Lipoteichoic acids from Lactobacillus strains elicit strong tumor necrosis factor alpha-inducing activities in macrophages through Toll-like receptor 2

Lactobacilli are nonpathogenic gram-positive inhabitants of microflora. At least some Lactobacillus strains have been postulated to have health beneficial effects, such as the stimulation of the immune system. Here we examined the stimulatory effects of lactobacilli on mouse immune cells. All six heat-killed Lactobacillus strains examined induced the secretion of tumor necrosis factor alpha (TNF-alpha) from mouse splenic mononuclear cells, albeit to various degrees. When fractionated subcellular fractions of Lactobacillus casei were tested for NF-kappaB activation and TNF-alpha production in RAW264.7, a mouse macrophage cell line, the activity was found to be as follows: protoplast > cell wall > polysaccharide-peptidoglycan complex. Both crude extracts and purified lipoteichoic acids (LTAs) from two Lactobacillus strains, L. casei and L. fermentum, significantly induced TNF-alpha secretion from RAW264.7 cells and splenocytes of C57BL/6, C3H/HeN, and C3H/HeJ mice but not from splenocytes of C57BL/6 TLR2(-/-) mice. Lactobacillus LTA induced activation of c-Jun N-terminal kinase activation in RAW264.7 cells. Furthermore, in HEK293T cells transected with a combination of CD14 and Toll-like receptor 2 (TLR2), NF-kappaB was activated in response to Lactobacillus LTA. Taken together, these data suggest that LTAs from lactobacilli elicit proinflammatory activities through TLR2.     

Effects of mandibular advancement on supine airway size in normal subjects during sleep

STUDY OBJECTIVES: To examine changes in the upper-airway dimension and its surrounding structures induced by mandibular advancement during sleep. DESIGN: Eleven nonapneic adult males participated in the study. A set of supine lateral cephalograms was taken for each subject at the end of expiration during stage 1 and 2 non-rapid-eye-movement sleep with and without a Klearway appliance (Great Lakes Orthodontics, NY, USA), which was adjusted to 67% of the maximum protrusion position. The Wilcoxon signed-rank test was used to compare changes in the anteroposterior width of the upper airway and the positions of the hyoid bone and third cervical vertebra with and without the appliance. SETTING: N/A. PATIENTS OR PARTICIPANTS: N/A. INTERVENTIONS: N/A. MEASUREMENTS AND RESULTS: The amount of jaw opening was significantly increased by wearing the titratable oral appliance, and the mandibular symphysis moved backward. The sagittal dimension of the superior pharyngeal airway was significantly increased; however, no significant changes were found in the middle and inferior pharyngeal airway. Significant posterior displacement of the hyoid bone and third cervical vertebra was seen. Moreover, significant inferior displacement of the hyoid bone was also seen. The relationship among the mandibular symphysis, the hyoid bone, and the third cervical vertebra remained constant. CONCLUSIONS: Mandibular advancement significantly increases the size of the upper airway in the velopharynx and results in posteroinferior displacement of the hyoid bone and posterior displacement of the third cervical vertebra during sleep.     

Clonality analysis of different histological components in combined small cell and non-small cell carcinoma of the lung

Combined small cell and non-small cell carcinoma is relatively rare in the lung. Examination of the clonal relationship of different components in this type of tumor may give a clue to the rarity. We retrieved 6 such tumors; all 6 had small cell carcinoma and adenocarcinoma components, and 3 had an additional squamous cell carcinoma component. We examined the point mutations in the p53 gene and allelic loss (ie, the loss of heterozygosity [LOH] pattern) of chromosome 3p in each component. p53 mutations were detected in the small cell carcinoma component of 5 tumors and in the non-small cell carcinoma components of 2 tumors. In 1 case, the squamous cell carcinoma component had a p53 mutation locus identical to that in the small cell carcinoma component, but in the other case, the adenocarcinoma component had a different mutation than that in the small cell carcinoma component. Chromosome 3p LOH loci in the squamous cell carcinoma component were present in the small cell carcinoma component in all 3 cases, but some LOH loci were not identical in the small cell carcinoma and adenocarcinoma components in 3 cases. These results suggest that the small cell and squamous cell carcinoma components of combined small cell lung carcinomas have an intimate clonal relationship. On the other hand, the adenocarcinoma component often may be derived from a separate clone or, more likely, undergo a progressive process separate from the squamous cell-small cell carcinoma beginning in a very early stage, that is, before the appearance of p53 and chromosome 3p abnormalities. This tumorigenesis process may explain the relative rarity of combined small cell and non-small cell carcinoma, which occurs primarily in the peripheral lung, an infrequent site of squamous cell carcinoma.     

