Application of real-time RT-PCR to quantifying gene expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human abdominal aortic aneurysm

BACKGROUND: The relative expression levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), key regulators in remodeling of extracellular matrix, are considered to play a pivotal role in the development of abdominal aortic aneurysm (AAA). However, few data exist regarding quantitative assessment of their expression in clinical settings. METHODS: In 22 patients with AAA who underwent graft replacement, tissue samples of the AAA and non-dilated aorta were obtained. Using a real-time RT-PCR method that enabled quantitative measurement of mRNA levels in small tissue samples, we determined gene expression levels of MMPs and TIMPs relative to that of glutaraldehyde 3-phosphate dehydrogenase in each sample. RESULTS: The expression levels of the MMP-1 and -3 genes were significantly augmented in AAA compared with non-dilated regions (4.48 +/- 2.01 versus 0.26 +/- 0.12, P < 0.01 and 1.89 +/- 1.00 versus 5.01 +/- 0.97, P < 0.05, respectively). Although genes for TIMP-1, -2 and -3 tended to be upregulated in AAA, relative expression levels of MMP-1 to TIMP-1, MMP-1 to TIMP-2, MMP-1 to TIMP-3, and MMP-3 to TIMP-2 were still higher in AAA than in non-dilated regions (1.12 +/- 0.63 versus 0.10 +/- 0.03, 4.13 +/- 1.12 versus 0.43 +/- 0.11, 1.61 +/- 0.59 versus 0.14 +/- 0.03, and 7.81 +/- 1.60 versus 2.56 +/- 0.76, respectively, P < 0.05). CONCLUSION: These results demonstrate that the present real-time RT-PCR method is reliable for the determination of mRNA levels in small samples of vascular tissue and that disproportional expression of both MMP-1 and MMP-3 relative to TIMPs relates pathologically to the evolution of AAA.     

Lactobacillus GG-fermented milk prevents DSS-induced colitis and regulates intestinal epithelial homeostasis through activation of epidermal growth factor receptor

Background

Fermented milk is considered one of the best sources for efficient consumption of probiotic strains by hosts to promote good health. The purpose of this study was to investigate the effects of orally administering LGG-fermented milk (LGG milk) on intestinal inflammation and injury and to study the mechanisms of LGG milk’s action.

Methods

LGG milk and non-LGG-fermented milk (non-LGG milk) were administered through gavage to mice before and during dextran sodium sulfate (DSS)-induced intestinal injury and colitis. Inflammatory/injury score and colon length were assessed. Intestinal epithelial cells were treated with the soluble fraction of LGG milk to detect its effects on the epidermal growth factor receptor (EGFR) and its downstream target, Akt activation, cytokine-induced apoptosis, and hydrogen peroxide (H₂O₂)-induced disruption of tight junctions.

Results

LGG milk treatment significantly reduced DSS-induced colonic inflammation and injury, and colon shortening in mice, compared to that in non-LGG milk-treated and -untreated mice. The soluble fraction of LGG milk, but not non-LGG milk, stimulated the activation of EGFR and Akt in a concentration-dependent manner, suppressed cytokine-induced apoptosis, and attenuated H₂O₂-induced disruption of tight junction complex in the intestinal epithelial cells. These effects of LGG milk were blocked by the EGFR kinase inhibitor. LGG milk, but not non-LGG milk, contained two soluble proteins, p40 and p75, that have been reported to promote survival and growth of intestinal epithelial cells through the activation of EGFR. Depletion of p40 and p75 from LGG milk abolished the effects of LGG milk on prevention of cytokine-induced apoptosis and H₂O₂-induced disruption of tight junctions.

Conclusions

These results suggest that LGG milk may regulate intestinal epithelial homeostasis and potentially prevent intestinal inflammatory diseases through activation of EGFR by LGG-derived proteins.     

Uremic Toxins Enhance Statin-Induced Cytotoxicity in Differentiated Human Rhabdomyosarcoma Cells

