Eritoran inhibits S100A8-mediated TLR4/MD-2 activation and tumor growth by changing the immune microenvironment.

S100A8/A9 is a major component of the acute phase of inflammation, and appears to regulate cell proliferation, redox regulation and chemotaxis. We previously reported that S100A8/S100A9 are upregulated in the premetastatic lung. However, the detailed mechanisms by which S100A8 contributes to tumor progression have not been elucidated. In this study, we investigated the TLR4/MD-2 dependency by S100A8 on tumor progression. We found that S100A8 (2-89) peptide stimulated cell migration in a manner dependent on TLR4, MD-2 and MyD88. The S100A8 (2-89) peptide also activated p38 and NF-κB in TLR4-dependent manner. The peptide induced the upregulation of both IL-6 and Ccl2 in peritoneal macrophages obtained from wild-type mice, but not TLR4-deficient mice. We then investigated the responsible region of S100A8 for TLR4/MD-2 binding by a binding assay, and found that C-terminal region of S100A8 binds to TLR4/MD-2 complex. To further evaluate the TLR4 dependency on tumor microenvironment, Lewis lung carcinoma-bearing mice were treated with Eritoran, an antagonist of TLR4/MD-2 complex. We found that both tumor volume and pulmonary recruitment of myeloid-derived suppressor cells were reduced with the treatment of Eritoran for five consecutive days. Eritoran reduced the development of tumor vasculature, and increased tumor-infiltration of CD8(+) T-cells. Taken together, S100A8 appears to play a crucial role in the activation of the TLR4/MD-2 pathway and the promotion of a tumor growth-enhancing immune microenvironment.

Awareness of early warning signs and help-seeking behaviours among patients with schizophrenia who utilize social rehabilitation facilities in Japan.

The purpose of this study was to examine the relationship between early warning signs (EWS) and early help-seeking behaviours (HSB) and to identify the characteristics of patients with schizophrenia who sought early help. A cross-sectional study was carried out in 2004 using a self-reported questionnaire. Participants were recruited from social rehabilitation facilities for the mentally ill; 224 subjects participated, 170 of whom had schizophrenia. The survey included questions about demographic characteristics, self-care behaviours (HSB, recognition of EWS and others) and current service utilization and satisfaction. Fisher's exact test and Student's t-test were used to compare the characteristics of study participants. Logistic regression analyses were used to examine the association between recognition of EWS and early HSB.We found that 96 (56.5%) of 170 patients with schizophrenia reported at least one occasion of early HSB during their deterioration. Early HSB were related to the following factors: recognition of EWS, consultation with non-professional and professional support persons during deterioration, consulting with public mental health workers and living with family. Care and support should be offered to patients with schizophrenia to enable them to recognize their own mental deterioration.

Modification of the damping rate of the oscillations of a dust particle levitating in a plasma due to the delayed charging effect.

Dependence of the damping rate of the oscillations of the dust particles levitating in the sheath on the plasma parameters is investigated both theoretically and experimentally. Significant deviations of the damping rate from the values predicted by the Epstein formula are found in the experiment. The delayed charging effect is applied for the theoretical explanation of the experimental results. Qualitative agreement between the theoretical and experimental data is obtained.

DNA vaccine-encapsulated virus-like particles derived from an orally transmissible virus stimulate mucosal and systemic immune responses by oral administration.

Delivery of foreign genes to the digestive tract mucosa by oral administration of nonreplicating gene transfer vectors would be a very useful method for vaccination and gene therapy. However, there have been few reports on suitable vectors. In the present study, we found that plasmid DNA can be packaged in vitro into a virus-like particle (VLP) composed of open reading frame 2 of hepatitis E virus, which is an orally transmissible virus, and that these VLPs can deliver this foreign DNA to the intestinal mucosa in vivo. The delivery of plasmid DNA to the mucosa of the small intestine was confirmed by the results of immunohistochemical analyses using an expression plasmid encoding human immunodeficiency virus env (HIV env) gp120. After oral administration of VLPs loaded with HIV env cDNA, significant levels of specific IgG and IgA to HIV env in fecal extracts and sera were found. Moreover, mice used in this study exhibited cytotoxic T-lymphocyte responses specific to HIV env in the spleen, Payer's patches and mesenteric lymph nodes. These findings suggest that VLPs derived from orally transmissible viruses can be used as vectors for delivery of genes to mucosal tissue by oral administration for the purpose of DNA vaccination and gene therapy.

