Effects of the acid polysaccharide fraction isolated from a cultivated Cordyceps sinensis on macrophages in vitro

The acid polysaccharide fraction (APSF) extracted from the mycelia of cultivated Cordyceps sinensis is water-soluble polysaccharide. In this study we evaluated the modulating effects of APSF on murine macrophage cell line RAW264.7. Phagocytotic assay by neutral red and FITC-dextran internalization showed that APSF stimulated the phagocytosis of macrophages. The nitrite levels in the culture supernatant determined using Griess reagent revealed the elevation of NO production after treatment with APSF. RT-PCR and immunocytochemistry assay indicated that APSF promoted both the mRNA and protein expressions of inducible nitric oxide synthase (iNOS). Furthermore, Western blotting demonstrated that NF-κB levels in nucleuses increased after APSF treatment, suggesting that APSF probably stimulated macrophage activities by activating the IκB-NF-κB pathway.     

A mushroom lectin from ascomycete Cordyceps militaris

A mushroom lectin has been purified from ascomycete Cordyceps militaris, which is one of the most popular mushrooms in eastern Asia used as a nutraceutical and in traditional Chinese medicine. This lectin, designated CML, exhibited hemagglutination activity in mouse and rat erythrocytes, but not in human ABO erythrocytes. SDS-PAGE of CML revealed a single band with a molecular mass of 31.0 kDa under both nonreducing and reducing conditions that was stained by silver nitrate, and a 31.4 kDa peak in a Superdex-200 HR gel-filtration column. The hemagglutination activity was inhibited by sialoglycoproteins, but not in by mono- or disaccharides, asialoglycoproteins, or de-O-acetylated glycoprotein. The activity was maximal at pH 6.0–9.1 and at temperatures below 50 °C. Circular dichroism spectrum analysis revealed that CML comprises 27% α-helix, 12% β-sheets, 29% β-turns, and 32% random coils. Its binding specificity and secondary structure are similar to those of a fungal lectin from Arthrobotrys oligospora. However, the N-terminal amino acid sequence of CML differs greatly from those of other lectins. CML exhibits mitogenic activity against mouse splenocytes.     

Deoxynucleic acids from Cryptococcus neoformans activate myeloid dendritic cells via a TLR9-dependent pathway

The mechanism of host cell recognition of Cryptococcus neoformans, an opportunistic fungal pathogen in immunocompromised patients, remains poorly understood. In the present study, we asked whether the DNA of this yeast activates mouse bone marrow-derived myeloid dendritic cells (BM-DCs). BM-DCs released IL-12p40 and expressed CD40 upon stimulation with cryptococcal DNA, and the response was abolished by treatment with DNase, but not with RNase. IL-12p40 production and CD40 expression were attenuated by chloroquine, bafilomycin A, and inhibitory oligodeoxynucleotides (ODN) that suppressed the responses caused by CpG-ODN. Activation of BM-DCs by cryptococcal DNA was almost completely abrogated in TLR9 gene-disrupted (TLR9(-/-)) mice and MyD88(-/-) mice, similar to that by CpG-ODN. In addition, upon stimulation with whole yeast cells of acapsular C. neoformans, TLR9(-/-) BM-DCs produced a lower amount of IL-12p40 than those from wild-type mice, and TLR9(-/-) mice were more susceptible to pulmonary infection with this fungal pathogen than wild-type mice, as shown by increased number of live colonies in lungs. Treatment of cryptococcal DNA with methylase resulted in reduced IL-12p40 synthesis by BM-DCs. Furthermore, using a luciferase reporter assay, cryptococcal DNA activated NF-kappaB in HEK293 cells transfected with the TLR9 gene. Finally, confocal microscopy showed colocalization of fluorescence-labeled cryptococcal DNA with CpG-ODN and the findings merged in part with the distribution of TLR9 in BM-DCs. Our results demonstrate that cryptococcal DNA causes activation of BM-DCs in a TLR9-dependent manner and suggest that the CpG motif-containing DNA may contribute to the development of inflammatory responses after infection with C. neoformans.     

