Immune and allergenic effects of the microalga Coccomyxa sp. strain KJ in healthy humans: A pilot study.

BACKGROUND: The Coccomyxa sp. strain KJ (Coccomyxa KJ), a microalga found in Japan, has a potential function in controlling viral infections. Recently, its dry powder has been marketed as a health food product. OBJECTIVES: This pilot study investigated the effects of Coccomyxa KJ powder tablet intake on allergic reactions and immune functions in healthy participants. MATERIAL AND METHODS: Nine healthy volunteers (4 males and 5 females) who expressed interest in foods containing Coccomyxa KJ, and were willing to undergo blood tests, were recruited. Each individual was asked to take 2 Coccomyxa KJ powder tablets (0.3 g) before breakfast once a day for 4 weeks. The salivary immunoglobulin A (IgA) level and blood parameters (white blood cell (WBC) count, eosinophil and lymphocyte counts and percentages, natural killer (NK) cell activity, interleukin (IL)-6 level, and T helper (Th)1/Th2 cell ratio) were evaluated at baseline and weeks 2 and 4. RESULTS: The 4-week intake of Coccomyxa KJ did not affect salivary IgA levels, WBC count, eosinophil and lymphocyte counts and percentages, or the Th1/Th2 ratio. There were significant differences in the NK cell activity after 4 weeks, with an average increase of 11.78 (95% confidence interval (95% CI): 6.80-16.76). None of the patients experienced adverse reactions during or after the study. CONCLUSIONS: Long-term Coccomyxa KJ intake improved NK cell activity without causing adverse effects on the indicators of local immunity, systemic inflammation and immune response balance. This study suggests that Coccomyxa KJ powder tablets can induce beneficial immune modifications without causing any adverse effects.

Anti-Influenza virus effects of Enterococcus faecalis KH2 and Lactobacillus plantarum SNK12 RNA.

Bacterial RNA has recently emerged as an immune-stimulating factor during viral infection. The immune response in an organism is directly related to the progression of virus infections. Lactic acid bacteria in particular have anticancer, bioprotective, and antiallergic effects by modulating immunity. Here, we aimed to demonstrate the effect of bacterial RNA on in vitro production of IL-12, a proinflammatory cytokine, and on in vivo activity against influenza A virus (IFV) infection. Oral administration of heat-killed Enterococcus faecalis KH2 (KH2) or Lactobacillus plantarum SNK12 (SNK) in IFV-infected mice suppressed viral replication and stimulated production of virus-specific antibodies. However, ribonuclease-treated KH2 or SNK abrogated the effect, reducing IL-12 production in vitro and anti-IFV effects in vivo. Taken together, KH2 or SNK showed antiviral effects in vivo when administered orally, and the RNAs of KH2 and SNK play a part in these effects, despite the phylogenetic differences between the bacteria.

[Surveillance of Cadmium Concentration in Chocolate and Cocoa Powder Products Distributed in Japan].

Chocolate and cocoa are manufactured from cacao beans produced by the cacao tree (Theobroma cacao). These products may contain cadmium (Cd), which originates from contaminated soil. Here, we surveyed the Cd concentrations in dark chocolate, milk chocolate, white chocolate and cocoa powder products purchased at retail stores in Japan, using inductively coupled plasma mass spectrometry. The Cd concentrations in these chocolate and cocoa powder products ranged from 0.00021 to 2.3 mg/kg and from 0.015 to 1.8 mg/kg, respectively. A weak positive correlation was found between the Cd concentration and the content of cocoa solids stated on the product labels. A comparison between these results and the maximum levels (MLs) set by the European Union revealed that the Cd concentrations in chocolate and cocoa powder products on the Japanese market exceeded the MLs for eight of the 180 chocolate products and 26 of the 140 cocoa powder products.

Membrane topology of Golgi-localized probable S-adenosylmethionine-dependent methyltransferase in tobacco (Nicotiana tabacum) BY-2 cells.

