Heat-killed Lactobacillus helveticus improves mood states: a randomised, double-blind, placebo-controlled study.

We investigated the effects of heat-killed Lactobacillus helveticus MCC1848 on daily mood states in healthy young adults. Participants (n=58) were randomised to receive heat-killed L. helveticus MCC1848 powder or placebo powder for 4 weeks. During the study period, adverse events were recorded in the participant diary. Mood states were assessed before and 2 and 4 weeks after initiation of the intervention. The primary outcomes were the shortened version of the Profile of Mood States 2 (POMS 2) scores. Secondary outcomes included other mood state (State-Trait Anxiety Inventory (STAI); visual analogue scale (VAS)), quality of life (acute form of the SF-36v2), sleep (Athens Insomnia Scale (AIS)) and fatigue (Chalder Fatigue Scale (CFS)) scores. Four weeks of heat-killed L. helveticus MCC1848 intake, compared to placebo, significantly improved the shortened version of the POMS 2 'friendliness' and the VAS 'relaxed' scores, which are two indicators of positive mood states. On the other hand, heat-killed L. helveticus MCC1848 intake had no significant effects on negative mood state items (e.g. anger, nervousness, confusion) assessed by the shortened version of the POMS 2, STAI and VAS. AIS and CFS scores also showed no significant differences. No adverse effects were observed with 4 weeks of heat-killed L. helveticus MCC1848 intake. These results suggest that daily consumption of heat-killed L. helveticus MCC1848 is safe and has the potential to improve positive mood states. UMIN Clinical Trial Registry: UMIN000043697.

Bioactive factors secreted by Bifidobacterium breve B-3 enhance barrier function in human intestinal Caco-2 cells.

Intestinal barrier function is closely related to intestinal health and diseases. Recent studies demonstrate that some probiotic and commensal bacteria secrete metabolites that are capable of affecting the intestinal functions. The present study examined an enhancing effect of bioactive factors secreted by Bifidobacterium breve strain B-3 on the intestinal tight junction (TJ) barrier integrity in human intestinal Caco-2 cells. Administration of conditioned medium obtained from B. breve strain B-3 (B3CM) to Caco-2 cells for 24 h increased trans-epithelial electrical resistance (TER), a TJ barrier indicator, across their monolayers. Immunoblot, immunofluorescence, and qPCR analyses demonstrated that B3CM increased an integral TJ protein, claudin-4 expression. In luciferase reporter assay, the administration of B3CM enhanced the claudin-4 promoter activity, indicating the transcriptional upregulation of claudin-4. Site-directed mutation of specificity protein 1 (Sp1) binding sites in the claudin-4 promoter sequence and suppression of Sp1 expression by siRNA technology clearly reduced the enhancing effect of B3CM on claudin-4 promoter activity. Liquid chromatography/mass spectrometry detected a significant amount of acetic acid in B3CM (28.3 mM). The administration of acetic acid to Caco-2 cells partially mimicked a B3CM-mediated increase in TER, but failed to increase claudin-4 expression. Taken together, bioactive factors secreted by B. breve B-3 enhanced the TJ barrier integrity in intestinal Caco-2 cells. Transcriptional regulation of claudin-4 through Sp1 is at least in part one of the underlying molecular mechanisms. In addition, acetic acid contributes to the B3CM-mediated barrier effect independently of claudin-4 expression.

Deciphering the influence of paraimmunobiotic bifidobacteria on the innate antiviral immune response of bovine intestinal epitheliocytes by transcriptomic analysis.

