Biological analysis of flexor tendon repair-failure stump tissue: A potential recycling of tissue for tendon regeneration.

OBJECTIVES: Re-rupture is common after primary flexor tendon repair. Characterization of the biological changes in the ruptured tendon stumps would be helpful, not only to understand the biological responses to the failed tendon repair, but also to investigate if the tendon stumps could be used as a recycling biomaterial for tendon regeneration in the secondary grafting surgery. METHODS: A canine flexor tendon repair and failure model was used. Following six weeks of repair failure, the tendon stumps were analyzed and characterized as isolated tendon-derived stem cells (TDSCs). RESULTS: Failed-repair stump tissue showed cellular accumulation of crumpled and disoriented collagen fibres. Compared with normal tendon, stump tissue had significantly higher gene expression of collagens I and III, matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and insulin-like growth factor (IGF). The stump TDSCs presented both mesenchymal stem and haematopoietic cell markers with significantly increased expression of CD34, CD44, and CD90 markers. Stump TDSCs exhibited similar migration but a lower proliferation rate, as well as similar osteogenic differentiation but a lower chondrogenic/adipogenic differentiation capability, compared with normal TDSCs. Stump TDSCs also showed increasing levels of SRY-box 2 (Sox2), octamer-binding transcription factor 4 (Oct4), tenomodulin (TNMD), and scleraxis (Scx) protein and gene expression. CONCLUSION: We found that a failed repair stump had increased cellularity that preserved both mesenchymal and haematopoietic stem cell characteristics, with higher collagen synthesis, MMP, and growth factor gene expression. This study provides evidence that tendon stump tissue has regenerative potential.Cite this article: C-C. Lu, T. Zhang, R. L. Reisdorf, P. C. Amadio, K-N. An, S. L. Moran, A. Gingery, C. Zhao. Biological analysis of flexor tendon repair-failure stump tissue: A potential recycling of tissue for tendon regeneration. Bone Joint Res 2019;8:232-245. DOI: 10.1302/2046-3758.86.BJR-2018-0239.R1.

Development and Evaluation of a Tissue-Engineered Fibrin-based Canine Mitral Valve Three-dimensional Cell Culture System.

Myxomatous mitral valve disease is the most common cardiac disease of the dog, but examination of the associated cellular and molecular events has relied on the use of cadaveric valve tissue, in which functional studies cannot be undertaken. The aim of this study was to develop a three-dimensional (3D) cell co-culture model as an experimental platform to examine disease pathogenesis. Mitral valve interstitial (VIC) and endothelial (VEC) cells were cultured from normal and diseased canine (VIC only) valves. VICs were embedded in a fibrin-based hydrogel matrix and one surface was lined with VECs. The 3D static cultures (constructs) were examined qualitatively and semiquantitatively by light microscopy, immunofluorescence microscopy and protein immunoblotting. Some constructs were manipulated and the endothelium damaged, and the response examined. The construct gross morphology and histology demonstrated native tissue-like features and comparable expression patterns of cellular (α-smooth muscle actin [SMA] and embryonic smooth muscle myosin heavy chain [SMemb]) and extracellular matrix associated markers (matrix metalloproteinase [MMP]-1 and MMP-3), reminiscent of diseased valves. There were no differences between constructs containing normal valve VICs and VECs (type 1) and those containing diseased valve VICs and normal valve VECs (type 2). Mechanical manipulation and endothelial damage (type 3) tended to decrease α-SMA and SMemb expression, suggesting reversal of VIC activation, but with retention of SMemb+ cells adjacent to the wounded endothelium consistent with response to injury. Fibrin-based 3D mitral valve constructs can be produced using primary cell cultures derived from canine mitral valves, and show a phenotype reminiscent of diseased valves. The constructs demonstrate a response to endothelial damage indicating their utility as experimental platforms.

Antrodia cinnamomea reduces obesity and modulates the gut microbiota in high-fat diet-fed mice.