S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) transfer methyl groups to substrates. In this study, a novel putative tobacco SAM-MTase termed Golgi-localized methyl transferase 1 (GLMT1) has been characterized. GLMT1 is comprised of 611 amino acids with short N-terminal region, putative transmembrane region, and C-terminal SAM-MTase domain. Expression of monomeric red fluorescence protein (mRFP)-tagged protein in tobacco BY-2 cell indicated that GLMT1 is a Golgi-localized protein. Analysis of the membrane topology by protease digestion suggested that both C-terminal catalytic region and N-terminal region seem to be located to the cytosolic side of the Golgi apparatus. Therefore, GLMT1 might have a different function than the previously studied SAM-MTases in plants.

Aptamer that binds to the gD protein of herpes simplex virus 1 and efficiently inhibits viral entry.

The ectodomain of the gD protein of herpes simplex viruses (HSVs) plays an important role in viral entry by binding to specific cellular coreceptors and mediating viral entry to the host cells. In the present study, we isolated RNA aptamers (aptamer-1 and aptamer-5) that specifically bind to the gD protein of HSV-1 with high affinity and are able to discriminate the gD protein of a different virus, HSV-2. Aptamer-1 efficiently interfered with the interaction between the gD protein and the HSV-1 target cell receptor (HVEM) in a dose-dependent manner. The 50% effective concentration (EC(50)) of aptamer-1 was estimated to be in the nanomolar range (60 nM). Furthermore, aptamer-1 was analyzed for anti-HSV-1 activity by using plaque assays, and it efficiently inhibited viral entry with an estimated K(i) of 0.8 µM. To expand the future applications of aptamer-1, a shorter variant was designed by using both mapping and boundary analyses, resulting in the mini-1 aptamer (44-mer). Compared to the full-length aptamer, mini-1 had at least as high an affinity, specificity, and ability to interfere with gD-HVEM interactions. These studies suggest that the mini-1 aptamer could be explored further as an anti-HSV-1 topical therapy designed to prevent the risk of acquiring HSV-1 infection through physical contact.

Immunostimulating effects of a sulfated galactan from Codium fragile.

A pyruvylated sulfated galactan from Codium fragile is a highly ramified polysaccharide consisting of 3-linked, 3,6-linked, and non-reducing terminal d-galactose with pyruvate and sulfate groups; the glycan exerts anti-herpes simplex virus type 2 effects in vitro and in vivo. This particular polysaccharide was found to stimulate the production of nitric oxide by inducing iNOS at the mRNA and protein levels. In addition, the polysaccharide also induced several cytokine mRNA expressions such as IL-1beta, IL-6, IL-10 and TNF-alpha. Therefore, it appears that the sulfated galactan might possess the immunostimulating effects via activation of macrophages.

[Evaluation and application of natural products for viral infections].

The limited efficacy and significant clinical toxicity of combination interferone and ribavirin therapy have generated strong interest in developing novel inhibitors of hepatitis C virus (HCV) replication. Recently, a growing understanding of the structure and function of critical viral enzymes and the development of HCV replicons have accelerated the development of highly specific candidate antiviral agents. In the life cycle of HCV, enveloped virions bind and penetrate into host cell using viral envelope glycoproteins. In the cytoplasm, the viral RNA genome serves as mRNA, and produces viral protein as a long polyprotein that is cleaved by both host and viral proteases. Progeny virions assemble by budding into ER/Golgi apparatus, where the glycoproteins maturate, and are released at the cell surface. All stages of replication cycle from the attachment of virus to the release of progeny should be antiviral targets. We have searched for antiviral candidates from natural resources for about 20 years. So far, we have found several classes of compounds with unique antiviral action. Among them, anionic substances interfere with virus attachment and/or entry, several substances inhibit the maturation of virus-specific glycoproteins, low molecules can inhibit the virus release from infected cells, glycerol derivatives reduce the pathogenicity of virus, and some compounds exert virucidal action that impairs the ability of virus to infect host cells. These substances might be worthy to be evaluated as novel anti-HCV agents by using HCV replication systems in cultured cell lines.