Previously, we reported that the non-viable immunomodulatory Bifidobacterium infantis MCC12 and Bifidobacterium breve MCC1274 strains (paraimmunobiotic bifidobacteria) were able to increase the protection against rotavirus infection in bovine intestinal epithelial (BIE) cells. In order to gain insight into the influence of paraimmunobiotic bifidobacteria on the innate antiviral immune response of BIE cells, their effect on the transcriptomic response triggered by Toll-like receptor 3 (TLR3) activation was investigated. By using microarray technology and qPCR analysis, we obtained a global overview of the immune genes involved in the innate antiviral immune response in BIE cells. Activation of TLR3 by poly(I:C) in BIE cells significantly increased the expression of interferon (IFN)-α and IFN-ß, several interferon-stimulated genes, cytokines, and chemokines. It was also observed that both paraimmunobiotic bifidobacteria differently modulated immune genes expression in poly(I:C)-challenged BIE cells. Most notable changes were found in genes involved in antiviral defence (IFN-ß, MX1, OAS1X, MDA5, TLR3, STAT2, STAT3), cytokines (interleukin (IL)-6), and chemokines (CCL2, CXCL2, CXCL6) that were significantly increased in bifidobacteria-treated BIE cells. B. infantis MCC12 and B. breve MCC1274 showed quantitative and qualitative differences in their capacities to modulate the innate antiviral immune response in BIE cells. B. breve MCC1274 was more efficient than the MCC12 strain to improve the production of type I IFNs and antiviral factors, an effect that could be related to its higher ability to protect against rotavirus replication in BIE cells. Interestingly, B. infantis MCC12 showed a remarkable anti-inflammatory effect. The MCC12 strain was more efficient to reduce the expression of inflammatory cytokines and chemokines (IL-16, IL-20, CX3CL1) when compared with B. breve MCC1274. These results provided valuable information for the deeper understanding of the antiviral immune response of intestinal epithelial cells as well as the host-paraimmunobiotic interaction in the bovine host.

Effects of paraprobiotic Lactobacillus paracasei MCC1849 supplementation on symptoms of the common cold and mood states in healthy adults.

We investigated the effects of paraprobiotic Lactobacillus paracasei MCC1849 (LAC-Shield™) on symptoms of the common cold and mood states in healthy young adults. A total of 241 participants were randomised to receive 1×1010 heat-killed L. paracasei MCC1849 cell powder (10LP), 3×1010 heat-killed L. paracasei MCC1849 cell powder (30LP), or placebo powder without any L. paracasei cells once daily for 12 weeks based on the incidence of the common cold in the previous year, so that the risk of the incidence was equal among the groups. The incidence and severity of common cold symptoms were rated daily in a subject diary. Salivary secretory immunoglobulin A concentrations and saliva flow rates were analysed at 0 and 6 weeks. The Profile of Mood States (POMS) was assessed using POMS 2 0, 6, and 12 weeks after the intervention. No significant differences were observed in the incidence of the common cold among the groups. In a prespecified subgroup of subjects who had the common cold in the previous year, the incidence, total number of days of symptoms, and symptom scores of the common cold significantly improved in the 10LP-intake group, and were slightly lower in the 30LP-intake group than in the placebo group. The level of deterioration in the positive mood state caused by stress was less in the MCC1849-intake group than in the placebo group. These results indicate that L. paracasei MCC1849 has the potential to improve resistance to common cold infections in susceptible subjects and maintain a desirable mood state, even under mental stress conditions. Further randomised controlled trials are needed in order to investigate the possible beneficial effects of paraprobiotic L. paracasei MCC1849 on the common cold in susceptible populations.

Fluctuation of fecal microbiota in individuals with Japanese cedar pollinosis during the pollen season and influence of probiotic intake.

BACKGROUND: We have previously reported the results of a randomized, double-blind, placebo-controlled trial that found the intake of yogurt supplemented with a probiotic strain, Bifidobacterium longum BB536, alleviates symptoms and affects blood parameters in individuals with Japanese cedar pollinosis (JCPsis) during the pollen season. OBJECTIVE: In the present study, fecal microbiota were investigated to examine whether any changes occur during the pollen season and whether any influence is exerted by probiotic intake. METHODS: Yogurt either with BB536 (BB536 yogurt) or without BB536 (placebo yogurt) was administered for 14 weeks at 2 x 100 g per day to 40 subjects (17 men, 23 women) with a clinical history of JCPsis. Fecal samples were obtained from 23 subjects (placebo group, n=13; BB536 group, n=10) before and during the intervention (weeks 4, 9 and 13) and fecal microbiota were analyzed using terminal-restriction fragment length polymorphism and real-time polymerase chain reaction (PCR) methods. RESULTS: From the fluctuation patterns of terminal-restriction fragments, the Bacteroides fragilis group and bifidobacteria were among the species that changed most with pollen dispersion. Real-time PCR analyses indicated that the cell numbers of the B fragilis group increased significantly along with pollen dispersion in both BB536 and placebo groups. Cell numbers of bifidobacteria were significantly higher in the BB536 group compared with the placebo group (P < .05 at weeks 4 and 9). The ratio of cell numbers of the B fragilis group to bifidobacteria increased significantly during the pollen season in the placebo group (P < .01 at weeks 9 and 14), but not in the BB536 group. An in vitro study using peripheral blood mononuclear cells from JCPsis subjects indicated that strains of the B fragilis group induced significantly more helper T cell (T(H)) type2 cytokines (interleukin [IL]-6) but fewer T(H)1 cytokines (IL-12 and interferon) compared with those of bifidobacteria. CONCLUSIONS: These results suggest a relationship between fluctuation in intestinal microbiota and pollinosis allergy. Furthermore, intake of BB536 yogurt appears to exert positive ihfluences on the formation of anti-allergic microbiota.