A single administration of interleukin-4 antagonistic mutant DNA inhibits allergic airway inflammation in a mouse model of asthma.

Interleukin 4 (IL-4) is essential for the switching of B cells to IgE antibody production and for the maturation of T helper (Th) cells toward the Th2 phenotype. These mechanisms are thought to play a crucial role in the pathogenesis of the allergic airway inflammation observed in asthma. In the present study, we examined the anti-inflammatory effects of DNA administration of murine IL-4 mutant Q116D/Y119D (IL-4 double mutant, IL-4DM), which binds to the IL-4 receptor alpha and is an antagonist for IL-4. Immunization of BALB/c mice with alum-adsorbed ovalbumin (OVA) followed by aspiration with aerosolized OVA resulted in the development of allergic airway inflammation. A single administration of IL-4DM DNA before the aerosolized OVA challenge protected the mice from the subsequent induction of allergic airway inflammation. Serum IgE level and extent of eosinophil infiltration in bronchoalveolar lavage (BAL) from IL-4DM DNA-administered mice were significantly lower than those in BAL from control plasmid-immunized mice. In our study, IL-4 or IL-4 mutants were not detected in sera from mice that had received a single administration of IL-4DM DNA. The results of this study provide evidence for the potential utility of IL-4 mutant antagonist DNA inoculation as an approach to gene therapy for asthma.

Induction of effective antitumor immune responses in a mouse bladder tumor model by using DNA of an alpha antigen from mycobacteria.

One of the main objectives of cancer immunotherapy is the activation and increase in number of antitumor effector cells. Recently, genetically modified tumor cell vaccines have been proposed for elicitation of antitumor effector cells. Native alpha antigen (alpha Ag) (also known as MPT59 and antigen 85B) of mycobacteria, which cross-reacts among mycobacteria species, may play an important biological role in host-pathogen interaction because it elicits various helper T-cell type 1 immune responses. To assess the induction of antitumor immune responses by alpha Ag, mouse tumor cell lines transfected with cDNA of alpha Ag from Mycobacterium kansasii were established, and the possibility of producing a tumor cell vaccine for induction of antitumor effects was explored. Transfection of tumor cell lines with an alpha Ag gene lead to primary tumor rejection and the establishment of protective immunity to nontransfected original tumor cell lines in Mycobacterium bovis bacillus Calmette-Gurin (BCG)-primed and unprimed mice. Mice immunized with tumor cell lines transfected with the alpha Ag gene showed delayed-type hypersensitivity responses in vivo and proliferative responses together with induction of interferon-gamma of spleen cells against nontransfected wild-type tumor cell lines in in vitro experiments. Moreover, immunization of mice with alpha Ag-expressing tumor cells elicited tumor-specific and cytotoxic T lymphocyte (CTL) epitope peptide-specific CD8+ CTLs. The results of this study provided evidence of the potential usefulness of alpha Ag in tumor cell vaccines.

Quintuple deglycosylation mutant of simian immunodeficiency virus SIVmac239 in rhesus macaques: robust primary replication, tightly contained chronic infection, and elicitation of potent immunity against the parental wild-type strain.

We previously generated a mutant of simian immunodeficiency virus (SIV) lacking 5 of a total of 22 N-glycans in its external envelope protein gp120 with no impairment in viral replication capability and infectivity in tissue culture cells. Here, we infected rhesus macaques with this mutant and found that it also replicated robustly in the acute phase but was tightly, though not completely, contained in the chronic phase. Thus, a critical requirement for the N-glycans for the full extent of chronic infection was demonstrated. No evidence indicating reversion to a wild type was obtained during the observation period of more than 40 weeks. Monkeys infected with the mutant were found to tolerate a challenge infection with wild-type SIV very well. Analyses of host responses following challenge revealed no neutralizing antibodies against the challenge virus but strong secondary responses of cytotoxic T lymphocytes against multiple antigens, including Gag-Pol, Nef, and Env. Thus, the quintuple deglycosylation mutant appeared to represent a novel class of SIV live attenuated vaccine.

Induction of virus-specific cytotoxic T lymphocytes by in vivo electric administration of peptides.