BACKGROUND: Obesity is associated with gut microbiota dysbiosis, disrupted intestinal barrier and chronic inflammation. Given the high and increasing prevalence of obesity worldwide, anti-obesity treatments that are safe, effective and widely available would be beneficial. We examined whether the medicinal mushroom Antrodia cinnamomea may reduce obesity in mice fed with a high-fat diet (HFD). METHODS: Male C57BL/6J mice were fed a HFD for 8 weeks to induce obesity and chronic inflammation. The mice were treated with a water extract of A. cinnamomea (WEAC), and body weight, fat accumulation, inflammation markers, insulin sensitivity and the gut microbiota were monitored. RESULTS: After 8 weeks, the mean body weight of HFD-fed mice was 39.8±1.2 g compared with 35.8±1.3 g for the HFD+1% WEAC group, corresponding to a reduction of 4 g or 10% of body weight (P<0.0001). WEAC supplementation reduced fat accumulation and serum triglycerides in a statistically significant manner in HFD-fed mice. WEAC also reversed the effects of HFD on inflammation markers (interleukin-1ß, interleukin-6, tumor necrosis factor-α), insulin resistance and adipokine production (leptin and adiponectin). Notably, WEAC increased the expression of intestinal tight junctions (zonula occludens-1 and occludin) and antimicrobial proteins (Reg3g and lysozyme C) in the small intestine, leading to reduced blood endotoxemia. Finally, WEAC modulated the composition of the gut microbiota, reducing the Firmicutes/Bacteroidetes ratio and increasing the level of Akkermansia muciniphila and other bacterial species associated with anti-inflammatory properties. CONCLUSIONS: Supplementation with A. cinnamomea produces anti-obesogenic, anti-inflammatory and antidiabetic effects in HFD-fed mice by maintaining intestinal integrity and modulating the gut microbiota.

[Expression and intratumoral heterogeneity of LN-5γ2 in esophageal squamous cell carcinoma].

OBJECTIVE: The present study aimed to evaluate the expression and intratumoral heterogeneity of LN-5γ2 in esophageal squamous cell carcinoma (ESCC). METHODS: The expression of LN-5γ2 protein was examined in 135 ESCC cases by immunohistochemistry, and to analyze its relationship with the clinical relevance of patients. The protein expressions in different regions in the same tumor as well as different nests in the same region were compared. RESULTS: Moderate and high expression of LN-5γ2 protein was detected in 40.0% (54/135) of tumor tissues. Positive immunohistochemical staining was observed in 31.1% (23/74) of early stage (stages Ⅰ/Ⅱ) cases and 50.8% (31/61) of late stage (stage Ⅲ) cases, with a significant difference between these two groups (P=0.023). There was no statistical association of LN-5γ2 expression with age, sex of patients, PT sage, lymph node metastasis and degree of tumor differentiation (P>0.05). However, differential expression of LN-5γ2 protein was found at different sampling sites in the same tumor and the same sampling site in different carcinomas. CONCLUSION: High expression of LN-5γ2 is positively correlated with tumor clinical stages and there existed intratumoral heterogeneity of LN-5γ2 expression in ESCC tissues.

Microtubule-associated protein 4 is an important regulator of cell invasion/migration and a potential therapeutic target in esophageal squamous cell carcinoma.

Cell invasion and migration significantly contribute to tumor metastasis. Microtubule-associated protein 4 (MAP4) protein is one member of microtubule-associate proteins family. It is responsible for stabilization of microtubules by modulation of microtubule dynamics. However, there is little information about the involvement of MAP4 in human cancer. Here we show that MAP4 serves as a regulator of invasion and migration in esophageal squamous cancer cells. By activating the ERK-c-Jun-vascular endothelial growth factor A signaling pathway, MAP4 promotes cell invasion and migration in vitro, tumor growth and metastasis in mouse models. Immunohistochemical staining of operative tissues indicated that MAP4 expression was associated with tumor stage, lymph node metastasis and shorter survival of the patients with esophageal squamous cell carcinoma (ESCC). Multivariate Cox regression analysis showed that MAP4 is an independent prognostic indicator. In the serial sections of ESCC tissues, there was a positive correlation between MAP4 and vascular endothelial growth factor A expression. Notably, an intratumoral injection of MAP4-small interfering RNA (siRNA) remarkably inhibited the growth of the tumors that formed by the MAP4-expressing ESCC cells in nude mice, and a combination of MAP4-siRNA and Bevacizumab significantly enhanced the inhibition effect. Our data suggest that MAP4 is probably a useful prognostic biomarker and a potential therapeutic target for the disease.