Development of an in vitro immunobiotic evaluation system against rotavirus infection in bovine intestinal epitheliocytes.

The bovine intestinal epithelial cell line (BIE cells) expresses the Toll-like receptor (TLR)3 and is able to mount an antiviral immune response after the stimulation with poly(I:C). In the present study, we aimed to further characterise the antiviral defence mechanisms in BIE cells by evaluating the innate immune response triggered by rotavirus (RV) infection. In addition, we attempted to determine whether immunobiotic bifidobacteria are able to confer protection of BIE cells against RV infection by beneficially modulating the antiviral immune response. RV OSU (porcine) and UK (bovine) effectively infected BIE cells, while a significant lower capacity to infect BIE cells was observed for human (Wa) and murine (EW) RV. We observed that viral infection in BIE cells triggered TLR3/RIG-I-mediated immune responses with activation of IRF3 and TRAF3, induction of interferon beta (IFN-ß) and up-regulation of inflammatory cytokines. Our results also demonstrated that preventive treatments with Bifidobacterium infantis MCC12 or Bifidobacterium breve MCC1274 significantly reduced RV titres in infected BIE cells and differentially modulated the innate immune response. Of note, both strains significantly improved the production of the antiviral factor IFN-ß in RV-infected BIE cells. In conclusion, this work provides comprehensive information on the antiviral immune response of BIE cells against RV, that can be further studied for the development of strategies aimed to improve antiviral defences in bovine intestinal epithelial cells. Our results also demonstrate that BIE cells could be used as a newly immunobiotic evaluation system against RV infection for application in the bovine host.

Effect of probiotic Bifidobacterium longum BB536 [corrected] in relieving clinical symptoms and modulating plasma cytokine levels of Japanese cedar pollinosis during the pollen season. A randomized double-blind, placebo-controlled trial.

Probiotic microorganisms have been shown to be effective in the treatment of allergic inflammation and food allergy, but their efficacy remains controversial. This study tested the effect of a yogurt supplemented with a probiotic strain Bifidobacterium longum BB536 in the treatment of Japanese cedar pollinosis (JCPsis). Forty subjects with a clinical history of JCPsis were given yoghurt either containing BB536 (BB536 yoghurt) or without BB536 (placebo yoghurt) at 2 X 100 g per day for 14 weeks, in a randomized, double-blind, placebo-controlled trial. Subjective symptoms and self-care measures were recorded daily and blood samples were taken before and during the intervention (at weeks 4, 9, and 14) to measure the blood parameter levels related to JCPsis. Yoghurt supplemented with BB536 significantly alleviated eye symptoms compared with placebo yoghurt (odds ratio 0.31; 95% confidence interval 0.10-0.97; p = 0.044). Although no statistically significant differences were detected, nasal symptoms such as itching, rhinorrhea, and blockage, as well as throat symptoms tended to be relieved with the BB536 yoghurt. BB536 tended to suppress the decreasing blood levels of interferon-gamma (IFN-y) and the increasing blood eosinophil rates; a significantly higher IFN-gamma level was observed for the difference from baseline at week 4. A decreased trend in the difference from baseline levels of JCP-specific IgE levels was also observed at week 4 in the BB536 group compared with the placebo group. In conclusion, these results suggest that intake of BB536-supplemented yoghurt may relieve JCPsis symptoms, probably through a modulating effect on Th balance.

Bifidobacterium breve prevents necrotising enterocolitis by suppressing inflammatory responses in a preterm rat model.