Generally, major histocompatibility complex (MHC) class I presentation of peptide antigens only occur for proteins' which are actively synthesized and processed intracellularly, so that immunization with a cytotoxic T lymphocyte (CTL) target peptide does not usually elicit effective CTL responses. In the present study, we explored the use of epitope peptides by in vivo electroporation to introduce directly into the cytoplasm for the vaccine elicitation of virus-specific CTLs in a mouse system. BALB/c mice were immunized with human immunodeficiency virus (HIV) env (P18, residues 311-320) or hepatitis C virus (HCV) NS5 (P17, residues 2423-2434) with or without electric pulses. Effector cells against peptide-labeled target cells were elicited in mice immunized with peptides with electric administration but not without electric administration. Moreover, cytolytic activities of CTL against peptide-labeled target cells were enhanced by the addition of plasmid having the immunostimulatory sequence (ISS) or cDNA of the B7-1 molecule in electric administration of peptides. The results of the present study suggest that a peptide vaccine against a virus using electric administration is effective in eliciting virus specific CTLs.

Experimental observation of vertically polarized transverse dust-lattice wave propagating in a one-dimensional strongly coupled dust chain.

Externally driven, vertically polarized transverse dust-lattice waves were observed in a one-dimensional strongly coupled dust chain levitated in the plasma-sheath boundary of a dc argon plasma at low gas pressure around 5 mtorr. Real and imaginary parts of the complex wave number were measured in the experiments. The experimental result clearly shows that the observed transverse dust-lattice wave propagates as a backward wave, which is in good agreement with the theoretical prediction.

Induction of hepatitis C virus-specific cytotoxic T lymphocytes in mice by an intrahepatic inoculation with an expression plasmid.

We assessed the possibility of intrahepatic inoculation with a plasmid encoding hepatitis C virus (HCV) proteins to elicit HCV-specific cytotoxic T lymphocytes (CTL) in mice as a conventional animal model of HCV infection. BALB/c mice were intrahepatically or intramuscularly inoculated with an expression plasmid DNA encoding HCV structural proteins under the control of the elongation factor 1-alpha promoter. Expressions of HCV-core protein and envelope proteins (E1 and E2) in hepatocytes were detected immunohistochemically 6 days after inoculation. CTL responses were examined using target cells either pulsed with a specific peptide or infected with a recombinant vaccinia virus expressing HCV structural protein. Both intrahepatically and intramuscularly DNA-inoculated mice developed CD8(+), MHC class I-restricted CTL responses that recognized the peptide pulsed as well as HCV proteins expressing target cells. These studies demonstrated the usefulness of a murine model of HCV infection induced by direct intrahepatic DNA inoculation for understanding the immunopathogenic mechanisms in HCV infection.

Detection of Borna disease virus in a pregnant mare and her fetus.

A pregnant mare showing pyrexia, reduced appetite, ataxia and paresis was euthanized and examined for the presence of Borna disease virus (BDV). Her brain, showing multiple neuronal degeneration and necrosis with hemorrhage, and the histologically normal brain of the fetus were both positive for BDV RNA. The BDV nucleotide sequences were identical in the mare and fetus in the second open reading frame (ORF). This is the first report of the possible vertical transmission of BDV in a horse.

Disseminated Trichosporonosis with Trichosporon asahii.

Trichosporon asahii fungemia was associated with multiple, purpuric, papular lesions on the abdomen and extremities in a 53-year-old man with acute myeloblastic leukemia. Histologically, budding yeasts were demonstrated in the dermis. The yeast-form fungus was identified as T. asahii.

Antiproliferative and apoptosis-inducing effects of an antitumor glycoprotein from Streptococcus pyogenes.

An acidic glycoprotein (SAGP) purified from an extract of Streptococcus pyogenes has been shown to inhibit the growth of methylcholanthrene-induced fibrosarcoma A (Meth A) cells via pertussis toxin-sensitive GTP-binding protein. The present study revealed that SAGP has activity to induce apoptosis in Meth A cells as assessed by DNA fragmentation and cell morphology with chromatin staining. The SAGP-induced DNA fragmentation in Meth A cells was augmented by herbimycin A, an inhibitor of protein tyrosine kinase, and prevented by orthovanadate, an inhibitor of protein tyrosine phosphatase. The growth inhibitory effect of SAGP on Meth A cells was reduced by orthovanadate, whereas the effect tended to be increased by herbimycin A. Western blotting analysis using antiphosphotyrosine antibody demonstrated that tyrosine phosphorylation of 170 kDa cellular protein was diminished in the cells incubated with SAGP. The inhibition of protein tyrosine phosphorylation was neither observed in the cells incubated with SAGP and orthovanadate nor in the cells incubated with heat-inactivated SAGP. These findings indicate that inhibition of tyrosine phosphorylation by protein tyrosine phosphatase(s) may be responsible for the SAGP-induced apoptosis and inhibition of cell growth.