Comparison of cellular changes in Cavalier King Charles spaniel and mixed breed dogs with myxomatous mitral valve disease.

INTRODUCTION: The aim of this study was to determine if there are differences in cellular changes in Cavalier King Charles spaniel (CKCS) myxomatous mitral valves compared to non-CKCS dogs. ANIMALS: Cavalier King Charles spaniels (n = 6) and age-matched mixed breed (n = 6) with severe myxomatous mitral valve disease (MMVD), and normal mixed breed (n = 4) dogs. MATERIALS AND METHODS: Immunohistochemistry staining and qualitative and quantitative analysis of mitral valves sections, examining for the presence of CD11c and CD45, vimentin, alpha smooth muscle actin (α-SMA) and embryonic smooth muscle myosin heavy chain (Smemb), von Willebrand factor and CD31 and Ki-67. RESULTS: Vimentin positive cell numbers were increased in the MMVD dogs and distributed throughout the valve with greatest density close to the endothelium. There were no significant differences in cell marker expression for the two diseased groups, but cell numbers were significantly increased compared to controls for α-SMA (CKCS only) and Smemb (CKCS and mixed breed: p < 0.05). Alpha smooth muscle actin+ cells were primarily located at the valve edge, with Smemb+ cells similarly located, but also present throughout the valve stroma. A small number of cells close to the valve edge co-expressed α-SMA and Smemb. Endothelial von Willebrand factor expression was identified in all valves, with evidence of disrupted endothelium in the diseased, but was also found in diseased valve stroma. There was no staining for CD11c, CD45 or CD31 in any valve. Ki-67+ cells formed linear clusters at the leaflet tip and were sparsely distributed throughout both myxomatous valve groups. CONCLUSIONS: The cellular changes notes with advanced stage MMVD appear similar for CKCS when compared to mixed breed dogs.

Extreme Hyperlactatemia After Heart Transplantation: One Center's Experience.

INTRODUCTION: Hyperlactatemia may occur early after cardiac surgery and is correlated with prognosis. This study was conducted to analyze the perioperative variables and postoperative outcomes among heart transplant recipients with extremely high lactate levels (>15 mmol/L). METHODS: The single-center medical records of heart transplantation from June 2006 to May 2013 were retrospectively reviewed for patient characteristics, perioperative hemodynamic variables, arterial blood gas analysis data, and postoperative mortality. RESULTS: Among 58 consecutive heart transplant recipients, lactate levels over the detectable upper limit (>15 mmol/L) were identified in 12 patients after intensive care unit admission, with peak time at 1.9 ± 2.0 (range 0-6.1) hours. The maximal preoperative lactate level was 3.1 mmol/L, and most (11/12) postoperative lactate levels returned to <4 mmol/L at 27.5 ± 12.8 hours after surgery (range 15-58, median 24), displaying a trend toward delayed extubation time in 10 recipients (P < .01). Blood glucose levels elevated significantly from preoperative 148.9 ± 45.2 to 375.7 ± 96.9 mg/dL at peak lactate level (P < .01). Four patients died in the ICU (range 5-32 days), 4 died after discharge (range 5-57 months), with 6 in total surviving over 1 year. CONCLUSION: Extreme hyperlactatemia commonly occurred early after heart transplantation and mostly recovered within 30 hours; however, with delayed extubation time after operation.

Gene network and canonical pathway analysis in canine myxomatous mitral valve disease: a microarray study.