Polysaccharide purified from Polyporus umbellatus (Per) Fr induces the activation and maturation of murine bone-derived dendritic cells via toll-like receptor 4

In this study, we report that a polysaccharide isolated from a Chinese medicinal herb, Zhu Ling (the sclerotium of Polyporus umbellatus (Per) Fr), induces phenotypic and functional maturation of murine bone-derived dendritic cells (BMDCs). Treatment of BMDCs with Polyporus polysaccharide (PPS) resulted in enhanced cell-surface expression of CD86, as well as enhanced production of both interleukin (IL)-12 p40 and IL-10 in a dose-dependent manner. In addition, treatment of BMDCs with PPS resulted in increased T cell-stimulatory capacity and decreased phagocytic ability. PPS-induced production of IL-12 p40 was inhibited by monoclonal antibodies to Toll-like receptor 4 (TLR4). Flow cytometric analysis showed that fluorescence-labeled PPS (f-PPS) bound specifically to BMDCs. This binding was blocked by both unlabeled PPS and anti-TLR4, but not by anti-TLR2 and anti-CR3 monoclonal antibodies. Taken together, our data show that PPS promotes the activation and maturation of murine BMDCs via TLR4.     

CpG oligodeoxynucleotides induce strong up-regulation of interleukin 33 via Toll-like receptor 9

We previously reported the strong immunostimulatory effects of a CpG oligodeoxynucleotide (ODN), designated MsST, from the lacZ gene of Streptococcus (S.) thermophilus ATCC19258. Here we show that 24 h of stimulation with MsST in mouse splenocytes and peritoneal macrophages strongly induces expression of interleukin (IL)-33, a cytokine in the IL-1 superfamily. Other IL-1 superfamily members, including IL-1α, IL-1β and IL-18, are down-regulated after 24 h of stimulation of MsST. We also found that MsST-induced IL-33 mRNA expression is inhibited by the suppressive ODN A151, which can inhibit Toll-like receptor 9 (TLR9)-mediated responses. This is the first report to show that IL-33 can be induced by CpG ODNs. The strong induction of IL-33 by MsST suggests that it may be a potential therapeutic ODN for the treatment of inflammatory disease. The presence of a strong CpG ODN in S. thermophilus also suggests that the bacterium may be a good candidate as a starter culture for the development of new physiologically functional foods.     

Effects of the mermithid nematode Ovomermis sinensis on the hemocytes of its host Helicoverpa armigera

Little is known about the mechanism by which mermithid nematodes avoid encapsulation responses of insect hosts. In this study, we investigated the influence of the mermithid nematode Ovomermis sinensis on host Helicoverpa armigera hemocyte number, encapsulation activity, spreading behavior and cytoskeleton. Parasitism by O. sinensis caused a significant increase in the total hemocyte counts (THC) and plasmatocyte numbers of H. armigera. However, in vivo encapsulation assays revealed that hemocyte encapsulation abilities of H. armigera were suppressed by O. sinensis. Moreover, parasitism by O. sinensis changed the spreading behavior and cytoskeletons of the host hemocytes. The results suggested that O. sinensis could actively suppress the hemocyte immune response of its host, possibly by destroying the host hemocyte cytoskeleton. This is the first report of a possible mechanism by which mermithid nematodes suppress encapsulation responses of insect hosts.     

Leishmania mexicana: Inhibition of camptothecin-induced apoptosis of monocyte-derived dendritic cells

Macrophages (M?) and dendritic cells (DC) are the major target cell populations of the obligate intracellular parasite Leishmania. Inhibition of host cell apoptosis is a method employed by multiple pathogens to ensure their survival in the infected cell. Leishmania has been shown to protect M? and neutrophils from both natural and induced apoptosis. As shown in this study, apoptosis in monocyte-derived dendritic cells (moDC) induced by treatment with camptothecin was downregulated by coincubation with L. mexicana, as detected by morphological analysis of cell nuclei, TUNEL assay, gel electrophoresis of low molecular weight DNA fragments, and annexin V binding to phosphatidylserine. The observed antiapoptotic effect was found to be associated with a significant reduction of caspase-3 activity in moDC. The capacity of L. mexicana to delay apoptosis induction in the infected moDC may have implications for Leishmania pathogenesis by favoring the invasion of its host and the persistence of the parasite in the infected cells.     

