manifestation of macrophagic antimycobacterial activity is needed. Furthermore, the effects of medicinal plants on macrophagic autophagy response to mycobacterial infection need to be clarified. We herein examined the significance of autophagic events in the manifestation of host immunity during mycobacterial infection, by performing a literature search using PubMed. Recent studies demonstrated that autophagy up-regulated macrophage functions related to the intracellular killing of mycobacteria, even when pathogens were residing within the cytoplasm of macrophages. The majority of medicinal plants potentiated macrophagic autophagy, thereby enhancing their antimycobacterial functions. In contrast, most medicinal plants down-regulate the development and activation of the Th17 cell population, which reduces macrophage antimycobacterial activity. These opposing effects of medicinal plants on macrophage autophagy (enhancement) and Th17 cell functions (inhibition) may provide a plausible explanation for the clinical observation of their modest efficacy in the treatment of mycobacterial infections.

Characteristics of suppressor macrophages induced by mycobacterial and protozoal infections in relation to alternatively activated M2 macrophages.

In the advanced stages of mycobacterial infections, host immune systems tend to change from a Th1-type to Th2-type immune response, resulting in the abrogation of Th1 cell- and macrophage-mediated antimicrobial host protective immunity. Notably, this type of immune conversion is occasionally associated with the generation of certain types of suppressor macrophage populations. During the course of Mycobacterium tuberculosis (MTB) and Mycobacterium avium-intracellulare complex (MAC) infections, the generation of macrophages which possess strong suppressor activity against host T- and B-cell functions is frequently encountered. This paper describes the immunological properties of M1- and M2-type macrophages generated in tumor-bearing animals and those generated in hosts with certain microbial infections. In addition, this paper highlights the immunological and molecular biological characteristics of suppressor macrophages generated in hosts with mycobacterial infections, especially MAC infection.

[Phagocytosis of Mycobacterium leprae down-regulates anti-microbial activity of murine macrophages against Mycobacterium intracellulare].

Patients with highly bacillated lepromatous leprosy (LL) essentially lack T cell-mediated immune responses specific to Mycobacterium leprae (ML) antigens, resulting in severely impaired host resistance to leprosy bacilli. Such type of immune unresponsiveness characteristic of LL patients is mainly attributable to markedly depressed T cell ability to activate/expand in response to ML antigens. In this study, we examined profiles of antimycobacterial activity of macrophages, which phagocytized leprosy bacilli, because there is another possibility that, in LL patients, host macrophages in the leprosy lesions are impaired in their antimicrobial activity due to their interaction with infected leprosy bacilli, particularly cellular events through binding with and/or internalization of the pathogens, thereby causing the reduction in host resistance to ML pathogens. The present study indicated the following. First, the anti-M. avium complex activity of murine peritoneal macrophages was significantly reduced when they had phagocytosed heat-killed leprosy bacilli. Second, infection of macrophages with leprosy bacilli did not affect macrophage-mediated suppressor activity against T cell proliferative response to Concanavalin A. These findings indicate that macrophage's intracellular signaling pathways that are up-regulated in response to phagocytosis of leprosy bacilli are linked to the signaling cascades participating in macrophage antimicrobial functions, but not cross-talk with those allowing the expression of macrophage's suppressor activity against T cell functions.

Development of new antituberculous drugs based on bacterial virulence factors interfering with host cytokine networks.

The worldwide increase in the prevalence of tuberculosis (TB), especially multidrug-resistant TB and extensively drug-resistant TB, is an important global health concern, and new effective drugs are urgently needed. Information on the genome of Mycobacterium tuberculosis (MTB) and various mycobacterial virulence genes is leading to the identification of genes that code for new drug targets. Mycobacterium tuberculosis (MTB) is resistant to the antimicrobial mechanisms of host macrophages and can survive and replicate in macrophages for long periods, resulting in a persistent infection. Mycobacterial virulence factors suppress macrophage bactericidal functions partly via their downregulatory effects on the host antimicrobial cytokine networks, consisting of proinflammatory, immunopotentiating, and Th1-inducing cytokines. Thus, for the development of unique drugs that exhibit antimycobacterial action through novel mechanisms, it is reasonable to search for targets among bacterial genes encoding virulence factors which interfere with the host cytokine responses protective to mycobacterial pathogens. In this review, we discuss the profiles of cytokine networks related to host resistance to mycobacteria, including the mechanisms of downregulation of host antimycobacterial immunity due to immunosuppressive cytokines, which are occasionally induced in the advanced stages of TB. We also highlight the development of antituberculous drugs based on bacterial virulence factors interfering with the host antimycobacterial cytokine network.

Immunoadjunctive Therapy against Bacterial Infections Using Herbal Medicines Based on Th17 Cell-mediated Protective Immunity.

