Effects of Bacillus subtilis on Epithelial Tight Junctions of Mice with Inflammatory Bowel Disease.

Intestinal mucosal barrier dysfunction associated with inflammatory bowel disease (IBD). Effects of Bacillus subtilis on epithelial tight junctions (TJs) and intrinsic regulatory mechanisms of the intestine were studied in pursuit of better treatments for IBD. Fifty Balb/c mice given 5% dextran sulfate sodium (DSS) in tap water ad libitum over a 7-day period (to induce colitis) were randomly assigned to 4 test groups [DSS, DSS+B. subtilis, DSS+5 amino salicylic acid (5ASA), and DSS+B. subtilis+5ASA] to compare with normal controls. In the test groups DSS was administered daily by oral gavage in normal saline (0.2 mL), adding B. subtilis (1 × 10(8) CFU), 5ASA (6 mg), or both for respective test groups. Defecation, body weight, colitis score, pathological features, epithelial TJs proteins [claudin-1, occludin, junctional adhesion molecule (JAM)-A, and zona occludens (ZO)-1], and various cytokines [interleukin (IL)-6, IL-17, IL-23, and tissue necrosis factor (TNF)-α] were evaluated. Relative to the DSS group, disease activity index scores, and graded histologic damage were all significantly reduced by B. subtilis intake. All parameters declined even further when B. subtilis and 5ASA were combined. Analytic testing (immunohistochemical, western blot, and PCR) revealed progressive increase in TJ protein (claudin-1, occludin, JAM-A, and ZO-1) expression in DSS, DSS+B. subtilis, DSS+5ASA, DSS+B. subtilis+5ASA, and normal control groups (P < 0.05), whereas cytokine (IL-6, IL-17, IL-23, and TNF-α) expression similarly declined (P < 0.05). B. subtilis intake upregulated expression of TJ proteins (claudin-1, occludin, JAM-A, and ZO-1), for improved barrier function, and downregulated cytokine expression (IL-6, IL-17, IL-23, and TNF-α) to reduce intestinal epithelial damage.

Detection of cellular damage by hydrogen peroxide using SV40-T2 cells on shear horizontal surface acoustic wave (SH-SAW) sensor.

The rat lung epithelial cell line SV40-T2 was used to develop a cellular biosensing system to assay for environmental toxicants. The novel approach on which this system is based involves direct attachment of cultured rat or human cells onto a cell-adhesive matrix on the device through which shear horizontal surface acoustic waves (SH-SAW) are transmitted using 50 MHz SAW resonator. This novel design enables sensitive monitoring of changes of the electrophysical characteristics of cells, such as their conductivity and relative permittivity. A time-dependent change of phase of SAW and change of insertion loss (change of amplitude) were observed when the cells were treated with 0.5 or 1.0 mM H2O2. The change of insertion loss was biphasic, with an early phase (1-3 h) and a late phase (3-6 h). The late phase coincided with the destruction of cell-cell tight junctions detected by measurement of the transepithelial electrical resistance and paracellular permeability; in contrast, the early phase coincided with the destruction of intracellular actin filaments by H2O2. The early-phase effect of H2O2 on phase shift may be attributable to the change of intracellular permittivity by a change of cellular polarity. Immunofluorescence microscopy showed the disappearance of zonula occludens protein 1 from the region of cell-cell contact. These results suggest the correlation between the change of insertion loss as an SAW parameter and the destruction of tight junctions of the cells on the SH-SAW device in the late phase.

Disruption of endothelial tight junctions in a patient with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS).

An electron microscopic study revealed disruption of capillary endothelial tight junctions (TJs) in both biopsied muscle, taken at 5 years and 1 month of age, and the autopsied brain, taken at 13 years and 6 months of age, in a patient with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) and mitochondrial DNA (mtDNA) point mutation A3243G. This endothelial barrier disruption might result in vasogenic edema and systemic lactic acidosis, possibly the critical pathology of MELAS.

Absence of tight junctions between microvascular endothelial cells in human cerebellar hemangioblastomas.

OBJECTIVE: Endothelial tight junctions form the main barrier of the blood-brain barrier (BBB). In human hemangioblastomas, cyst formation is a common and important clinical manifestation. Although most researchers consider that the cyst formation in hemangioblastomas may be caused by the breakdown of the BBB, the underlying molecular mechanisms for cyst formation remain unknown. At present, there are few reports about the change of tight junctions in microvessel endothelium of human hemangioblastomas. The purpose of this research is to investigate the change of tight junction and its major molecular components in microvessel endothelium of human hemangioblastomas. METHODS: Twenty-four consecutive patients with cerebellar hemangioblastomas were studied. Tight junctions in the microvessels of hemangioblastomas and the control brain were examined by electron microscopy. Immunohistochemistry and double immunofluorescent microscopy were used to analyze the expression of CLN5 and its relationship with astrocytic endfeet in the control brain and hemangioblastomas. Quantitative real-time reverse-transcriptase polymerase chain reaction and Western blots were used to investigate the expression level of CLN5 in hemangioblastomas. Triple immunofluorescent microscopy was used to analyze the coexpression of vascular endothelial growth factor, vascular endothelial growth factor-R1, and placenta growth factor on microvessels of hemangioblastomas. Clinical and experimental data were correlated and analyzed by the one-way analysis of variance, Kruskal-Wallis test, and Spearman rank correlation test. RESULTS: In the control brain, the paracellular cleft between adjacent endothelial cells is sealed by continuous strands of tight junctions. In cystic hemangioblastomas, a significant paracellular cleft could be found between adjacent endothelial cells. Some endothelial cells were connected with adherens junction and no tight junction was found between them. Compared with the control brain, expression of CLN5 was decreased in cystic hemangioblastomas (P < 0.05). Phosphorylated CLN5 was detected in most hemangioblastomas, but not in the control brain. Microvessels in hemangioblastomas showed a significant absence of astrocytic endfeet. Coexpression of vascular endothelial growth factor, vascular endothelial growth factor-R1, and placenta growth factor was detected in the endothelial cells. The Spearman rank correlation test showed a significant correlation between a greater degree of CLN5 expression and less morphological cystic formation in these patients studied (correlation coefficient = -0.520; P = 0.009). CONCLUSION: The continuity of tight junctions of the BBB is interrupted in human cerebellar hemangioblastomas. Significant absence of astrocytic endfeet and tight junctions can be found in microvessels of hemangioblastomas, which may lead to the breakdown of the BBB in these tumors. These findings suggest that the absence of tight junctions might play a role in cyst formation of hemangioblastomas.