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.

IL-1ß in eosinophil-mediated small intestinal homeostasis and IgA production.

Eosinophils are multifunctional leukocytes that reside in the gastrointestinal (GI) lamina propria, where their basal function remains largely unexplored. In this study, by examining mice with a selective deficiency of systemic eosinophils (by lineage ablation) or GI eosinophils (eotaxin-1/2 double deficient or CC chemokine receptor 3 deficient), we show that eosinophils support immunoglobulin A (IgA) class switching, maintain intestinal mucus secretions, affect intestinal microbial composition, and promote the development of Peyer's patches. Eosinophil-deficient mice showed reduced expression of mediators of secretory IgA production, including intestinal interleukin 1ß (IL-1ß), inducible nitric oxide synthase, lymphotoxin (LT) α, and LT-ß, and reduced levels of retinoic acid-related orphan receptor gamma t-positive (ROR-γt(+)) innate lymphoid cells (ILCs), while maintaining normal levels of APRIL (a proliferation-inducing ligand), BAFF (B cell-activating factor of the tumor necrosis factor family), and TGF-ß (transforming growth factor ß). GI eosinophils expressed a relatively high level of IL-1ß, and IL-1ß-deficient mice manifested the altered gene expression profiles observed in eosinophil-deficient mice and decreased levels of IgA(+) cells and ROR-γt(+) ILCs. On the basis of these collective data, we propose that eosinophils are required for homeostatic intestinal immune responses including IgA production and that their affect is mediated via IL-1ß in the small intestine.

A new objective histological scale for studying human photoaged skin.

BACKGROUND: A quantitative understanding of the histological alteration of the skin is important for assessing the severity of photoaging. METHODS: We performed Elastica-van Gieson staining and immunohistochemistry for decorin on 34 facial skin sections. We evaluated the alteration of collagen fibers and decorin (a modulator for collagen fibrillogenesis), according to the 5 grades of morphological change in elastic fibers that was established by Kligman (1969). The objectivity of a stage (Stages I-VI), which was established in this study, was evaluated using weighted kappa statistical analysis based on the degree of agreement in stage determination by 11 observers using a blind procedure. Correlation between the crow's-feet-area wrinkles grades of another 26 women and stages was also analyzed. RESULTS: The initial alteration of elastic fibers was observed in the deep dermis. Decorin was not detected in very severely altered skin. Based on the combination of changes in the elastic fibers, collagenic fibers, and decorin, skin tissues were categorized into 6 stages according to severity. The statistical analysis showed almost perfect agreement between observers. Significant positive correlation between stages and wrinkle scores was found. CONCLUSIONS: We propose a new objective histological scale that is useful for assessing the severity of photoaging.

The endoscopic diagnosis of nonerosive reflux disease using flexible spectral imaging color enhancement image: a feasibility trial.

Nonerosive reflux disease (NERD) is classified into grade M (minimal change, endoscopically; erythema without sharp demarcation, whitish turbidity, and/or invisibility of vessels due to these findings) and grade N (normal) in the modified Los Angeles classification system in Japan. However, the classification of grades M and N NERD is not included in the original Los Angeles system because interobserver agreement for the conventional endoscopic diagnosis of grades M or N NERD is poor. Flexible spectral imaging color enhancement (FICE) is a virtual chromoendoscopy technique that enhances mucosal and vascular visibility. The aim of this study is to evaluate whether the endoscopic diagnosis of grades M or N NERD using FICE images is feasible. Between April 2006 and May 2008, 26 NERD patients and 31 controls were enrolled in the present study. First, an experienced endoscopist assessed the color pattern of minimal change in FICE images using conventional endoscopic images and FICE images side-by-side and comparing the proportion of minimal change between the two groups. Second, three blinded endoscopists assessed the presence or absence of minimal change in both groups using conventional endoscopic images and FICE images separately. Intraobserver variability was compared using McNemar's test, and interobserver agreement was described using the kappa value. Minimal changes, such as erythema and whitish turbidity, which were detected using conventional endoscopic images, showed up as navy blue and pink-white, respectively, in color using FICE images in the present FICE mode. The NERD group had a higher proportion of minimal change, compared with the control group (77% and 48%, respectively) (P= 0.033). In all three readers, the detection rates of minimal change using FICE images were greater than those using conventional endoscopic images (P= 0.025, <0.0001, and 0.034 for readers A, B, and C, respectively). The kappa values for all pairs of three readers using FICE images were between 0.683 and 0.812, while those using conventional endoscopic images were between 0.364 and 0.624. Thus, the endoscopic diagnosis of grades M or N NERD using FICE images is feasible and may improve interobserver agreement.