Myxomatous mitral valve disease (MMVD) is the single most common acquired heart disease of the dog and is particularly common in small pedigree breed dogs such as the Cavalier King Charles spaniel (CKCS). There are limited data on the mitral valve transcriptome and the aim of this study was to use the microarray technology in conjunction with bioinformatics platforms to analyse transcript changes in MMVD in CKCS compared to normal dogs (non-CKCS). Differentially expressed genes (n = 5397) were identified using cut-off settings of fold change, false discovery rate (FDR) and P <0.05. In total, 4002 genes were annotated to a specific transcript in the Affymetrix canine database, and after further filtering, 591 annotated canine genes were identified: 322 (55%) were up-regulated and 269 (45%) were down-regulated. Canine microRNAs (cfa-miR; n = 59) were also identified. Gene ontology and network analysis platforms identified between six and 10 significantly different biological function clusters from which the following were selected as relevant to MMVD: inflammation, cell movement, cardiovascular development, extracellular matrix organisation and epithelial-to-mesenchymal (EMT) transition. Ingenuity Pathway Analysis identified three canonical pathways relevant to MMVD: caveolar-mediated endocytosis, remodelling of epithelial adherens junctions, and endothelin-1 signalling. Considering the biological relevance to MMVD, the gene families of importance with significant difference between groups included collagens, ADAMTS peptidases, proteoglycans, matrix metalloproteinases (MMPs) and their inhibitors, basement membrane components, cathepsin S, integrins, tight junction cell adhesion proteins, cadherins, other matrix-associated proteins, and members of the serotonin (5-HT)/transforming growth factor -ß signalling pathway.

Culture and characterisation of canine mitral valve interstitial and endothelial cells.

Valve interstitial cells (VICs) have an important role in the aetiopathogenesis of myxomatous mitral valve disease (MMVD) in the dog. Furthermore, there is evidence that valve endothelial cells (VECs) also contribute to disease development. In addition to examining native valve tissue to understand MMVD, another strategy is to separately examine VIC and VEC biology under in vitro culture conditions. The aim of this study was to isolate and characterise canine mitral VICs and VECs from normal dog valves using a combination of morphology, immunohistochemistry and reverse transcription PCR (RT-PCR). Canine mitral VECs and VICs were isolated and cultured in vitro. The two cell populations exhibited different morphologies and growth patterns. VECs, but not VICs, expressed the endothelial markers, platelet endothelial cell adhesion molecule (PECAM-1 or CD31) and acetylated low density lipoprotein (Dil-Ac-LDL). Both VECs and VICs expressed vimentin and embryonic non-smooth muscle myosin heavy chain (SMemb), an activated mesenchymal cell marker. The myofibroblast marker, alpha smooth muscle actin (α-SMA), was detected at the mRNA level in both VEC and VIC cultures, but only at the protein level in VIC cultures. The morphological heterogeneity and expression of non-endothelial phenotypic markers in VEC cultures suggested that a mixture of cell types was present, which might be due to cell contamination and/or endothelial-mesenchymal transition (EndoMT). The use of a specific endothelial culture medium for primary VEC cultures enhanced the endothelial properties of the cells and reduced α-SMA and SMemb expression.

Smoking behaviours of adolescents, influenced by smoking of teachers, family and friends.