[False-positive reaction of urinary ketone bodies caused by bucillamine].

In many patients taking bucillamine, an anti-rheumatic drug, urinary examinations show false-positive findings for ketone on urine dipsticks, because of the SH radicals in bucillamine. Our group examined the frequency of false-positive ketone testing and the causative factors in 179 outpatients being treated by the Department of Rheumatology in our hospital. The samples that remained purple even after boiling were determined to be false-positives, because boiling volatilizes urinary ketones while leaving bucillamine unchanged. Forty-six of 49 patients taking bucillamine tested false-positive for ketone, whereas there were no false-positive reactions among patients not receiving bucillamine. The patients showing false-positive reactions had significantly lower serum albumin levels, higher specific gravity of urine and more acidic urine. Moreover, we could predict whether the reaction was true-positive or bucillamine-induced false-positive by observing differences between the colors and levels of discoloration of the urine test paper. In the evaluation of ketone findings on urinary dipstick, the physician must ascertain whether the patient is taking bucillamine.

[Consideration of preventive free-flowing IV infusion method for vinorelbine].

Vinorelbine is administered to treat solid tumors such as non-small cell lung cancer and breast cancer, and good treatment results have been reported. Although this agent is known to cause phlebitis, some studies indicated that its administration over 5 minutes or less decreased the incidence of this adverse effect to approximately 5%. However,most studies employed bolus injection, and no study has reported completing drip infusion within 5 minutes. In the present study,we investigated the preventive effects on phlebitis of administering this agent over 5 minutes or less by drip infusion,which is simpler and more useful than intravenous injection. We administered vinorelbine 35 times to 6 patients with breast cancer or non-small cell lung cancer. The mean administration period was 3 minutes and 59 seconds +/-22 seconds, and the incidence of phlebitis was 5.7%. Our administration method involving drip infusion prevented phlebitis as markedly as by intravenous injection. In addition,there were no marked differences in the incidences of adverse effects other than phlebitis. The administration method employed in this study (drip infusion within five minutes) prevented vinorelbine-induced phlebitis, and was simpler than intravenous injection.

Long-circulating liposome-encapsulated ganciclovir enhances the efficacy of HSV-TK suicide gene therapy.

To enhance the efficacy of ganciclovir/herpes simplex virus thymidine kinase (GCV/HSV-TK) suicide gene therapy for nasopharyngeal cancer KB, we developed long-circulating liposome-encapsulated GCV, and evaluated cytotoxicity in vitro and in vivo. PEGylated liposome-encapsulated GCV (PEG-GCV-lipo) was prepared by the freeze-thawing method. In vitro experiments demonstrated that GCV from liposomes was gradually released over a period of 3 days. The in vitro cytotoxicity of PEG-GCV-lipo was similar to that of GCV solution in human cervical carcinoma HeLa cells expressing HSV-TK. Pharmacokinetics studies in mice showed that, compared with GCV solution, intravenous and intraperitoneal injection of PEG-GCV-lipo (10 mg/kg) led to long circulation in plasma; the area under the curve was 36-fold or 32-fold higher than that of GCV solution, respectively. In GCV/HSV-TK suicide gene therapy, the HSV-TK gene complexed with nanoparticle vector was directly injected into KB xenografts, and PEG-GCV-lipo or GCV solution was injected intravenously in mice once a day (25 mg/kg/day every 2nd day, 4 times). PEG-GCV-lipo was significantly 3-fold more effective than GCV solution in inhibiting tumor growth and produced durable complete tumor remissions on day 11 after injection. These findings demonstrate that long-circulating liposome-encapsulated GCV is a new approach to drug carriers to enhance the efficacy of suicide gene therapy.

Liposome vector containing biosurfactant-complexed DNA as herpes simplex virus thymidine kinase gene delivery system.