Primary mucinous carcinoma of the skin: a case of metastasis after 10 years of disease-free interval.

Primary mucinous carcinoma of the skin (MCS) is a rare neoplasm. Clinically, it has a high local recurrence rate, but it is known to be a slow-growing benign tumor with a rare incidence of distant metastases. We present a case of primary MCS on the jaw that underwent tumor resection twice and was disease-free for 10 years after the second surgery. The patient had no evidence of local recurrence and distant metastasis until his 11th year follow-up. At that time, he was diagnosed with lung and bone metastasis and died 3 years after this. To our knowledge, this is the first case of MCS that presented with metastasis with more than 10-year disease-free interval. Since MCS is a slow-growing asymptomatic tumor, distant metastasis is difficult to diagnose without detailed radiological examination. We believe that computed tomography and resonance imaging should be performed for early diagnosis of metastasis even for cases with long-term disease-free interval, especially cases of local recurrence.

Expression and role of mannose receptor/terminal high-mannose type oligosaccharide on osteoclast precursors during osteoclast formation.

Osteoclasts are formed from hematopoietic precursors via cell-cell fusion. We have previously reported that mannose residues are expressed on the outer membranes of monocytes during osteoclast differentiation. In the present study, we have attempted to demonstrate the pattern of expression levels of terminal high-mannose type oligosaccharide and to show that the mannose receptor is expressed on osteoclast precursor cells. Osteoclasts were formed using three different systems, namely mouse bone marrow cell culture, co-culture of mouse spleen cells with stromal cells, and RAW264.7 cell cultures. During osteoclast differentiation, the expression of terminal high-mannose type oligosaccharide gradually increased and then peaked at the stage of fusion in all three systems. Expression of the mannose receptor gradually increased during osteoclast differentiation in bone marrow cells and the co-culture system. In contrast, that in RAW264.7 cells had already been detected in the absence of the soluble receptor activator of NF-kappaB ligand and did not change during osteoclast differentiation. To ascertain whether expression of high-mannose type oligosaccharide is involved in tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cell (MNC) formation, glycosidase inhibitors were used on RAW264.7 cell culture. Castanospermine, an inhibitor of glucosidase I, inhibited the TRAP-positive MNCs, and deoxymannojirimycin, an inhibitor of alpha-mannosidase I, increased the TRAP-positive MNC formation. These results indicate that the binding of terminal high-mannose and mannose receptor is important for the process of cellular fusion in osteoclast formation.

Preventive effect of sulphated colominic acid on P-selectin-dependent infiltration of macrophages in experimentally induced crescentic glomerulonephritis.

Leucocytes infiltrate into renal tissue and are involved in the pathogenesis of crescentic glomerulonephritis. The initial event in the process of leucocyte infiltration is characterized by selectin-mediated leucocyte rolling on endothelial surface. Role of selectins in pathogenesis of glomerulonephritis has still been controversial. Sulphated glycolipids and sulphated polysaccharides interfere with the binding of P- and L-selectin with carbohydrate ligands on endothelial cells or on leucocytes. Here we evaluated the role of selectins and the preventive effects of sulphated colominic acid (SCA), a synthetic sulphated polysaccharide, on experimental crescentic glomerulonephritis in Wistar-Kyoto (WKY) rats. Crescentic glomerulonephritis was induced by injection of nephrotoxic serum (NTS) in WKY rats. Rats subsequently received intraperitoneal injection of saline, neutralizing or non-neutralizing monoclonal antibody (mAb) to rat P-selectin and L-selectin, SCA (5 or 10mg/kg/day) or nonsulphated colominic acid (CA) (10mg/kg/day) for 2 weeks. Localization of P-, E-selectin, ligands for L-selectin and intraglomerular leucocytes was examined by immunohistochemistry. Gene expression of platelet-derived growth factor (PDGF) B chain in glomeruli was quantified using real-time RT-PCR. P-selectin was highly expressed on glomerular endothelial cells after injection of NTS, whereas E-selectin and L-selectin ligands were not detected. Anti-P-selectin mAb, but not anti-L-selectin mAb, significantly reduced glomerular infiltration of macrophages, crescent formation, and proteinuria. SCA also reduced proteinuria, macrophage infiltration, and crescent formation in a dose-dependent manner. Furthermore, SCA suppressed gene expression of PDGF B chain in glomeruli. Our results indicate that P-selectin partially mediates glomerular infiltration of macrophage in experimental crescentic glomerulonephritis. Moreover, SCA may inhibit intraglomerular infiltration of macrophages by interfering with P-selectin-dependent adhesion pathway, and progression of experimental crescentic glomerulonephritis.