Necrotising enterocolitis (NEC) is associated with inflammatory responses and barrier dysfunction in the gut. In this study, we investigated the effect of Bifidobacterium breve M-16V on factors related to NEC development using an experimental rat model. Caesarean-sectioned rats were given formula milk with or without B. breve M-16V by oral gavage thrice daily, and experimental NEC was induced by exposing the rats to hypoxic conditions. Naturally delivered rats that were reared by their mother were used as healthy controls. The pathological score of NEC and the expression of molecules related to inflammatory responses and the barrier function were assessed in the ileum. B. breve M-16V reduced the pathological scores of NEC and resulted in some improvement in survivability. B. breve M-16V suppressed the increased expression of molecules related to inflammation and barrier function that resulted from NEC induction. B. breve M-16V normalised Toll-like receptor (TRL)4 expression and enhanced TLR2 expression. Our data suggest that B. breve M-16V prevents NEC development by modulating TLR expressions and suppressing inflammatory responses in a rat model.

Egr-1 mRNA expression is independent of regulatory proliferative responses in the immature B cell line WEHI-231.

We have recently reported cellular growth arrest induced following crosslinking of surface IgM (sIgM) but not surface IgD (sIgD) in the WEHI-231 cell line, representative of the immature B cell stage, and its delta heavy chain (delta) transfectant. An initial report has indicated WEHI-231.7, a subclone of WEHI-231, failed to express Egr-1 mRNA following sIgM crosslinking, in contrast to significant up-regulation found in mature B lymphocytes. The implication for linkage between selective surface immunoglobulin (sIg) signal transduction, expression of immediate/early genes and control of cellular growth imposes an attractive model for induction of immature B cell tolerance. Our investigations examined the relationships between Egr-1 mRNA expression and growth regulation in WEHI-231, WEHI-231.7 and their respective delta-transfectants (WEHI-delta, WEHI-delta 7). We report sIgM and sIgD crosslinking leads to a rapid increase of Egr-1 mRNA expression in WEHI-231 and WEHI-delta but not in the subclone WEHI-231.7 and WEHI-delta 7. Nevertheless, both WEHI-231, WEHI-231.7 and their delta-transfectants demonstrate the ability to induce growth arrest following sIgM but not sIgD crosslinking. Furthermore, we found Egr-1 expression could be achieved by direct activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) circumventing the classical sIg activated phosphatidylinositol signal transduction pathway. Our results suggest Egr-1 expression does not directly participate in growth regulation of immature B cell clones but rather is a consequence of signal transduction through sIg.

Effect of Bifidobacterium breve B-3 on skin photoaging induced by chronic UV irradiation in mice.

Probiotics have been shown to have a preventative effect on skin photoaging induced by short term UV irradiation, however, the underlying mechanisms and the effect of probiotics on skin photoaging induced by chronic UV irradiation remain unclear. In this study, we investigated the effect of Bifidobacterium breve B-3 on skin photoaging induced by chronic UV irradiation in hairless mice. Mice were irradiated with UVB three times weekly and orally administered B. breve B-3 (2×10(9) cfu/mouse /day) for 7 weeks. Nonirradiated mice and UVB-irradiated mice without probiotic treatment were used as controls. B. breve B-3 significantly suppressed the changes of transepidermal water loss, skin hydration, epidermal thickening and attenuated the damage to the tight junction structure and basement membrane induced by chronic UVB irradiation. Administration of B. breve B-3 tended to suppress the UV-induced interleukin-1ß production in skin (P=0.09). These results suggest that B. breve B-3 could potentially be used to prevent photoaging induced by chronic UV irradiation.

Expression of exogenous p59fyn modulates signaling in an immature B cell line, WEHI-231.

The WEHI-231 B lymphoma line is representative of immature B cells, which undergo growth arrest/apoptosis following cross-linking of surface immunoglobulin M (sIgM). In B cells, sIgM engagement has been shown to induce immediate (within seconds) activation of src family protein tyrosine kinases (PTKs) such as p53lyn/56lyn, p55blk, p56lck and p59fyn which are associated with B cell antigen receptor (BCR) complex. However, p59fyn expression is very low in both normal immature B cells and apoptosis-prone B cell lines, including WEHI-231. Such a finding prompted us to investigate the effects of ectopic expression of p59fyn in growth regulation of WEHI-231 cells. We have obtained WEHI-231 transfectants expressing the exogenous p59fyn by retroviral mediated gene transfer method. The transfectants demonstrated increased [Ca2+]i level in both the non-stimulated condition and sIgM cross-linking. The expression of ectopic p59fyn also increased the sensitivity of the transfectants to growth arrest signal by sIgM cross-linking. The results suggest that p59fyn can modulate signal transduction and growth regulation when expressed in the immature B cell line.