One of the major health concerns in the world is the global increase in intractable bacterial infectious diseases due to the emergence of multi- and extensively drug-resistant bacterial pathogens as well as increase in compromised hosts around the world. Particularly, in the case of mycobacteriosis, the high incidence of tuberculosis in developing countries, resurgence of tuberculosis in industrialized countries, and increase in the prevalence of Mycobacterium avium complex infections are important worldwide health concerns. However, the development of novel antimycobacterial drugs is currently making slow progress. Therefore, it is considered that devising improved administration protocols for clinical treatment against refractory mycobacteriosis using existing chemotherapeutics is more practical than awaiting the development of new antimycobacterial drugs. The regulation of host immune responses using immunoadjunctive agents may increase the efficacy of antimicrobial treatment against mycobacteriosis. The same situations also exist in cases of intractable infectious diseases due to common bacteria other than mycobacteria. The mild and long-term up-regulation of host immune reactions in hosts with intractable chronic bacterial infections, using herbal medicines and medicinal plants, may be beneficial for such immunoadjunctive therapy. This review describes the current status regarding basic and clinical studies on therapeutic regimens using herbal medicines, useful for the clinical treatment of patients with intractable bacterial infections. In particular, we focus on immunoadjunctive effects of herbal medicines on the establishment and manifestation of host antibacterial immunity related to the immunological roles of Th17 cell lineages.

Properties of immunosuppressive macrophages generated by Mycobacterium intracellulare infection in M. intracellulare-susceptible and resistant mice.

Splenic macrophages (M(phi)s) generated in Mycobacterium intracellulare (Min)-infected mice exhibit suppressor activity against T cell mitogenesis. We examined profiles of the Min-induced generation of immunosuppressive M(phi)s (Min-M(phi)s) in Min-susceptible BALB/c (bcg(s)) and resistant CBA/JN (bcg(r)) mice. Min infection in BALB/c mice caused a more rapid generation of the immunosuppressive M(phi)s, which expressed M(phi) markers such as CD11b and F4/80, exhibited an increased ability to generate reactive oxygen intermediates, and inhibited IL-2 receptor expression by mitogenic T cells, than did Min infection in CBA/JN mice. Thus, the bcg gene may be related to the generation of Min-M(phi)s in host mice.

Clinical and Basic Studies on Therapeutic Efficacy of Herbal Medicines against Mycobacterial Infections.

The high incidence of tuberculosis (TB) in developing countries, the resurgence of TB in industrialized countries, and the worldwide increase in the prevalence of Mycobacterium avium complex infections are important global health concerns. However, the development of novel antimycobacterial drugs is currently making very slow progress. Therefore, it is considered that devising improved administration protocols for clinical treatment against intractable mycobacteriosis using existing chemotherapeutics is more practical than awaiting the development of new antimycobacterial drugs. The regulation of host immune responses using immunoadjunctive agents may increase the efficacy of antimicrobial treatment against mycobacteriosis. In particular, the mild and long-term up-regulation of host immune reactions against mycobacterial pathogens using herbal medicines may be beneficial for such immunoadjunctive therapy. This review focuses on the current status regarding basic and clinical studies on protocols using herbal medicines, including medicinal plants, useful for the clinical treatment of intractable mycobacterial infections.

Tropoelastin regulates chemokine expression in fibroblasts in Costello syndrome.

Costello syndrome is a multiple congenital anomaly associated with growth and mental retardation, cardiac and skeletal anomalies, and a predisposition to develop neoplasia. Comprehensive expression analysis revealed remarkable up-regulation of several cytokines and chemokines including Gro family proteins, interleukin-1beta (IL-1beta), IL-8 and MCP-1 but down-regulation of extracellular matrix components including collagens and proteoglycans of skin fibroblasts derived from a Japanese Costello syndrome patient characterized by significantly reduced tropoelastin mRNA, impaired elastogenesis and enhanced cell proliferation. In contrast, decreases in these chemokines and IL-1beta expression were observed in Costello fibroblastic cell lines stably expressing the bovine tropoelastin (btEln) gene and in restored elastic fibers. These results strongly suggest that the human TE gene (ELN) transfer could be applicable for the gene therapy of a group of Costello syndrome patients with reduced ELN gene expression.

Identification of Novel Mycobacterial Inhibitors Against Mycobacterial Protein Kinase G.