Locally expressed CTLA4-Ig in a pancreatic beta-cell line suppresses accelerated graft rejection response induced by donor-specific transfusion.

AIMS/HYPOTHESIS: This study examined whether locally expressed CTLA4-Ig can suppress the accelerated islet allograft rejection that is induced by donor-specific transfusion. METHODS: CTLA4-Ig-transfected or parental MIN6 cells were transplanted subcutaneously into the right flank of streptozotocin-induced diabetic C3H/Hej mice with or without donor-specific transfusion. For donor-specific transfusion, spleen cells from C57BL/6 mice were injected i.v. at the time of transplantation. In other experiments, CTLA4-Ig-transfected and parental MIN6 cells were transplanted separately into each flank, together with donor-specific transfusion. Rejection was defined as a blood glucose concentration of more than 300 mg/dl in two consecutive measurements, and graft survival was confirmed by hyperglycaemia after the grafts were removed. The effect of an anti-CTLA4 antibody on the survival of CTLA4-Ig-transfected MIN6 cells was also examined. RESULTS: In 7 of 12 donor-specific transfusion sensitised mice, CTLA4-Ig-transfected MIN6 cells remained viable 20 days after grafting, whereas all parental MIN6 cells ( n = 10) were rejected promptly, within 14 days. The prolonged allograft survival was observed even in the absence of detectable levels of serum CTLA4-Ig, while the surviving allografts continued to produce CTLA4-Ig in situ. This protection was abrogated by an anti-CTLA4 antibody, but not by a control antibody. Furthermore, six animals that maintained normoglycaemia after the separate transplantation of parental and CTLA4-Ig-transfected MIN6 cells into each flank all showed abrupt hyperglycaemia after the CTLA4-Ig/MIN6 graft was removed, suggesting that this protection operated locally. CONCLUSION/INTERPRETATION: A beta-cell line genetically engineered to secrete CTLA4-Ig can protect a graft locally from the alloimmune response induced by donor-specific transfusion.

Involvement of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the pathogenesis of granulomatous colitis in rats.

Although increased expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) has been demonstrated in inflammatory sites of various diseases, its role in colitis remains unknown. In this study, we examined whether MAdCAM-1 is involved in the pathogenesis of granulomatous colitis induced by peptidoglycan-polysaccharide (PG-PS). Experimental colitis was induced by intramural injection of PG-PS to rat colon. After 3 weeks the colon was removed and the mucosal inflammation was assessed. The area of MAdCAM-1-positive venules and the subsets of infiltrating cells were determined in colonic mucosa by immunohistochemistry. In another experiment, monoclonal antibody against MAdCAM-1 was administered intraperitoneally to examine its attenuating effect on colitis. The intramural injection of PG-PS induced significant colonic inflammation with granuloma formation. The submucosa was drastically thickened with the infiltration of CD4 positive lymphocytes and ED-1 positive macrophages. Intense MAdCAM-1 expression was observed on endothelium of the submucosal venules in inflamed mucosa. Administration of anti-MAdCAM-1 antibody significantly attenuated the PG-PS-induced colonic damage and cell infiltration. Enhanced expression of MAdCAM-1 was demonstrated in venular endothelium of the inflamed colon in PG-PS-induced colitis. The attenuating effect of anti-MAdCAM-1 suggests the importance of the MAdCAM-1-dependent process in the formation of chronic granulomatous colitis.

The role of leukocytes during acute phase inflammation in crystalline silica-induced lung injury.

Silicosis is characterized by progressive granulomatous and fibrogenic response in the lung. Inhaled crystalline silica (Qt) induces activation of pulmonary macrophages and leukocyte infiltration in the lung of Qt-treated animals. We investigated the role of leukocyte infiltration and L-selectin during the acute phase of inflammation in developing chronic lung injury in Qt-treated rats. Seventy Wistar male rats were treated with a single transtracheal instillation of Qt (25 mg/kg). Rats were treated intraperitoneally with anti L-selectin monoclonal antibody (mAb), F(ab')2 HRL-3 (HRL-3, a blocking mAb), or RF(ab')2 HRL-2 (HRL-2, a non-blocking mAb)for 4 days before and after Qt injection. Administration of HRL-3 reduced approximately 50% of leukocyte infiltration in the BAL, whereas HRL-2 treatment prior to Qt stimulation showed time-dependent increase of BAL leukocytes. CINC and GRO levels as well as peripheral blood cell counts were similar in HRL-2- or HRL-3-treated animals in the first 4 days of the study. Three months after Qt treatment, extensive granuloma-containing macrophages and leukocytes developed in the lung of the HRL-3-treated rats as compared with the HRL-2-treated rats. Ratio of CD4+ to CD8+ T cells in granulomas did not differ between the HRL-3 and HRL-2 groups. Results suggest that an early phase of leukocyte activation was diminished by blocking L-selectin with the antibody, but treatment with anti-L-selectin increased the formation of granulomas in the Qt-treated rats.