BACKGROUND: Smoking tobacco is a global health problem, and this study highlights adolescent smoking in Taiwan. Smoking was completely banned on campuses under the Tobacco Hazards Prevention Act and School Health Regulations. Few have investigated the association between personnel smoking/school smoking policies and adolescent smoking in Taiwan. The smoking rate has gradually increased for senior high school students in Taiwan from 10.7% in 1994 to 14.7% in 2011. AIM: This study examined the influence of family and friends' smoking on the association between the presence of teachers smoking and each stage of adolescents' smoking behaviour. METHODS: A cross-sectional survey was conducted in nine high schools (n = 921). Logistic regression analysis was used to examine the association between smoking stage, father smoking, mother smoking, sibling smoking, friends smoking and the presence of smoking teachers. RESULTS: After adjusting for gender, school type and grade, fathers', siblings' and friends' smoking were significantly associated with adolescents' ever and current smoking behaviours, but mothers' smoking was only associated with adolescents' current smoking behaviours. Friends' smoking was strongly associated with being a smoker. While there was an interaction between friends' smoking and the presence of smoking teachers on current smoking behaviours indicating the effect of the presence of smoking teachers was increased when friends did not smoke. DISCUSSION: The results suggest that teachers smoking on school may increase the likelihood of being a current smoker as their friends do not smoke. Family and friends smoking are associated with adolescent smoking. CONCLUSION: The Taiwanese government has regulated a comprehensive smoking ban in schools to prohibit both student and staff smoking on all school premises. To achieve this, schools should make tobacco control communication efforts. Nurses could address the combined influence of family and friends as well as the effect of school smoking restrictions to help prevent adolescents from smoking.

Effect of DNA damage response by quinazolinone analogue HMJ-38 on human umbilical vein endothelial cells: evidence for γH2A.X and DNA-PK-dependent pathway.

The present study aims to explore the mechanism of quinazolinone analogue HMJ-38-induced DNA damage in endothelial cells in vitro. We attempt to evaluate the antiangiogenetic response utilizing human umbilical vein endothelial cells (HUVECs). Herein, the results demonstrated that HMJ-38 incubation triggered DNA damage behavior and showed a longer DNA migration in HUVECs based on the comet assay and the analysis of DNA agarose gel electrophoresis to contact DNA smears. We further gained to determine a marker of DNA double strand breaks, phosphorylated histone H2A.X (Ser139) (γH2A.X), in HMJ-38-treated HUVECs by flow cytometry and Western blotting assay. We consider that HMJ-38 has caused an increase in γH2A.X, and DNA damage seemed to mediate through DNA-dependent serine/threonine protein kinase (DNA-PK) binding to Ku70/Ku80 as well as advanced activated p-Akt (Ser473) and stimulated phosphorylated glycogen synthase kinase-3ß (p-GSK-3ß) conditions in HUVECs. Importantly, the effect of above DNA damage response was prevented by N-acetyl-l-cysteine (a reactive oxygen species scavenger), and NU7026 (a DNA-PK inhibitor) could attenuate DNA-PK catalytic subunit and phosphorylation of H2A.X on Ser139 expression in comparison with HMJ-38 alone treated HUVECs. Therefore, HMJ-38-provoked DNA damage stress in HUVECs probably led to the activation of γH2A.X/DNA-PK/GSK-3ß signaling. In summary, our novel finding provides more information addressing the pharmacological approach of newly synthesized HMJ-38 for further development and therapeutic application in antiangiogenetic effect of cancer chemotherapy.

Comparison of the clinical and radiological outcomes of three minimally invasive techniques for total knee replacement at two years.

Minimally invasive total knee replacement (MIS-TKR) has been reported to have better early recovery than conventional TKR. Quadriceps-sparing (QS) TKR is the least invasive MIS procedure, but it is technically demanding with higher reported rates of complications and outliers. This study was designed to compare the early clinical and radiological outcomes of TKR performed by an experienced surgeon using the QS approach with or without navigational assistance (NA), or using a mini-medial parapatellar (MP) approach. In all, 100 patients completed a minimum two-year follow-up: 30 in the NA-QS group, 35 in the QS group, and 35 in the MP group. There were no significant differences in clinical outcome in terms of ability to perform a straight-leg raise at 24 hours (p = 0.700), knee score (p = 0.952), functional score (p = 0.229) and range of movement (p = 0.732) among the groups. The number of outliers for all three radiological parameters of mechanical axis, frontal femoral component alignment and frontal tibial component alignment was significantly lower in the NA-QS group than in the QS group (p = 0.008), but no outlier was found in the MP group. In conclusion, even after the surgeon completed a substantial number of cases before the commencement of this study, the supplementary intra-operative use of computer-assisted navigation with QS-TKR still gave inferior radiological results and longer operating time, with a similar outcome at two years when compared with a MP approach.