Protein kinase G (PknG) is a eukaryotic-like serine/threonine kinase that is expressed by Mycobacterium tuberculosis and promotes survival of mycobacteria in host macrophages by suppressing phagosome-lysosome fusion. Thus, compounds showing inhibitory activity against PknG are promising anti-mycobacterial agents. We therefore aimed to develop anti-mycobacterial agents by identifying new PknG inhibitors. A luciferase-based PknG kinase assay was used to screen potential inhibitors of PknG. We found that four compounds, namely AZD7762, R406, R406-free base, and CYC116, inhibited PknG activities. AZD7762, R406, and R406-free base promoted transfer of mycobacteria to lysosomes. These compounds also inhibited survival of M. bovis Bacillus Calmette-Guérin (BCG) inside human macrophages. Furthermore, R406 and R406-free base showed bactericidal activity against BCG in infected human macrophages without cytotoxicity. The PknG inhibitors identified in this study by the luciferase-based PknG kinase assay may be promising leads for the development of anti-mycobacterial agents.

Effects of picolinic acid on the antimicrobial functions of host macrophages against Mycobacterium avium complex.

Picolinic acid (PA) potentiates macrophage (MPhi) antimicrobial activity against intracellular Mycobacterium avium complex (MAC). Here, we studied the mechanisms of this phenomenon using human THP-1 MPhis. First, when PA-treated MAC-infected MPhis were cultured in the presence or absence of reactive oxygen intermediate (ROI) scavengers, nitric oxide synthase (NOS) inhibitors or phospholipase A(2) (PLA(2)) inhibitors, none of these agents blocked the activity of PA in potentiating MPhi anti-MAC activity. Second, when PA was added to the in vitro anti-MAC bactericidal system consisting of either ROIs, reactive nitrogen intermediates (RNIs) or free fatty acid (FFA) molecules, which are the major MPhi antimicrobial effectors, PA inhibited the activity of ROIs and conversely potentiated the activity of RNIs; PA did not affect the activity of FFAs. Third, PA reduced mRNA expression of NADPH oxidase and beta-defensin-1 by MAC-infected MPhis, whilst neither cytosolic PLA(2) nor CAP37 mRNA expression was affected. Notably, inducible NOS and secretory PLA(2) mRNA expression was not detected for MAC-infected MPhis even when given PA treatment. These findings suggest that ROIs, RNIs, FFAs and beta-defensin-1 do not play important roles in the PA-induced potentiation of MPhi anti-MAC activity.

[Profiles of intracellular expression, distribution, and activation of phospholipase A2 in host macrophages infected with Mycobacterium avium complex].

Our recent studies have shown that type IV cytosolic phospholipase A2 (cPLA2) plays an important role in the expression of macrophage (MPh) antimicrobial activity against the Mycobacterium avium complex (MAC). To clarify the modes of cPLA2 participation in MPh anti-MAC antimicrobial function in detail, we studied intracellular profiles of phospholilase A2, focusing on cPLA2, in MAC-infected MPhs, with the following findings: In murine peritoneal MPhs, cPLA2 was constitutively expressed even in uninfected MPhs, and MAC infection did not increase intramacrophage cPLA2 expression. In an RAW264.7 mouse MPh cell line (RAW264.7 MPhs) infected with MAC, a portion of intracellular cPLA2 was concentrated in MAC organisms residing within MPh phagosomes. MAC infection upregulated intramacrophage cPLA2 expression and induced its phosphorylation. These findings suggest that MAC infection of RAW264.7 MPhs may induce the activation of intracellular cPLA2, translocating it to phagosomes engulfing infected MAC organisms.

Host-Directed Therapeutics against Mycobacterial Infections.

The high incidence of tuberculosis (TB) in the world, especially in developing countries, the resurgence of TB in industrialized countries, and the global increase in the prevalence of Mycobacterium avium complex infections in immunocompromised hosts have prompted the quest for novel antimycobacterial drugs. However, the development of such antimicrobial chemotherapeutics is currently making very slow progress even with using the bioinformatics-based methodology for drug design. It thus appears that devising improved administration protocols for clinical treatment against intractable mycobacterial infections using existing chemotherapeutics is more practical than awaiting the development of new antimycobacterial drugs. The potentiation of host immune responses using immunoadjunctive agents, alternatively called host-directed therapeutics (HDTs), may increase the efficacy of antimycobacterial regimens against mycobacteriosis. Particularly, the modulation of host immunity relating to macrophage antimicrobial functions may be beneficial to the immunoadjunctive therapy. This review will deal with the current status and future prospects regarding the development of HDTs useful for the clinical control of intractable mycobacterial infections.

Anti-Mycobacterium avium complex activity of clarithromycin, rifampin, rifabutin, and ethambutol in combination with adenosine 5'-triphosphate.