Localization and expression of chondromodulin-I in the rat cornea

The localization and expression in the rat cornea of chondromodulin-I (ChM-I), an inhibitory angiogenesis factor, were examined by immunohistochemistry, Western blot analysis, ribonuclease protection assay, and real-time PCR assay. We found immunoreactivity for ChM-I in the epithelial layer but not the stromal layer or endothelial layer in the cornea, in addition to the positive ChM-I immunoreactivity in other sites in the eye such as the sclera, retina, and ciliary body. The ChM-I immunoreactivity was most intense at the outside of the basal cells and in their cytoplasm while the intensity of the immunoreactivity decreased gradually from the wing cells to the superficial cells in the corneal epithelial layer. No reactivity however, was detected in the Bowman's membrane or conjunctival epithelial cells which had continuity with the corneal epithelial cells. The expression of ChM-I mRNA was demonstrated in the cornea at one-third less intensity than that in the sclera with choroids and retinal pigment epithelium by ribonuclease protection assay and real-time PCR. ChM-I in the corneal epithelial layer may prevent neovascularization and maintain avascularity in the cornea.     

Deposition of complement protein C3b on mixed self-assembled monolayers carrying surface hydroxyl and methyl groups studied by surface plasmon resonance

Since complement activation is recognized as a common response of the host defense system when an artificial medical device is applied to a patient, great effort has been devoted to studies on the interaction of the complement system with artificial materials. However, some uncertainties remain, partially because of the lack of well characterized surfaces and suitable analytic methods for study of the surface phenomena that occur on artificial materials under physiologic conditions. In this study, we employed self-assembled monolayers (SAMs) and the surface plasmon resonance (SPR) technique to study interactions of the serum complement with well characterized surfaces. Self-assembled monolayers carrying various concentrations of hydroxyl groups were prepared using 11-mercapto-1-undecanol (C11-OH) and one of n-nonanethiol, n-dodecanethiol, and n-hexadecanethiol. The amount of NHS deposition on the SAMs increased with increasing C11-OH content of the SAMs, and the amount of anti-C3b antibody immobilization formed on the NHS deposition layers increased with increasing C11-OH content of the SAMs. These results clearly demonstrate that a large amount of C3b, produced through the activation of the complement system, binds covalently to and is adsorbed by hydroxyl-group-rich surfaces. The combination of SAMs and the SPR technique is suitable for studying the interaction of the complement system with solid surfaces, and the results should give basic information needed for a rational design of biocompatible surfaces on synthetic materials.     

Development of early apopotosis and changes in lymphocyte subsets in lymphoid organs of mice orally inoculated with nivalenol

Development of early apoptosis and changes in lymphocyte subsets were examined in lymphoid organs of female BALB/c mice after oral administration of 15 mg/kg b.w. of nivalenol (NIV), the major type B trichothecene mycotoxin, by FACS analysis. Judging from the results of viable cell count and apoptotic cell index, NIV attacked Peyer's patches first and thymus most severely. In thymus, selective damage in CD4(+)CD8(+) cells was observed at 12 and 24 h after inoculation (HAI), following the peak of apoptosis at 9 HAI. CD4(+) cells were clearly suppressed at 3 HAI in Peyer's patches, at and after 9 HAI in mesenteric lymph nodes, and 3 to 12 HAI in spleen, respectively. CD8(+) cells were also suppressed at 24 HAI in mesenteric lymph nodes and at 12 HAI in spleen, respectively. As to changes in B cell subsets, IgG(+) cells significantly decreased from 3 to 12 HAI and all B cell subsets at 24 HAI in mesenteric lymph nodes. In spleen, IgM(+) cells were suppressed at 9 HAI. On the other hand, in Peyer's patches, following clear decrease in the numbers of pan-T and pan-B cells and viable cells at 3 HAI, all B cell subsets, especially IgA(+) cells, showed a significant increase in their numbers at 9 HAI, and the numbers of IgA(+) and IgM(+) cells remained higher values than controls thereafter. Taken together, in the course of recovery from NIV-induced prominent damage in Peyer's patches at 3 HAI, interaction of NIV with Peyer's patches might result in in vivo stimulation of interleukin production at this site and result in increased proliferation and differentiation of IgA-secreting B cells at and after 9 HAI.     