The corpus-predominant gastritis index may serve as an early marker of Helicobacter pylori-infected patients at risk of gastric cancer.

BACKGROUND: To eradicate Helicobacter pylori before the occurrence of precancerous changes is important to prevent gastric carcinogenesis. AIM: To validate whether the corpus-predominant gastritis index (CGI) can serve as an early marker to identify the H. pylori-infected patients at risk of gastric carcinogenesis. METHODS: This study enrolled 188 subjects, including 43 noncardiac gastric cancer patients, 63 of their first-degree relatives and 82 sex- and age-matched duodenal ulcer patients as controls. All received endoscopy to provide topographic gastric specimens to test for H. pylori infection and its related histological features, translated into the operative link on gastritis assessment (OLGA), operative link on gastric intestinal metaplasia assessment (OLGIM) stages, and the presence of CGI. Spasmolytic polypeptide-expressing metaplasia (SPEM) was assessed by immunohistochemistry staining of trefoil factor 2. RESULTS: Gastric cancer patients had higher prevalence of CGI and OLGIM stage II-IV, but not OLGA stage II-IV, than the controls (P = 0.001, OR = 3.4[95% CI: 1.4-8.1] for CGI; OR = 5.0[95% CI: 2.0-12.8] for OLGIM). In patients with the combined presence of CGI and OLGIM stage II-IV, the risk of gastric cancer increased to 9.8 (P < 0.001). The first-degree relatives of the gastric cancer patients had a higher rate of the presence of CGI, but not OLGA or OLGIM stage II-IV than the duodenal ulcer controls (P = 0.001). Of the first-degree relatives, the presence of CGI increased the risk of SPEM (P = 0.003, OR = 5.5[95% CI: 1.8-17.0]). CONCLUSION: The corpus-predominant gastritis index, which is highly correlated to SPEM, may serve as an early marker to identify the H. pylori-infected patients at a higher risk of gastric cancer.

The effect of pre-procedure anxiety on sedative requirements for sedation during colonoscopy.

This study investigated the effects of pre-procedural anxiety (assessed using the Beck Anxiety Inventory) on sedative requirements in 135 patients undergoing sedation for colonoscopy. Deep sedation was defined as loss of consciousness and no response to colonoscopy, and was achieved by target-controlled infusion of propofol. Patients' characteristics, baseline haemodynamic profiles, Beck Anxiety Inventory scores, effect-site propofol concentration at loss of consciousness and characteristics of recovery were recorded. No correlations were found between Beck Anxiety Inventory scores and effect-site propofol concentration at loss of consciousness or baseline haemodynamic profiles. There was no statistical difference in the characteristics of recovery among patients with different levels of anxiety. In conclusion, in patients receiving deep sedation for colonoscopies, the level of pre-procedural anxiety did not relate to the sedative requirement or post-procedural recovery characteristics.

Comparison of clinical outcome of single-incision laparoscopic surgery using a simplified access system with conventional laparoscopic surgery for malignant colorectal disease.

AIM: Instrument crowding is encountered in single-incision laparoscopic surgery (SILS). Our aim was to compare the results of SILS with those of conventional laparoscopic surgery (CLS) for malignant colorectal disease. METHODS: The records of 27 patients who received SILS for the treatment of malignant disease using a home-made multiple-port system were compared with those of 68 patients who received CLS performed in a standard manner using four to five trocar sites. RESULTS: There were no significant differences in age, gender, disease stage, tumour location or tumour size between the SILS and CLS groups. The most common surgery was high anterior resection in both groups (SILS, 63.0%vs CLS, 58.8%). There were no significant differences between the groups in types of surgery performed, length of bowel resected, resection margin, blood loss, duration of surgery or postoperative complications. Postoperative pain scores were significantly higher in the SILS group than in the CLS group (3.07 ± 1.14 vs 2.41 ± 0.63, respectively, P < 0.001). CONCLUSIONS: SILS is as effective as CLS, and is not associated with increased duration of surgery, blood loss or complications.