We previously reported that adenosine 5'-triphosphate (ATP) inhibited the growth of various bacteria, including mycobacteria, Staphylococcus, and Pseudomonas, without damaging bacterial surface structures. Notably, ATP's antibacterial activity was found to be attributable to its iron-chelating ability. ATP exhibited combined effects with some antimicrobials against Mycobacterium intracellulare and methicillin-resistant S. aureus, suggesting its usefulness as an adjunctive drug in the chemotherapy against certain intractable infections. In this study, we examined detailed profiles of the anti-Mycobacterium avium complex (MAC) activity of some antimicrobial agents, including clarithromycin (CLA), rifampin (RIF), rifabutin (RBT), and ethambutol (EMB), in combination with ATP. It was found that the anti-MAC activity of CLA+RIF, CLA+RBT, and CLA+EMB was markedly potentiated in a strain-dependent manner. In this case, the onset of the regrowth of antimicrobial agent-treated mycobacteria during cultivation was significantly delayed in the presence of ATP, indicating the usefulness of ATP as an adjunctive drug in chemotherapy against MAC infections.

Proteomic alteration in gastic adenocarcinomas from Japanese patients.

BACKGROUND: Gastric adenocarcinomas comprise one of the common types of cancers in Asian countries including Japan. Comprehensive protein profiling of paired surgical specimens of primary gastric adenocarcinomas and nontumor mucosae derived from Japanese patients was carried out by means of two-dimensional gel electrophoresis (2D-EP) and liquid chromatography-electrospray ionic tandem mass spectrometry (LC-ESI-MS) to establish gastric cancer-specific proteins as putative clinical biomarkers and molecular targets for chemotherapy. RESULTS: Relatively common alterations in protein expression were revealed in the tumor tissues. Increases in manganese dismutase and nonhistone chromosomal protein HMG-1 (HMG-1) were observed, while decreases in carbonic anhydrases I and II, glutatione-S-transferase and foveolin precursor (gastrokine-1) (FOV), an 18-kDa stomach-specific protein with putative tumor suppressor activity, were detected. RT-PCR analysis also revealed significant down-regulation of FOV mRNA expression in tumor tissues. CONCLUSION: A possible pathological role for down-regulation of FOV in gastric carcinogenesis was demonstrated. Evaluation of the specific decreases in gene and protein expression of FOV in patients may be utilized as clinical biomarkers for effective diagnosis and assessment of gastric cancer.

Significant decrease in tropoelastin gene expression in fibroblasts from a Japanese Costello syndrome patient with impaired elastogenesis and enhanced proliferation.

Costello syndrome is a connective tissue disorder associated with sparse, thin, and fragmented elastic fibers in tissues. In this study we demonstrated a significant decrease in the expression of tropoelastin mRNA in fibroblasts derived from a Japanese Costello syndrome patient with impaired elastogenesis and enhanced proliferation. In contrast, there were no changes in expression of the Harvey ras (HRAS), fibrillin-1, fibulin-5, microfibril-associated glycoprotein-1 (MAGP-1), lysyl oxidase (LOX), or 67-kDa non-integrin elastin-binding protein (EBP) gene. The proliferative activity of the Costello fibroblasts was about 4-fold higher than that of the normal and pathological control ones. However, no mutations were detected in the coding region of HRAS mRNA. Transduction of the bovine tropoelastin (bTE) gene with the lentiviral vector restored the elastic fiber formation and decreased the growth rate in the Costello fibroblasts. These results strongly suggest that the defect of human tropoelastin (hTE) gene expression should induce the impaired elastogenesis and enhanced proliferation of Costello fibroblasts, and that a primary cause other than the HRAS gene mutation should contribute to the pathogenesis in the present Costello case.

Elastogenesis in cultured dermal fibroblasts from patients with lysosomal beta-galactosidase, protective protein/cathepsin A and neuraminidase-1 deficiencies.

The human GLB1 gene encodes a lysosomal beta-galactosidase (beta-Gal) and an elastin-binding protein (EBP). Defect of the EBP as a chaperon for tropoelastin and a component of receptor complex among neuraminidase-1 (NEU1) and protective protein/cathepsin A (PPCA) is suggested responsible for impaired elastogenesis in autosomal recessive beta-Gal, PPCA and NEU1 deficiencies. The purpose of this study is to determine effects of GLB1, PPCA and NEU1 gene mutations on elastogenesis in skin fibroblasts. Elastic fiber formation and the EBP mRNA expression were examined by immunofluorescence with an anti-tropoelastin antibody and RT-PCR selective for EBP in skin fibroblasts with these lysosomal enzyme deficiencies. Apparently normal elastogenesis and EBP mRNA expression were observed for fibroblasts from Morquio B disease cases with the GLB1 gene alleles (W273L/W273L, W273L/R482H and W273L/W509C substitutions, respectively), a galactosialidosis case with the PPCA allele (IVS7+3A/IVS7+3A) and a sialidosis case with the NEU1 allele (V217M/G243R) as well as normal subject. In this study, the W273L substitution in the EBP could impossibly cause the proposed defect of elastogenesis, and the typical PPCA splicing mutation and the V217M/G243R substitutions in the NEU1 might hardly have effects on elastic fiber formation in the dermal fibroblasts.