Neospora caninum excreted/secreted antigens trigger CC-chemokine receptor 5-dependent cell migration

Neospora caninum, the causative agent of neosporosis, is an obligate intracellular parasite considered to be a major cause of abortion in cattle throughout the world. Most studies concerning N. caninum have focused on life cycle, seroepidemiology, pathology and vaccination, while data on host-parasite interaction, such as host cell migration, mechanisms of evasion and dissemination of this parasite during the early phase of infection are still poorly understood. Here we show the ability of excreted/secreted antigens from N. caninum (NcESAs) to attract monocytic cells to the site of primary infection in both in vitro and in vivo assays. Molecules from the family of cyclophilins present on the NcESAs were shown to work as chemokine-like proteins and NcESA-induced chemoattraction involved G_i protein signaling and participation of CC-chemokine receptor 5 (CCR5). Additionally, we demonstrate the ability of NcESAs to enhance the expression of CCR5 on monocytic cells and this increase occurred in parallel with the chemotactic activity of NcESAs by increasing cell migration. These results suggest that during the first days of infection, N. caninum produces molecules capable of inducing monocytic cell migration to the sites of infection, which will consequently enhance initial parasite invasion and proliferation. Altogether, these results help to clarify some key features involved in the process of cell migration and may reveal virulence factors and therapeutic targets to control neosporosis.     

A novel cold-regulated gene from Camellia sinensis, CsCOR1, enhances salt- and dehydration-tolerance in tobacco

In present research, the full-length cDNA and the genomic sequence of a novel cold-regulated gene, CsCOR1, were isolated from Camellia sinensis L. The deduced protein CsCOR1 contains a hydrophobic N-terminus as a signal peptide and a hydrophilic C-terminal domain that is rich in glycine, arginine and proline. Two internal repetitive tridecapeptide fragments (HSVTAGRGGYNRG) exist in the middle of the C-terminal domain and the two nucleotide sequences encoding them are identical. CsCOR1 was localized in the cell walls of transgenic-tobaccos via CsCOR1::GFP fusion approach. The expression of CsCOR1 in tea leaves was enhanced dramatically by both cold- and dehydration-stress. And overexpression of CsCOR1 in transgenic-tobaccos improved obviously the tolerance to salinity and dehydration.     

Genetic diversity of the Chinese liver fluke Clonorchis sinensis from Russia and Vietnam

Clonorchiasis is a parasitic disease of high public health importance in many countries in southeastern Asia and is caused by the Chinese liver fluke Clonorchis sinensis. However, the genetic structure and demographic history of its populations has not been sufficiently studied throughout the geographic range of the species and available data are based mainly on partial gene sequencing. In this study, we explored the genetic diversity of the complete 1560 bp cytochrome c oxidase subunit 1 (cox1) gene sequence for geographically isolated C. sinensis populations in Russia and Vietnam, to our knowledge for the first time. The results demonstrated low nucleotide and high haplotype differentiation within and between the two compared regions and a clear geographical vector for the distribution of genetic diversity patterns among the studied populations. These results suggest a deep local adaptation of the parasite to its environment including intermediate hosts and the existence of gene flow across the species’ range. Additionally, we have predicted an amino acid substitution in the functional site of the COX1 protein among the Vietnamese populations, which were reported to be difficult to treat with praziquantel. The haplotype networks consisted of several region-specific phylogenetic lineages, the formation of which could have occurred during the most extensive penultimate glaciations in the Pleistocene Epoch. The patterns of genetic diversity and demographics are consistent with population growth of the liver fluke in the late Pleistocene following the Last Glacial Maximum, indicating the lack of a population bottleneck during the recent past in the species’ history. The data obtained have important implications for understanding the phylogeography of C. sinensis, its host-parasite interactions, the ability of this parasite to evolve drug resistance, and the epidemiology of clonorchiasis under global climate change.     