Hepatocyte growth factor/scatter factor is implicated in the mode of stromal invasion of uterine squamous cervical cancer.

OBJECTIVE: The purpose of this study was to examine the relationship of hepatocyte growth factor/scatter factor (HGF/SF) to cell motility and invasion in uterine cervical cancer. METHODS: We examined the expression of HGF/SF and its receptor, c-met, in cervical cancer cell lines SKG-IIIa (squamous cell carcinoma) and Hela-S3 (adenocarcinoma) and in stromal cells of the cervical cancer tissue by reverse transcription-polymerase chain reaction. We studied the effect of HGF/SF on invasiveness of SKG-IIIa and Hela-S3 in an invasion model of the modified Boyden chamber method and by electron microscopy. SKG-IIIa cells were also seeded on the thick Matrigel-coated layer to evaluate the invasion patterns in three-dimensional directions. To investigate the mechanism of an inductive effect of HGF/SF on the invasiveness of SKG-IIIa, we examined the effect of HGF/SF on the expression of intercellular adhesion molecule E-cadherin, cell-substrate adhesion molecules CD44, alpha2beta1, and alpha6beta1, and intracellular skeleton fiber actin in SKG-IIIa in cell enzyme-linked immunosorbent assay (ELISA) and immunofluorescence staining. RESULTS: HGF/SF messenger RNA (mRNA) was detected in stromal cells, and c-met mRNA was detected in SKG-IIIa and Hela-S3. Hela-S3 that initially showed weak intercellular contact freely invaded the Matrigel-coated multiporous membrane without the addition of HGF/SF. In contrast, SKG-IIIa that initially showed strong intercellular adhesion could invade the membrane after the addition of HGF/SF. The same results were represented by an addition of HECD-1, an anti-human E-cadherin antibody. In an experiment with cell culture in a thick Matrigel layer, control SKG-IIIa showed a mirror-ball-like invasion pattern, whereas HGF/SF-stimulated SKG-IIIa spread horizontally over the membrane and migrated through the membrane holes, presenting a tentacular invasion pattern. Migration of SKG-IIIa under the membrane was confirmed by scanning and transmission electron microscopy. The addition of HGF/SF in cell ELISA assay decreased the expression of E-cadherin and actin in SKG-IIIa, but it did not change the expression of CD44, alpha2beta1, and alpha6beta1. Immunofluorescence staining revealed that the expression of E-cadherin in cell membrane was disturbed by HGF/SF. CONCLUSIONS: Our data indicate that HGF/SF produced by stromal cells influences the mode of stromal invasion of squamous cervical cancer by selectively decreasing the expression of both E-cadherin and actin.

A nanometer-sized high-spin polyradical: poly(4-phenoxyl-1,2-phenylenevinylene) planarily extended in a non-Kekulé fashion and its magnetic force microscopic images.

A pi-conjugated, but non-Kekulé- and nondisjoint-type poly(1,2-phenylenevinylene) network bearing 4-substituted di-tert-butylphenoxyls was synthesized through a one-pot polycondensation of the star-shaped subpart and the subsequent oxidation, which was persistent even at room temperature. The polyphenoxyl radical with a spin concentration of 0.4 displayed an average S of 10/2. The polyradical with the molecular weight of 3.2 x 10(4) gave a disklike image of ca. 35 x 0.6 nm with both an atomic and a magnetic force microscopy: the molecular image was examined as a nanoscale and single-molecular-based magnetic dot.

CD44 binds a chondroitin sulfate proteoglycan, aggrecan.