Hepatitis B virus DNA integration in hepatocellular carcinomas and their adjacent non-neoplastic liver tissues.

Hepatitis B virus (HBV) DNA integration was studied in 24 hepatocellular carcinoma (HCC) tissues obtained at operations or autopsies. In 11 cases whose sera were positive for hepatitis B virus surface antigen (HBsAg), HBV DNA integration was demonstrated by Southern blot analysis. Only one of 6 cases whose sera were positive for hepatitis B surface antibody (HBsAb) but negative for HBsAg revealed the integration and the other 5 cases revealed no HBV DNA integration. HBV DNA amplification was noted in 4 of these 6 cases in which HBV DNA integration was found when compared with the adjacent liver tissues. The integration pattern of HBV DNA was different in one case between primary HCC tissue and a metastasized lymph node. It is suggested that HBV DNA amplification is not directly related to the development of HCC and that there are polyclonal tumor cells which have different patterns of virus genome integrations.

Expression of hepatitis B surface antigen in Chang cells transfected with hepatitis B virus DNA.

To investigate hepatitis B virus (HBV) biology in vitro, the transfection of recirculized HBV DNA into Chang cell line was performed. Linear HBV DNA was isolated from recombinant HBV DNA, pHBR105, which includes the whole genome of HBV and was recirculized. Chang cells were transfected with this recirculized HBV DNA by the two different procedures of calcium/phosphate coprecipitation and electroporation. After the transfection, the presence of large nucleated cells with multinuclei and ground-glass cytoplasma were noticed and these cells seemed to proliferate faster than untreated Chang cells. Transient expression of hepatitis B virus surface antigen (HBsAg) was demonstrated in cytoplasma of transfected cells by indirect immunofluorescence. HBsAg was not detected in the culture supernatants by radioimmunoassay. The extra-chromosomal HBV DNA was detected in the transfected cells by both procedures 7 weeks after the transfection by Southern blot analysis but it was lost 4 weeks after that. It was demonstrated that it was possible to transfect Chang cells with HBV DNA and that DNA was functioning to express HBsAg transiently.

Extensive, obstructing sodium urate deposition on wall of isolated ileum for replacement of entire ureter.

We report a rare case of obstructing sodium urate stones occurring in the form of extensive deposition on the wall of isolated ileum for replacement of the entire ureter. The stones were disintegrated and removed with a rigid ureteroscope and an electrohydraulic lithotriptor. Hyperuriaciduria, excessive alkalization of urine and urinary stagnation due to reflux, mucous retention, and prostatic hyperplasia were considered as the etiologic factors in the stone formation.

Antibody-dependent cell-mediated cytotoxicity against cell lines generated by liver-specific idiotype-bearing antibody.

We produced a murine monoclonal antibody (mAb), designated H2-mAb, against a fractionated soluble phase of human liver homogenate which antibody reacted with human liver cells. A human antibody possessing the same idiotype as the H2-mAb, designated LSIA (liver-specific idiotype-bearing antibody), can be measured by a sandwich enzyme-linked immunosorbent assay, using the anti-H2 idiotype antibody. The serum level of LSIA in patients with histologically proven chronic hepatitis (CH) was significantly higher than that in healthy subjects and it was also higher than that in subjects with other diseases, including systemic lupus erythematosus. In a comparison between patients with CH type B and those with CH type C, there was no significant difference in serum levels of LSIA. It was possible to purify LSIA from the sera of patients with CH. The purified LSIA bound to the human cell lines Chang and HCC-M, derived from liver cells and a hepatoma respectively, but not to HeLa cells, a uterine carcinoma derivative. The reactivity of this mAb to HCC-M was weaker than that to Change. Moreover, the presence of LSIA caused an antibody-dependent cell-mediated cytotoxic challenge against Change cells in vitro.

Assessment of histological features and outcome of interferon therapy in chronic hepatitis C.