Immunohistochemical characteristics of duodenal adenomas in familial adenomatous polyposis with special reference to cell kinetics

The duodenum is the second most frequent site of cancer in patients with familial adenomatous polyposis (FAP). The main objective of this study was to evaluate the cell kinetics in duodenal and ampullary adenomas in FAP. The endoscopic and biopsy findings of duodenal adenomas in 22 FAP subjects and 18 non-FAP subjects were compared. Adenomas in FAP included 15 ampullary adenomas and 17 nonampullary adenomas. The cell kinetics was evaluated by immunohistochemistry for Ki-67, p53, bcl-2, and cyclooxygenase 2 (COX2), and the apoptotic index (AI) as determined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) method. Any correlations between the indices for cell kinetics and the endoscopic findings were identified. All 50 adenomas were histologically verified to be tubular adenoma with low-grade dysplasia. Neither the expression of Ki-67, p53, bcl-2, and COX2 nor the AI differed substantially between FAP and non-FAP subjects. In patients with FAP, duodenal adenoma tended to have a higher Ki-67-labeling index than the ampullary adenoma (54.3 +/- 11.3 versus 46.8 +/- 12.7; .05 < P < .1). In addition, the Ki-67-labeling index in endoscopically normal or slightly enlarged ampullary adenoma was significantly higher than that in markedly enlarged ampullary adenoma (51.8 +/- 11.4 versus 39.4 +/- 11.3; P < .05). Duodenal adenoma in FAP subjects was not found to have a higher proliferative activity or a smaller degree of apoptosis compared with those in non-FAP subjects. The smaller proliferative activity in larger ampullary adenoma may thus be related to the static nature of ampullary adenoma in FAP.     

Astroglial responses against Abeta initially occur in cerebral primary cortical cultures: species differences between rat and cynomolgus monkey.

In the present study, we investigated how amyloid beta (Abeta) peptides initially affect neuronal cells in primary cerebral cortical cultures from rat and cynomolgus monkey. In these cultures, complicated interactions between glial and neuronal cells occur; moreover, synaptic interactions similar to those observed in vivo also occur between neuronal cells in these cultures. In this study, we applied low concentrations of Abeta to these well-characterized primary cultures to investigate how Abeta initially affects neurons or astroglial cells. In both rat and monkey cortical cultures, treatment with low concentrations of Abeta failed to drastically change or damage of neurons. Abeta treatment, however, significantly activated astrocytes, resulting in increased apolipoprotein E (ApoE) production. Rat astrocytes were more sensitive to Abeta than monkey astrocytes, and responded to Abeta via a different mechanism. In monkey astrocyte cultures, only direct treatment with Abeta increased ApoE production. In rat astrocyte cultures, however, treatment with conditioned media from cortical cultures grown with Abeta increased ApoE production, indicating that some sort of neuron-derived soluble factor(s) was also involved in activating rat astrocytes. These species differences suggest that monkey cortical cultures would be more useful as an in vitro model system to understand the details of how Abeta accumulates in the human brain, since monkeys are phylogenetically more similar to humans.     

A new method for assaying adhesion of cancer cells to the greater omentum and its application for evaluating anti-adhesion activities of chemically synthesized oligosaccharides

A new ex vivo method for assaying adhesion of cancer cells to the greater omentum has been developed using mouse greater omentum and [³H]labelled human gastric and mouse colorectal cancer cells. Since the adhesion rates were found to increase up to 18 h and labelled cells seemed to be stable during the period, the present method could be useful for investigating adhesion of cancer cells to the greater omentum, which must occur at the first step of the peritoneal dissemination. The adhesion of cancer cells to the greater omentum was inhibited by a series of chemically synthesized oligosaccharides and Galβ1,3[3OMeGalβ1,4GlcNAcβ1,6]αBn was found to be the best inhibitor. The anti-tumor effect of this novel tetrasaccharide in vivo was shown in preliminary experiments using Balb/c mice and colon26 cells.