Classical CD11c dendritic cells, not plasmacytoid dendritic cells, induce T cell responses to Plasmodium chabaudi malaria

Dendritic cells play an important role in the development of immune responses in malaria, but the contribution of plasmacytoid dendritic cells (pDC) to CD4 T cell activation and immunopathology is unknown. We have investigated pDC in a Plasmodium chabaudi infection in mice. During infection, pDC increased in number and transiently up-regulated expression of Major Histocompatibility Complex class II and co-stimulatory molecules. However, in contrast to classical CD11c^high DC, pDC could not phagocytose parasites or process parasite proteins, to activate CD4 T cells. Activation of naïve pDC, but not CD11c^high DC, by infected red blood cells induced IFNα in vitro, which was dependent on the Toll-like receptor, TLR9. However, inactivation of TLR9 in knock-out mice had no effect on a P. chabaudi infection suggesting that TLR9 was not crucial for parasite elimination or pathology. Neither pDC nor IFNαβ were essential for parasite clearance as mice depleted of pDC or IFNαβ Receptor-knock-out mice could control infection. However, these mice lost significantly more weight than untreated or wild-type mice. We conclude that classical DC are the major antigen-presenting cells for CD4 T cells in this infection, but that pDC and IFNαβ may play minor roles in controlling the magnitude of acute stage pathology.     

Toxoplasma gondii: Impaired maturation and pro-inflammatory response of dendritic cells in MIF-deficient mice favors susceptibility to infection

Macrophage migration inhibitory factor (MIF) has been found to be involved in host resistance to several parasitic infections. To determine the mechanisms of MIF-dependent responses to Toxoplasma gondii, we investigated host resistance in MIF−/− mice (BALB/c background) during natural oral infection. We focused on the potential involvement of MIF in Dendritic Cell (DC) maturation and IL-12 production. Following oral T. gondii infection, wild type mice developed a strong IL-12 response with an adequate maturation of their draining mesenteric lymph node DC (MLNDC) population and were resistant to challenge with either 40 or 100 cysts (ME49 strain). In contrast, similarly infected MIF−/− mice mounted a weak IL-12 response, displayed immature MLNDCs in the early phases of infection and rapidly succumbed to both type of challenges. Lack of maturation and IL-12 production of DCs in response to T. gondii antigens was confirmed by in vitro studies, and these effects were reversed following treatment with recombinant MIF. These findings demonstrate that MIF-induced early DC maturation and IL-12 production mediate resistance to T. gondii infection.     

Detecting and differentiating Theileria sergenti and Theileria sinensis in cattle and yaks by PCR based on major piroplasm surface protein (MPSP)

Theileria sergenti and Theileria sinensis are closely related members of benign Theileria species found in cattle and yaks in China. They are morphologically indistinguishable. A polymerase chain reaction (PCR) targeting major piroplasm surface protein of T. sergenti and T. sinensis was developed in this study. The newly developed oligonucleotide primer set was able to specifically amplify the DNA of T. sinensis and in conjunction with primers for T. sergenti and these two species could be detected and distinguished. Specificity testing also revealed that there was no cross-reaction with the other tick-borne diseases Theileria annulata, Babesia ovata, Anaplasma marginale as well as bovine white blood cells. Phylogenetic analysis based on the MPSP gene sequences confirmed the specificity of PCR assays. The sensitivity of the methods was 0.1 pg DNA for the T. sergenti PCR and 1 pg DNA for T. sinensis PCR. Two hundred and thirty-six field blood samples from of cattle and yaks were collected from five different geographical regions in China where benign Theileria species have been found. T. sergenti was found in all five provinces but was absent from one county in Gansu Province. T. sinensis was only found in Gansu Province. In both counties in Gansu where the parasites co-existed, mixed infections were detected. Our results indicate that the PCR methods developed in this study are suitable for the detection and differentiation of T. sergenti and T. sinensis.     

Toll-like receptor recognition of Toxoplasma gondii

Toxoplasma gondii potently stimulates IFN-gamma production by both the innate and adaptive immune system as part of its host adaptation. This response is known to be dependent on an Myeloid Differentiation factor 88 signaling pathway used by Toll-like receptors (TLRs), a family of proteins involved in the recognition of microbial molecular patterns. In the following review, we summarise the evidence for specific TLR function in host resistance to T. gondii focusing on the recent discovery in the parasite of a profilin-like ligand that potently stimulates TLR11 and regulates the production of IL-12, a cytokine necessary for the protective IFN-gamma response. In addition, we discuss the hypothesis that TLR11 may have evolved as a general pattern recognition receptor for apicomplexan protozoa and that as highly conserved proteins associated with actin-based motility, profilins are logical ligand targets for this form of pathogen detection. Finally, we review the evidence for involvement of other TLR and TLR ligands in host resistance to T. gondii and discuss how such receptors might synergise with TLR11 in the innate response to the parasite.     