For injectable-sized liposome complexed with DNA (lipoplexes) with high transfection efficiency of genes, we initially prepared small-sized liposomes by addition of biosurfactant. For selectivity of gene expression, the thymidine kinase (MK-tk) gene controlled by midkine was used for herpes simplex virus thymidine kinase (HSV-tk) gene therapy. Liposomes composed of 3([N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), L-dioleoylphosphatidylethanolamine (DOPE), and a biosurfactant, such as beta-sitosterol beta-D-glucoside (Sit-G) for Sit-G-liposomes and mannosylerythrytol lipid A (MEL) for MEL-liposomes, produced about 300-nm-sized lipoplexes. Sit-G- and MEL-liposomes showed higher transfection efficiency of the luciferase marker gene and thymidine kinase activity in the presence of serum in the cells. The treatment with transfection of MK-tk gene by Sit-G-liposome and injection of ganciclovir significantly reduced tumor growth in a solid tumor model, compared with that by Sit-G-liposome alone. This finding suggested that Sit-G-liposome is a potential vector for HSV-tk gene therapy.

Antiviral sulfated polysaccharide from Navicula directa, a diatom collected from deep-sea water in Toyama Bay.

A sulfated polysaccharide named naviculan was isolated from a diatom, Navicula directa (W. SMITH) RALFS, collected in deep sea water from Toyama Bay. The polysaccharide consisted of fucose, xylose, galactose, mannose, rhamnose and sulfate with an apparent molecular weight of 220000. It showed antiviral activities against herpes simplex viruses type 1 and 2, and influenza A virus with selectivity indices (CC50/IC50) of 270, 510 and 32, respectively. Naviculan also showed an inhibitory effect on cell-cell fusion between CD4-expressing and human immunodeficiency virus (HIV) gp160-expressing cells that was used as a model system of infection with HIV.

The role of a HSV thymidine kinase stimulating substance, scopadulciol, in improving the efficacy of cancer gene therapy.

BACKGROUND: The most extensively investigated strategy of suicide gene therapy for treatment of cancer is the transfer of the herpes simplex virus thymidine kinase (HSV-TK) gene followed by administration of antiviral prodrugs such as acyclovir (ACV) and ganciclovir (GCV). The choice of the agent that can stimulate HSV-TK enzymatic activity is one of the determinants of the usefulness of this strategy. Previously, we found that a diterpenoid, scopadulciol (SDC), produced a significant increase in the active metabolite of ACV. This suggests that SDC may play a role in the HSV-TK/prodrug administration system. METHODS: The anticancer effect of SDC was evaluated in HSV-TK-expressing (TK+) cancer cells and nude mice bearing TK+ tumors. In vitro and in vivo enzyme assays were performed using TK+ cells and tumors. The phosphorylation of ACV monophosphate (ACV-MP) was measured in TK- cell lysates. The pharmacokinetics of prodrugs was evaluated by calculating area-under-the-concentration-time-curve values. RESULTS: SDC stimulated HSV-TK activity in TK+ cells and tumors, and increased GCV-TP levels, while no effect of SDC was observed on the phosphorylation of ACV-MP to ACV-TP by cellular kinases. The SDC/prodrug combination altered the pharmacokinetics of the prodrugs. In accord with these findings, SDC enhanced significantly the cell-killing activity of prodrugs. The bystander effect was also significantly augmented by the combined treatment of ACV/GCV and SDC. CONCLUSIONS: SDC was shown to be effective in the HSV-TK/prodrug administration system and improved the efficiency of the bystander effect of ACV and GCV. The findings will be considerably valuable with respect to the use of GCV in lower doses and less toxic ACV. This novel strategy of drug combination could provide benefit to HSV-TK/prodrug gene therapy.

Enzymatic and biochemical properties of a novel human serine dehydratase isoform.