The correlation between the histological features of liver biopsy specimens before interferon (IFN) treatment and the clinical effect of IFN administration on chronic hepatitis C was investigated. A study of the relation between several histological features that were graded in 60 liver biopsy specimens from chronic hepatitis C patients before IFN treatment disclosed that the grade of portal fibrosis was positively correlated with the grade of other inflammatory features, including piecemeal necrosis and portal and lobular inflammation. The degree of portal fibrosis adversely affected the rate of normalization of ALT levels in chronic hepatitis C during and after IFN treatment. We reexamined 36 liver biopsy specimens that showed a moderate degree of portal fibrosis, and found that the degree of piecemeal necrosis was inversely correlated with the extent of lymphoid follicle formation in the portal tracts. During IFN therapy, the group of chronic hepatitis C patients who showed marked piecemeal necrosis and less lymphoid follicle formation in the liver specimens had a poor response to IFN treatment, whereas another group that showed marked lymphoid follicle formation and little piecemeal necrosis in the liver specimens had a good response to IFN. These relationships gradually disappeared after the completion of IFN treatment.

Aspergillus aneurysm of the middle cerebral artery causing a fatal subarachnoid hemorrhage.

A rare case of Aspergillus aneurysm of the central nervous system (CNS) leading to subarachnoid hemorrhage (SAH) is reported. An 83-year-old woman developed visual disturbance and headache. Computed tomographic scans showed no evidence of aneurysm or tumor in the intracranium. She suddenly died from SAH. Autopsy revealed massive SAH due to ruptured Aspergillus aneurysm of the middle cerebral artery. Aspergillus was suggested to have extended from the paranasal sinuses. Aspergillosis of CNS should be considered in patients with neurological symptoms such as visual disturbance and trigeminal neuralgia, especially in cases of the aged or immunocompromised.

Adhesion and releasing of Poliovirus to activated sludge of wastewater purifying plants.

Pathogenic enteric viruses are released from infected persons through domestic wastewater to the environment. From that point of view, the knowledge of the viral behavior in wastewater purifying process is important: it is, however, still poorly understood. In this study, we reported the adhesion of Poliovirus to activated sludge samples taken from wastewater purifying plants by using a model system. More than 10(6) particles adhered to one gram (wet) of activated sludge, and the adhered viral particles maintained infectivity for longer period of time and showed higher thermo-resistant than the free viral particles. The adhered viral particles were released by increase of salt concentration or alkaline pH buffer as infectious particles. The data suggest that pathogenic viruses could be enriched and maintain the infectivity in the activated sludge, and released to environments under certain conditions.

[A case of toxic shock-like syndrome due to Streptococcus pyogenes].

A case of toxic-shock syndrome due to Streptococcus pyogenes is reported. A 76-year-old female was admitted with complaints of fevers and chills. She had been suffering from cellulitis on her right dorsum pedis for 7 months. Laboratory data on admission showed elevated values of WBC, CRP, and dysfunction of the liver and kidney. She was diagnosed as sepsis due to the cellulitis, and was treated with PIPC and FMOX. However, several hours after admission, her blood pressure decreased and oliguria appeared. Bacteriological examinations from the blood and the cellulitis revealed group A beta-hemolytic Streptococcus which gave streptococcal pyrogenic exotoxin (T-28, SPE.B + C). She died 23 hours after her admission in spite of changing antibiotics to a high-dose of PC-G therapy. This is one of the rare cases of toxic shock-like syndrome due to Streptococcus pyogenes from the cellulitis of the dorsum pedis.

[A case of carcinomatous peritonitis for which combination therapy of 5-FU, leucovorin and cisplatin was effective].

This case report describes a case of carcinomatous peritonitis which showed a good response to the combination therapy of 5-FU, leucovorin and cisplatin. A 55-year-old man was admitted with complaints of meteorism due to ascites fluid. Ascitic examination showed a high concentration of CEA and CA19-9, and adenocarcinoma cells. Laboratory and radiological examination did not reveal the origin of the carcinoma, and laparotomy suggested an appendiceal origin. The combination chemotherapy with 5-FU, leucovorin and cisplatin was conducted for a total of 11 courses for 2 years with good QOL until his death. This therapy was suggested to be useful for inoperable carcinomatous peritonitis.