Anti-HCV activity of the Chinese medicinal fungus Cordyceps militaris

Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although the sustained virologic response rate in the treatment of genotype 1 using new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has been improved by more than 70%, several severe side effects such as skin rash/ageusia and advanced anemia have become a problem. Under these circumstances, a new type of anti-HCV oral drug with few side effects is needed. Our recently developed HCV drug assay systems, including the HuH-7 cell line-derived OR6 and AH1R, and the Li23 cell line-derived ORL8 and ORL11, allow genome-length HCV RNAs (several strains of genotype 1b) encoding renilla luciferase to replicate efficiently. Using these systems as anti-HCV candidates, we have identified numerous existing medicines that can be used against HCV with few side effects, such as statins and teprenon. To obtain additional anti-HCV candidates, we evaluated a number of oral health supplements, and found that the capsule but not the liquid form of Cordyceps militaris (CM) (Ascomycotinanorth, North Chinese caterpillar fungus), which is used as a Chinese herbal medicine, exhibited moderate anti-HCV activity. In combination with interferon-α or ribavirin, CM exhibited an additive inhibitory effect. Among the main components of CM, cordycepin, but not ergosterol, contributed to the anti-HCV activity of CM. In consideration of all these results, we suggest that CM would be useful as an oral anti-HCV agent in combination with interferon-α and/or ribavirin.     

Expression pattern and substrate specificity of Clonorchis sinensis tyrosinases

Tyrosinase (TYR) is a copper-containing glycoenzyme that mediates hydroxylation of tyrosine into dihydroxyphenylalanine and oxidation of dihydroxyphenylalanine into dihydroxyphenylalanine quinone. TYRs play pivotal roles in eggshell sclerotisation of trematode parasites, while their comprehensive biochemical properties remain elusive. We characterised genes encoding four TYRs (CsTYR1–4) of Clonorchis sinensis, a causative agent of human hepatobiliary disease. These genes shared tightly conserved amino acid residues, two copper binding catalytic motifs and a cysteine-rich epidermal growth factor-like domain. The native and recombinant CsTYRs showed high reactivity against diphenol compounds, especially those with hydroxyl groups in ortho-positions (catechol and l-dihydroxyphenylalanine), but showed minimal activity toward monophenol compounds. Diphenolase activity was enhanced by increased pH of the reaction buffer from 5.0 to 7.0. The temporal induction of CsTYR expression coordinated with the sexual maturation of the worm; enzyme activity was mainly in the vitelline glands and intrauterine immature eggs proximal to the ovary. The primary structures and functional domains of CsTYRs showed significant similarities to those of the vertebrate orthologs, whereas the amino acids shared with the nematode and insect proteins were largely restricted in the bicopper active center. Unlike highly diverged TYR homologs in vertebrates, multiple paralogs have not yet evolved into the separate lineages in trematode genomes, suggesting that duplication of TYR genes might relate to increased genic dosage/redundancy in trematodes. In vitro treatment of copper chelator, diethyldithiocarbamic acid, inhibited generation of phenotypically normal egg. TYR proteins are essential for C. sinensis reproduction, thus might be targeted for therapeutic and vaccine strategies against clonorchiasis, which is prevalent in several Asian countries and is one of the most important predisposing factors for human cholangiocarcinoma. The close phylogenetic relationships between trematode and vertebrate homologs also provide a molecular clue to understand the multifaceted evolutionary pathway of TYR homologs across animal taxa.     

rDNA-targeted PCR primers and FISH probe in the detection of Ophiocordyceps sinensis hyphae and conidia