Here we report that CD44 binds a chondroitin sulfate (CS) proteoglycan, aggrecan, a major component of cartilage. Soluble CD44-IgG and CD44(+) cells bound to aggrecan from rat chondrosarcoma and bovine cartilage, immobilized on microtiter plates. In both cases, binding was blocked by a neutralizing anti-CD44 mAb or by the pretreatment of aggrecan with chondroitinase, but not hyaluronidase or keratanase, indicating that CD44 binds aggrecan in a manner dependent on CS side chains of aggrecan and that hyaluronic acid is not involved in the binding. Structural analysis showed that glycosaminoglycans of aggrecan from rat chondrosarcoma and bovine articular cartilage consist of mainly CS A and a mixture of CS A and C respectively. When immobilized on microtiter plates, both CS A and C bound CD44-IgG, and the reaction was specifically inhibited by an anti-CD44 mAb. In addition, aggrecan augmented apoptosis in cells expressing CD44-Fas chimeric molecules in synergy with a non-blocking anti-CD44 mAb IRAWB14.4, suggesting that CD44-aggrecan interaction can induce oligomerization of the chimeric molecules. These results suggest that aggrecan interacts with CD44 to mediate cell adhesion and to trigger oligomerization of CD44 molecules, which may lead to intracellular signaling.

Versican interacts with chemokines and modulates cellular responses.

We previously reported that versican, a large chondroitin sulfate proteoglycan, isolated from a renal adenocarcinoma cell line, ACHN, binds L-selectin. Here we report that versican also binds certain chemokines and regulates chemokine function. This binding was strongly inhibited by the chondroitinase digestion of versican or by the addition of soluble chondroitin sulfate (CS) B, CS E, or heparan sulfate. Furthermore, these glycosaminoglycans (GAGs) could bind directly to the chemokines that bind versican. Thus, versican appears to interact with chemokines via its GAGs. We next examined if versican or GAGs affect secondary lymphoid tissue chemokine (SLC)-induced integrin activation and Ca(2+) mobilization in lymphoid cells expressing a receptor for SLC, CC chemokine receptor 7. Interestingly, whereas heparan sulfate supported both alpha(4)beta(7) integrin-dependent binding to mucosal addressin cell adhesion molecule-1 (MAdCAM-1)-IgG and Ca(2+) mobilization induced by SLC, versican or CS B inhibited these cellular responses, and the extent of inhibition was dependent on the dose of versican or CS B added. These findings suggest that different proteoglycans have different functions in the regulation of chemokine activities and that versican may negatively regulate the function of SLC via its GAG chains.

A case of rupture of the internal jugular vein caused by postoperative infection of functional neck dissection.

A 68-year old diabetic man with gingival cancer of the lower jaw underwent resection of the mandible and functional neck dissection. Swabs of a postoperative wound infection revealed methicillin-resistant Staphylococcus aureus (MRSA). The wound was irrigated, and antibiotics administered. The pathogens isolated were sensitive to the antibiotics used, but the infection failed to respond to treatment. Bleeding ensued on the 14th postoperative day (#14POD), when the wound was opened to reveal thrombosis and rupture of the right internal jugular vein. The patient's condition improved after ligation and surgical debridement of the right internal jugular vein. Our experience underlines the importance of early radiological investigation for possible thrombus formation in the internal jugular vein in cases of postoperative wound infection follows functional neck dissection with conservation of the internal jugular vein. It is also important to actively treat this condition surgically, including ligation of internal jugular vein for suppressing inflammation.

The failure of oral tolerance induction is functionally coupled to the absence of T cells in Peyer's patches under germfree conditions.

Although intestinal bacterial flora has been thought to play a role in the induction of oral tolerance, the mechanism has yet to be elucidated. We therefore examined the bacterial flora-dependent acquisition of susceptibility to oral tolerance induction using a gnotobiotic murine model. Germ-free (GF) mice exhibited a significant shortage of T cells in the PPs in comparison to SPF mice. A recovery in the number of such T cells was accomplished in the gnotobiotic mice associated with Bifidobacterium infantis or Escherichia coli but not in the gnotobiotic mice with Clostridium perfringens or Staphylococcus aureus. To examine the susceptibility to oral tolerance induction, these mice were orally given ovalbumin (OVA) as a tolerogen and then injected i.p. with the Ag. The Ag-specific IgG1 in the serum remained at a low level in both SPF and those gnotobiotic mice groups containing a sufficient number of T cells in the PPs. However, no such unresponsiveness in the Ab response was observed in GF or the other gnotobiotic mice groups containing only a few T cells in the tissues. Adoptive cell transfer analysis clearly showed that a sufficient number of T cells in the PPs is required for the induction of oral tolerance. Furthermore, the reduced expression of SLC (secondary lymphoid-tissue chemokine), which is responsible for T-cell migration to lymphoid organs, was observed in the PPs of GF mice, resulting in a shortage of T cells in the tissues. However, the reduced expression of SLC was restored even in the GF mice after conventionalization, thus suggesting that the failure of oral tolerance induction is functionally coupled to the innate absence of T cells under the GF condition.