The risk of myopathy and rhabdomyolysis is considerably increased in statin users with end-stage renal failure (ESRF). Uremic toxins, which accumulate in patients with ESRF, exert cytotoxic effects that are mediated by various mechanisms. Therefore, accumulation of uremic toxins might increase statin-induced cytotoxicity. The purpose of this study was to determine the effect of four uremic toxins—hippuric acid, 3-carboxy-4-methyl-5-propyl-2-furanpropionate, indole-3-acetic acid, and 3-indoxyl sulfate—on statin-induced myopathy. Differentiated rhabdomyosarcoma cells were pre-treated with the uremic toxins for seven days, and then the cells were treated with pravastatin or simvastatin. Cell viability and apoptosis were assessed by viability assays and flow cytometry. Pre-treatment with uremic toxins increased statin- but not cisplatin-induced cytotoxicity (p < 0.05 vs. untreated). In addition, the pre-treatment increased statin-induced apoptosis, which is one of the cytotoxic factors (p < 0.05 vs. untreated). However, mevalonate, farnesol, and geranylgeraniol reversed the effects of uremic toxins and lowered statin-induced cytotoxicity (p < 0.05 vs. untreated). These results demonstrate that uremic toxins enhance statin-induced apoptosis and cytotoxicity. The mechanism underlying this effect might be associated with small G-protein geranylgeranylation. In conclusion, the increased severity of statin-induced rhabdomyolysis in patients with ESRF is likely due to the accumulation of uremic toxins.     

Primary orbital neuroblastoma in a 1‐month‐old boy

Neuroblastoma is a malignant tumor predominantly occurring in children and usually arising from the adrenal gland or sympathetic ganglia. We describe a neuroblastoma in a 1‐month‐old boy arising from his left orbital cavity. This tumor was refractory to chemotherapy or radiotherapy, requiring enucleation of the left eye for complete removal of the intraorbital tumor. Thereafter, he received high‐dose chemotherapy followed by autologous peripheral blood stem cell transplantation, and has been in complete remission for 3 years. Unlike neuroblastomas arising from the adrenal gland or sympathetic ganglia, primary orbital neuroblastoma may be refractory even in early infancy.     

One-pot synthesis of conjugated triphenylamine macrocycles and their complexation with fullerenes

Triphenylamine derivates have been utilized as building blocks in hole-transporting materials. Herein, we describe the synthesis of three octyl-derived conjugated triphenylamine macrocycles with different sizes, and a 4-(2-ethylhexyloxy)-substituted cyclic triphenylamine hexamer using a palladium-catalyzed C–N coupling reaction. These conjugated triphenylamine macrocycles not only have interesting structures, but also are capable of complexing with C₆₀, C₇₀ and PC₆₁BM. Their binding stoichiometries with fullerenes were all determined to be 1 : 1 by an emission titration method. The association constants of these complexes were measured to be in the range of 0.115–1.53 × 10⁵ M⁻¹ depending on the cavity size of the triphenylamine macrocycles and the volume of the fullerenes. The space-charge-limited current properties of the complexes were further investigated using the fabricated ITO/PEDOT:PSS/active layer/Au devices.

Cyclic triphenylamine (TPA) oligomers synthesized by C–N coupling were found to be capable of complexing with fullerenes, and the applications in optoelectronic devices were investigated by using the fabricated ITO/PEDOT:PSS/active layer/Au devices.     

The treatment outcomes of synchronous and metachronous esophageal squamous cell carcinoma and head and neck squamous cell carcinoma

Background

The treatment outcomes of patients with esophageal squamous cell carcinoma (ESCC) and head and neck squamous cell carcinoma (HNSCC) have been poorly documented.

Patients and methods

We investigated 50 patients with synchronous and metachronous ESCC and HNSCC. We focused on the treatment results of 20 patients with synchronous ESCC and HNSCC who received simultaneous chemoradiotherapy (CRT).

Results

There were 34 patients (68.0?%) with stage 0?CI ESCC and 40 patients (80.0?%) with stage II?CIV HNSCC. A total of 13 (26.0?%) patients underwent endoscopic mucosal resection and 28 (56.0?%) underwent CRT for ESCC, and 35 (70.0?%) of the patients with HNSCC were treated with CRT. The 5-year overall survival rates of the 50 patients with synchronous and metachronous ESCC and HNSCC was 57.8?%. For the 20 patients with synchronous ESCC and HNSCC who received simultaneous CRT, the CRT was completed in 19 (95.0?%) patients. Although grade 3?C4 adverse events were observed in five (25.0?%) patients, there were no therapy-related deaths. Complete responses (CRs) of both ESCC and HNSCC were observed in ten (50.0?%) patients. The 5-year overall survival rate of the 20 patients was 60.0?%. CRs of both ESCC and HNSCC were obtained in seven (58.3?%) patients by using a cisplatin/5-FU regimen (n?=?12), and in the other three (37.5?%) patients by a platinum-based monotherapy regimen (n?=?8).

Conclusion

The surveillance of double cancer and the use of radical treatment contributed to the favorable outcome of the patients with ESCC and HNSCC. The optimal chemotherapy regimen for simultaneous CRT remains to be determined.