Ophiocordyceps sinensis (Berk.) Sung, Sung, Hywel-Jones & Spatafora (syn. Cordyceps sinensis) one of the entomopathogenic fungi, is a rare Traditional Chinese Medicine (TCM) found in the Qinghai-Tibetan Plateau. Polymerase Chain Reaction (PCR) and Fluorescence in situ hybridization (FISH) methods are necessary to identify the mycelia or spores of O. sinensis from its habitat and to monitor its dispersal, colonization and infectivity. To develop both primers and probe specific to O. sinensis, ribosomal DNA (rDNA) amplified with universal primers from O. sinensis genomic DNA and seven closely related fungi were sequenced. According to these sequences, the upper and lower primers (OsT-F and OsT-R) were designed within internal transcribed spacer region 1 (ITS1) and ITS2 and flanked by universal primers ITS5 and ITS4, respectively. The designed primers were used for general PCR, touchdown PCR, or both together with the universal primers for nested-touchdown PCR. The results showed that only the extracted DNA of O. sinensis was specifically amplified. The sensitivity of nested-touchdown PCR with extracted DNA of O. sinensis is as low as 10⁻¹⁴ g (10 fg) and at least 1000 times higher than the other PCR methods. In addition, Cy5-labeled probe (OsLSU) for cytoplasmic LSU rRNA was hybridized with the ascospores of O. sinensis. It showed a strong red fluorescence throughout the whole cell but did not cross-react with other entomopathogenic fungi. Taken together, these methods were useful for studying the biology and ecology of O. sinensis.     

Effects of the aqueous extract of white tea (Camellia sinensis) in a streptozotocin-induced diabetes model of rats

White tea (WT) is very similar to green tea (GT) but it is exceptionally prepared only from the buds and young tea leaves of Camelia sinensis plant while GT is prepared from the matured tea leaves. The present study was investigated to examine the effects of a 0.5% aqueous extract of WT in a streptozotocin-induced diabetes model of rats. Six-week-old male Sprague-Dawley rats were divided into 3 groups of 6 animals in each group namely: normal control (NC), diabetic control (DBC) and diabetic white tea (DWT). Diabetes was induced by an intraperitoneal injection of streptozotocin (65 mg/kg BW) in DBC and DWT groups except the NC group. After 4 weeks feeding of 0.5% aqueous extracts of WT, the drink intake was significantly (P < 0.05) increased in the DWT group compared to the DBC and NC groups. Blood glucose concentrations were significantly decreased and glucose tolerance ability was significantly improved in the DWT group compared to the DBC group. Liver weight and liver glycogen were significantly increased and serum total cholesterol and LDL-cholesterol were significantly decreased in the DWT group compared to the DBC group. The food intake, body weight gain, serum insulin and fructosamine concentrations were not influenced by the consumption of WT. Data of this study suggest that the 0.5% aqueous extract of WT is effective to reduce most of the diabetes associated abnormalities in a steptozotocin-induced diabetes model of rats.     

Leishmanicidal activity of synthetic antimicrobial peptides in an infection model with human dendritic cells

Different species of Leishmania are responsible for cutaneous, mucocutaneous or visceral leishmaniasis infections in millions of people around the world [14]. The adverse reactions caused by antileishmanial drugs, emergence of resistance and lack of a vaccine have motivated the search for new therapeutic options to control this disease. Different sources of antimicrobial molecules are under study as antileishmanial agents, including peptides with antimicrobial and/or immunomodulatory activity, which have been considered to be potentially active against diverse species of Leishmania [7] and [39]. This study evaluated the cytotoxicity on dendritic cells, hemolytic activity, leishmanicidal properties on Leishmania panamensis and Leishmania major promastigotes and effectiveness on parasite intracellular forms (dendritic cells infected with L. panamensis and L. major promastigotes), when each parasite in culture was exposed to different concentrations of a group of synthetic peptides with previously reported antimicrobial properties, which were synthesized based on their naturally occurring reported sequences. Dermaseptin, Pr-2 and Pr-3 showed inhibitory activity on the growth of L. panamensis promastigotes, while Andropin and Cecropin A (with a selectivity index of 4 and 40, respectively) showed specific activity against intracellular forms of this species. The activities of Andropin and Cecropin A were exclusively against the intracellular forms of the parasite, therefore indicating the relevance of these two peptides as potential antileishmanial agents. In the case of L. major promastigotes, Melittin and Dermaseptin showed inhibitory activity, the latter also showed a selectivity index of 8 against intracellular forms. These findings suggest Andropin, Cecropin A and Dermaseptin as potential therapeutic tools to treat New and Old World cutaneous leishmaniasis.