A cDNA clone similar to human serine dehydratase (SDH) is deposited in the GenBank/EMBL databases, but its structural and functional bases remain unknown. Despite the occurrence of mRNA, the expected protein level was found to be low in cultured cells. To learn about physicochemical properties of the protein, we expressed the cDNA in Escherichia coli, and compared the expressed protein with that of a hepatic SDH. The purified protein showed l-serine and l-threonine dehydratase activity, demonstrating to be an isoform of SDH. However, their Km and Vmax constants were different in a range of two-order. Removal of Pro128 from the hepatic SDH consisting of 328 residues, which is missing in the corresponding position of the isoform consisting of 329 residues, significantly changed the Michaelis constants and Kd value for pyridoxal 5'-phosphate, whereas addition of a proline residue to the isoform was without effect. These findings suggest the difference in the structures of the active sites of the two enzymes. Another striking feature was that the expressed level of the isoform in E. coli was 7-fold lower than that of the hepatic SDH. Substitution of Val for Leu287 in the isoform dramatically increased the protein level. The high yield of the mutated isoform was also confirmed by the in vitro transcription and translation experiment. The poor expression of the isoform could be explained by the more stable secondary structure of the mRNA than that of the hepatic SDH mRNA. The present findings may provide a clue as to why the protein level in cultured cells is low.

Isolation of an antiviral polysaccharide, nostoflan, from a terrestrial cyanobacterium, Nostoc flagelliforme.

A novel acidic polysaccharide, nostoflan, was isolated from a terrestrial cyanobacterium, Nostoc flagelliforme. Nostoflan exhibited a potent anti-herpes simplex virus type 1 (HSV-1) activity with a selectivity index (50% cytotoxic concentration/50% inhibitory concentration against viral replication) of 13,000. Sugar composition and methylation analyses revealed that it was mainly composed of -->4)-D-Glcp-(1-->, -->6,4)-D-Glcp-(1-->, -->4)-D-Galp-(1-->, -->4)-D-Xylp-(1-->, D-GlcAp-(1-->, D-Manp-(1--> with a ratio of ca. 1:1:1:1:0.8:0.2. Two pyridylaminated oligosaccharides were prepared by partial acid hydrolysis and pyridylamination. On the basis of MALDI-TOF-MS and NMR analyses, they were found to be beta-D-Glcp-(1-->4)-D-Xyl-PA and beta-D-GlcAp-(1-->6)-beta-D-Glcp-(1-->4)-D-Gal-PA. From these results, nostoflan might be mainly composed of the following two types of sugar sequence: -->4)-beta-D-Glcp-(1-->4)-D-Xylp-(1--> and -->4)-[beta-D-GlcAp-(1-->6)-]-beta-D-Glcp-(1-->4)-D-Galp-(1-->. Besides anti-HSV-1 activity, nostoflan showed potent antiviral activities against HSV-2, human cytomegalovirus, and influenza A virus, but no activity against adenovirus and coxsackie virus was observed. Therefore, nostoflan has a broad antiviral spectrum against enveloped viruses whose cellular receptors are carbohydrates. Furthermore, nostoflan showed no antithrombin activity, unlike sulfated polysaccharides.

Evaluation of scopadulciol-related molecules for their stimulatory effect on the cytotoxicity of acyclovir and ganciclovir against Herpes simplex virus type 1 thymidine kinase gene-transfected HeLa cells.

Herpes simplex virus type 1 thymidine kinase (HSV TK) is involved in both antiherpetic therapy and cancer gene therapy with acyclovir (ACV) and ganciclovir (GCV). Enhanced sensitivity to these drugs is advantageous in their clinical use. In the present study, scopadulciol (SDC) and its related compounds were evaluated for their stimulatory effect on the cytotoxicity of ACV and GCV by determination of selective toxicities against HSV TK-expressing HeLa cells. Although SDC remarkably potenciated the cytotoxicity of ACV and GCV, the other tested compounds showed only weak selectivity, except for compound 34.

Nonsuppurative myocarditis associated with so-called fowl glioma.