[Endoscopic treatment of malignant stenosis in home care].

At our institute, we have tried to increase the patient's quality of life (QOL) by endoscopic stenting for upper intestinal stenosis due to malignant tumor, which could not be treated by surgery or chemotherapy. We report the endoscopic stenting and home therapy for those patients. The subjects were 44: (esophageal stenosis: 13 cases, biliary stenosis: 31 cases) out of 60 patients who had intestinal stenosis or obstruction, which could not be treated by surgery or chemotherapy. Esophageal stenosis was treated mainly by stenting and laser cautery, and biliary stenosis was treated by drainage using stenting. The home stay period, effective treat period, life survival period, and complications were analyzed in each case. It was found that the mean home stay period, mean effective treatment period, and mean life survival period were 3.4 months, 4.0 months, and 5.5 months, respectively, with esophageal stenosis, and 3.7 months, 4.4 months, 5.5 months with biliary stenosis. Mean home stay period/life survival period was 62% in cases of esophageal stenosis, and 67% in cases of biliary stenosis. Complications were observed in 40.0% of patients with esophageal stenting and in 12.5% with biliary stenting. Esophageal stenting showed a higher incidence of complications; however improvements in the stenting instrument will reduce the number of complications. Endoscopic stenting is thus effective for upper intestinal stenosis due to malignant tumor, especially in increasing the patient's QOL when curative therapy is not indicated, and the patient stays at home. We believe patients with uncurable malignant disease should have home treatment as early as possible.

New antifungal and cytotoxic steroidal saponins from the bulbs of an elephant garlic mutant.

Saponins in bulbs of a mutant of elephant garlic were investigated, and three new steroidal saponins named yayoisaponins A-C were obtained together with the known dioscin and aginoside. Their structures, including the relative stereochemistry, were elucidated by spectral data interpretation, while the absolute stereochemistry of the sugar moieties was assigned on the basis of a chiral gas chromatographic analysis of the acid hydrolysates. Yayoisaponins A-C and aginoside exhibited not only in vitro cytotoxicity against P388 cells at 2.1 micrograms/ml, but also antifungal activity against Mortierella ramanniana at 10 micrograms/disk.

Enhancement of anti-tumor activity of natural killer cells by BALL-1, a B cell lymphoma line.

The anti-tumor activity of human peripheral blood mononuclear cells (PBMC) against various tumor cell line cells (K562, Daudi, KMG-2, and KATOIII) was enhanced by coculture with irradiated BALL-1, but not with other irradiated B cell line cells (NALM-1, Namalwa, and Daudi). PBMC cocultured with BALL-1, however, failed to exhibit evident cytotoxicity against autologous concanavalin A-induced lymphoblasts. The enhancement of the anti-tumor activity seemed not to be correlated with EBNA and HLA-DR expression on B cell line cells. Monoclonal antibodies (mAbs) against interleukin (IL)-2, interferon-gamma, IL-12, IL-15, tumor necrosis factor-alpha and lymphotoxin showed little or no suppression of the anti-tumor activity of PBMC treated with irradiated BALL-1. Furthermore, the culture supernatants of BALL-1 failed to enhance the anti-tumor activity of PBMC, suggesting no involvement of soluble factors in the induction of the anti-tumor activity. The anti-tumor activity of PBMC treated with BALL-1 was synergistically enhanced by an additional IL-2 stimulation. Periodate-lysine-paraformaldehyde-fixed, but not ethanol- or acetone-fixed, BALL-1 could significantly enhance the anti-tumor activity. Furthermore, BALL-1-derived membrane fraction, but not that of Daudi, enhances the anti-tumor activity. It was thus suggested that some membrane glycoproteins on the cell surface of BALL-1 play a crucial role in the induction of the anti-tumor activity. By analysis using mAbs against human leukocytes, we found that depletion of CD11b, CD16, and CD56-positive cells resulted in decreased anti-tumor activity, suggesting that the main effector cells in the BALL-1-induced anti-tumor activity were natural killer (NK) cells. The present results thus raise the possibility that BALL-1, probably via membrane glycoproteins, modulates NK cell-mediated anti-tumor activity.