Involvement of the cyclic-AMP-dependent protein kinase A pathway in thyroxine effects on calcitonin secretion from TT cells.

Previous studies have demonstrated that plasma calcitonin is lower in hypothyroid patients and that thyroxine stimulates the human thyroid to release calcitonin. Therefore, thyroid hormones may regulate the secretion of calcitonin, but further work is needed to address this possibility in more detail. TT cells, a model of human thyroid C cells, were incubated in a medium containing vehicle, thyroxine, or thyroxine methyl-hemisuccinate-bovine serum albumin (BSA-L-T(4), thyroxine was immobilized and linked to BSA); then, the levels of secreted calcitonin (hCT), calcitonin mRNA, and cAMP were measured. To study links that connect the cAMP-dependent protein kinase A (PKA) pathway to the observed thyroxine effects, cells were treated with either vehicle or thyroxine plus SQ22536 [an adenylyl cyclase (AC) inhibitor], KT5720 (a PKA inhibitor), or 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor). The activity levels of AC and PKA, and secreted calcitonin were then measured. The results indicate that thyroxine increases calcitonin secretion, cellular cAMP accumulation, and the activities of AC and PKA, but does not increase hCT mRNA levels in TT cells. BSA-L-T(4) also increases calcitonin secretion. These effects are inhibited by SQ22536, and KT5720 and suggest that the nongenomic thyroxine effects that stimulate calcitonin secretion from TT cells involve the cAMP-dependent PKA pathway.

Safrole induces apoptosis in human oral cancer HSC-3 cells.

Phytochemicals have been used as potential chemopreventive or chemotherapeutic agents. However, there are data suggesting a mutagenic effect of some phytochemicals. We hypothesized that safrole would have anticancer effects on human oral squamous cell carcinoma HSC-3 cells. Safrole decreased the percentage of viable HSC-3 cells via induction of apoptosis by an increased level of cytosolic Ca(2+) and a reduction in the mitochondrial membrane potential (ΔΨ(m)). Changes in the membrane potential were associated with changes in the Bax, release of cytochrome c from mitochondria, and activation of downstream caspases-9 and -3, resulting in apoptotic cell death. In vivo studies also showed that safrole reduced the size and volume of an HSC-3 solid tumor on a xenograft athymic nu/nu mouse model. Western blotting and flow cytometric analysis studies confirmed that safrole-mediated apoptotic cell death of HSC-3 cells is regulated by cytosolic Ca(2+) and by mitochondria- and Fas-dependent pathways.

Mixed infections of Helicobacter pylori: tissue tropism and histological significance.

Mixed infections with Helicobacter pylori facilitate interstrain gene transfer and the maintenance of genetic diversity for adaptation to the gastric environment, but whether mixed infections with histological significance and tissue tropism occur in the human stomach is still unclear. Helicobacter pylori was isolated from the antrum and the corpus of 30 dyspeptic patients. Four to eight colonies were randomly collected from each site. The genetic diversity of each isolate was evaluated by comparing random amplified polymorphic DNA banding patterns. The prevalence of mixed infections was 23.3% (7/30), and different dominant strains were isolated from the antrum and the corpus specimens. In the 23 patients infected with a single strain, the acute inflammation (AI) score, chronic inflammation (CI) score, atrophy (AT) score and lymphoid follicle (LF) score of the antrum were usually higher than those of the corpus (p 0.05). Moreover, the patients with mixed infections had marginally higher CI and HPD scores than those with single-strain infection (p 0.062 and p 0.095, respectively) in the corpus and had a significantly higher rate of appearance of intestinal metaplasia (IM) in the antrum (p 0.005). These data show that H. pylori tissue tropism was found in the human stomach, and suggest that mixed infections could change the histological features in the antrum and in the corpus, and that they could be associated with the appearance of IM in the antrum.