C/O specific pathogen-free White Leghorn chickens were intracerebrally inoculated at one day of age with a brain homogenate of Japanese bantams (Gallus gallus domesticus) affected with fowl glioma. Histologically, six of eight inoculated chickens developed nonsuppurative meningoencephalitis in cerebrum and two of them had the characteristic lesions of fowl glioma. Hyperplastic lymphoid foci concomitantly developed in many organs of these birds, especially in the heart. Apart from these lymphoid foci, lymphocytic myocarditis was observed in all inoculated birds. Matrix inclusions were also noted in myocardial cells. Immunohistochemically, avian leukosis virus antigens were detected in reticular cells in the lymphoid foci, mesangial cells of the kidney, smooth muscle cells of the blood vessels, and myocardial cells. Of these tissues, the myocardium of all inoculated birds consistently showed strong reactivity for this antigens. The matrix inclusions were also positive for the antigens. These results suggest that the causal virus of fowl glioma has a high propensity to replicate, especially in myocardium and nonsuppurative myocarditis occurs associated with so-called fowl glioma.

Liver targeting liposomes containing beta-sitosterol glucoside with regard to penetration-enhancing effect on HepG2 cells.

The aim of this study was to examine the interaction of soybean-derived sterylglucoside (SG) with the human hepatoblastoma cell line HepG2 with regard to the penetration-enhancing effect of beta-sitosterol glucoside (Sit-G) to clarify the accumulation of SG-containing liposomes (SG-liposomes) to the liver in vivo. The approach was based on measurement of the association of SG-liposomes labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (Dil) in terms of asialoglycoprotein receptor (ASGP-R)-mediated endocytosis, affinity of Sit-G using lAsys and the association of FITC-dextran 4400 (FD-4) increased by Sit-G with the cells. The association of SG-liposomes was decreased by addition of asialofetuin, suggesting that SG-liposomes might be taken up via ASGP-R. Sit-G showed higher affinity with HepG2 cells than HeLa cells, and enhanced the association of FD-4 depending on the incubation time and Sit-G concentrations. Significant positive correlations were found between Sit-G and FD-4 association with the cells, indicating that Sit-G enhanced the drug penetration by distribution in cell membranes. The high degree of liver association of SG-liposomes in vivo might be related to recognition of glucose residues of SG by ASGP-R and to the high affinity and penetration-enhancing effect of Sit-G with hepatocytes.

Contribution of a combination of ponicidin and acyclovir/ganciclovir to the antitumor efficacy of the herpes simplex virus thymidine kinase gene therapy system.

We have previously reported that ponicidin (PND), isolated from Rabdosia ternifolia, potentiates the cell-killing activity of antiherpes prodrugs acyclovir (ACV) and ganciclovir (GCV) in human cancer cells expressing herpes simplex virus thymidine kinase (HSV-TK). To extend these in vitro results to in vivo situations, HSV-TK-expressing HeLa cells were injected into nude mice. The in vivo growth of TK(+) HeLa cells was significantly inhibited by coadministration of PND and ACV, or of PND and GCV, compared with single use of ACV or GCV in spite of lower doses of 1 or 0.25 mg/mouse, respectively. These results indicate that there is a good correlation between this in vivo efficacy and previously reported in vitro efficacy. Because of the insufficiency of incorporation of genes into tumors, bystander cell killing has attracted special interest. In the present study, we determined the ability of PND to potentiate the bystander effects of ACV and GCV in both in vitro and in vivo systems. In vitro combined use of PND with ACV or GCV rendered tumor cells more sensitive to the prodrugs, demonstrating a 1.8- to 97-fold or 2.8- to 26-fold reduction in IC(50) compared with ACV or GCV only, respectively, in 1 to 20% of HSV-TK(+) cells. In the in vivo experiments using nude mice injected with 3 or 10% HSV-TK(+) cells, tumor volume was lower in mice treated with a combination of PND and ACV/GCV than in those treated with ACV or GCV only. No toxicity of PND was seen in mice even at a dose 10-fold higher than that used in the in vivo experiments. These novel strategies could provide benefit to ablative cancer gene therapy by making it feasible to use toxic GCV at lower doses and relatively nontoxic ACV.