Characterization of a streptococcal antitumor glycoprotein (SAGP).

An acidic antitumor glycoprotein (SAGP) was purified from a crude extract of Streptococcus pyogenes, Su strain. Intraperitoneal injection with SAGP (20 mg protein/kg/day for 4 consecutive days) prolonged the life span of mice inoculated i.p. with Ehrlich ascite carcinoma cells and methylcholanthrene-induced fibrosarcoma cells (Meth A) up to 244% and 169% of that of the control mice, respectively. These in vivo antitumor effects were reduced in immunosuppressed mice. The effector spleen cells from the Meth A-inoculated and SAGP-injected mice showed a considerable cytostatic activity on Meth A cells in vitro, and immunosuppression studies suggested that carrageenan-sensitive and/or asialo-GM1 positive spleen cells are responsible for the in vivo antitumor effect of SAGP. SAGP inhibited the cell growth of cultured cell lines including transformed hamster embryonic lung cells, murine leukemia L 1210, Meth A and human promyelocytic leukemia HL60 cells. The IC50s for the cell growth of these cells were all below 0.1 microg protein/ml. SAGP inhibited the incorporation of nucleic acid precursors into Meth A cells. It seems that sulfhydryl groups of the SAGP molecule are essential for the expression of the antitumor action of SAGP. The cell growth-inhibitory activity of SAGP was diminished in Meth A cells preincubated with pertussis toxin (IAP), whereas it was augmented in the cells preincubated with cholera toxin (CTX), suggesting the involvement of toxin-sensitive GTP (G)-proteins in the SAGP-action. IAP and CTX-catalyzed ADP ribosylation assays confirmed that SAGP augmented the activity of IAP-sensitive G-protein. In addition, this augmentation was detected neither in Meth A cells incubated with heat-inactivated SAGP nor in SAGP-insensitive L929 cells. SAGP induced apoptosis in Meth A and HL60 cells as assessed by DNA fragmentation. A single dose injection of SAGP (100 mg protein/kg, i.v., s.c., or i.p.) into mice produced no toxic signs except occasional pain responses observed for one week after the injection. Thus, SAGP is a low toxic substance that shows in vivo antitumor activity by modulating immune responses of the host, and also exhibits in vitro cell-growth inhibition through IAP-sensitive G-protein.

[Death at home: home care for terminal cancer patients].

For the past seven years, we provided home hospice care for 26 terminal cancer patients. Seven patients died at home and nineteen patients were readmitted and died in our hospital. There was no difference in the mean age, sex, primary disease, nutrition management, pain control, informing of disease between the patients who died at home and the patients who died in the hospital was longer than that of patients who died at home. According to our questionnaire on home care, the satisfaction of patients and their families with home care was more than 80% in both groups. But patients who died at home needed more care than patients who died in the hospital because the former had more care helpers than the latter. In fact, only patient's families as care helpers and often took care of their patient. Both the physical and mental burden of patient's families increased, at last it was too difficult for them to continue home care. Therefore the home care system must be arranged support both the mental and physical status of the patients and their families.

[A case of elderly home care after surgical treatment for gastric cancer--elderly patient with decubitus].

As the elderly population grows, the number of elderly suffering from intestinal cancer has been increasing. After surgical treatment of intestinal cancer, occasionally the quality of life (QOL) of a patient is difficult due to pain and anxiety. Especially elderly patients experience these problems more than younger patients. Recently, we have experienced a case of an elderly patient operated for gastric cancer whose going down the level of QOL and performance status (PS) were decreasing because of lower nutrition and severe infectious decubitus. But with familial cooperation, his QOL and PS have been improving with long time Home Care.

Hepatoprotective principles of Swertia japonica Makino on D-galactosamine/lipopolysaccharide-induced liver injury in mice.

The n-BuOH extract of Swertia japonica showed a significant hepatoprotective effect on D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced liver injury in mice. The activity-guided fractionation led to the isolation of a new tetrahydroxanthone derivative, tetrahydroswertianolin (1), as well as two known iridoids, gentiopicroside (2) and sweroside (3). Their structures were elucidated by spectroscopic methods and chemical reactions. Of the three compounds, 2 and 3 possessed mild hepatoprotective activity at a dose range of 25-50 mg/kg, whereas, 1 exhibited potent activity in a dose-dependent manner. The hepatoprotective effect of tetrahydroswertianolin (1) was stronger than that of glycyrrhizin which was used as a positive control.