Shorter pregnant women restrained in the rear seat of a car are at risk for serious neck injuries: Biomechanical analysis using a pregnant crash test dummy.

INTRODUCTION: When considering seat belt contacts to the neck in pregnant woman of shorter height sitting in the rear seat of a vehicle, subsequent injuries after a collision must be understood in the context of both maternal and fetal outcomes. To determine likely injuries to a pregnant woman sitting in the rear seat, we determined the kinematics of a "pregnant" crash test dummy by measuring neck compression forces and biomechanical parameters acting on the head and neck. METHODS: Sled tests using a shorter-height pregnant woman crash test dummy (Maternal Anthropometric Measurement Apparatus, ver. 2B) were performed at the HYGE sled test facility representing full frontal impact at target velocities of 29km/h and 48km/h. Kinematics of the dummy and biomechanical parameters of the head, neck, and chest were measured. Pressure to the neck was measured using Prescale (Fujifilm, Tokyo, Japan). RESULTS: During frontal collision tests, the shoulder belt compressed the neck at a pressure >12.8MPa, even during the low-velocity impact. In addition to neck flexion, right side bending and the head and chest moving in opposite directions were observed, with maximum differences of 42.4mm at high velocity and 33.7mm at low velocity. CONCLUSIONS: This study provides data on the kinematics of pregnant women of short height sitting in the rear seat during a frontal collision using a pregnant woman crash test dummy. The knowledge gathered from this study should be useful for determining pregnant women passengers' kinematics at the time of collision and evaluating the relationship between the vehicle collision and fetal outcomes.

Seatbelt paths of the pregnant women sitting in the rear seat of a motor vehicle.

PURPOSE: Seatbelt use during pregnancy is important to improve maternal and fetal survival after motor vehicle collisions. However, because the rear seatbelt of a motor vehicle tends to make contact with the neck, even if it is adequately used, some pregnant women sitting in the rear seat opt not to fasten the belt. The purpose of this study is to explore seatbelt-neck contact for pregnant women sitting in the rear seat of a motor vehicle. METHODS: We carried out an anthropometric study. Japanese women who were ≥30 weeks pregnant (n = 12) sat in the left side of the rear seat of a typical mid-size passenger sedan and fastened the seatbelt. Seating posture was investigated by measuring the coordinates of the anthropometric data points of the pregnant women (head, shoulder, hip joint, and knee joint). The belt path was analyzed by measuring the clearance between the belt and the sternum or navel. RESULTS: Among the 12 pregnant women at 33.9 week ± 3.3 week gestation, the shoulder belt deviated to the right side and subsequently contacted to the neck in four pregnant women (Contact group). The height of the Contact group was significantly shorter than that of Non-contact group (152.3 cm ± 3.0 cm vs. 159.0 cm ± 3.3 cm, p = 0.008). Regarding the relative position of the seatbelt to the subject's body, the distances from the top of the sternum to the center of the shoulder belt were significantly shorter in Contact group (3.9 cm ± 3.5 cm) than that in the Non-contact group (8.0 cm ± 1.6 cm, p = 0.03). However, no significant difference was found for the distance from the umbilicus to the center of the lap belt. CONCLUSION: Our findings show that because of short height and late term of pregnancy with protrusion of the abdomen, the shoulder belt deviates to the right or left, avoiding the protruded uterus, and subsequently makes contact with the neck. Seatbelt systems for rear seats need to be developed to improve passenger safety, especially for pregnant women.

Concomitant loss of p120-catenin and ß-catenin membrane expression and oral carcinoma progression with E-cadherin reduction.

The binding of p120-catenin and ß-catenin to the cytoplasmic domain of E-cadherin establishes epithelial cell-cell adhesion. Reduction and loss of catenin expression degrades E-cadherin-mediated carcinoma cell-cell adhesion and causes carcinomas to progress into aggressive states. Since both catenins are differentially regulated and play distinct roles when they dissociate from E-cadherin, evaluation of their expression, subcellular localization and the correlation with E-cadherin expression are important subjects. However, the same analyses are not readily performed on squamous cell carcinomas in which E-cadherin expression determines the disease progression. In the present study, we examined expression and subcellular localization of p120-catenin and ß-catenin in oral carcinomas (n = 67) and its implications in the carcinoma progression and E-cadherin expression using immunohitochemistry. At the invasive front, catenin-membrane-positive carcinoma cells were decreased in the dedifferentiated (p120-catenin, P < 0.05; ß-catenin, P < 0.05) and invasive carcinomas (p120-catenin, P < 0.01; ß-catenin, P < 0.05) and with the E-cadherin staining (p120-catenin, P < 0.01; ß-catenin, P < 0.01). Carcinoma cells with ß-catenin cytoplasmic and/or nuclear staining were increased at the invasive front compared to the center of tumors (P < 0.01). Although the p120-catenin isoform shift from three to one associates with carcinoma progression, it was not observed after TGF-ß, EGF or TNF-α treatments. The total amount of p120-catenin expression was decreased upon co-treatment of TGF-ß with EGF or TNF-α. The above data indicate that catenin membrane staining is a primary determinant for E-cadherin-mediated cell-cell adhesion and progression of oral carcinomas. Furthermore, it suggests that loss of p120-catenin expression and cytoplasmic localization of ß-catenin fine-tune the carcinoma progression.

Progression of oral squamous cell carcinoma accompanied with reduced E-cadherin expression but not cadherin switch.

The cadherin switch from E-cadherin to N-cadherin is considered as a hallmark of the epithelial-mesenchymal transition and progression of carcinomas. Although it enhances aggressive behaviors of adenocarcinoma cells, the significance and role of cadherin switch in squamous cell carcinomas (SCCs) are largely controversial. In the present study, we immunohistochemically examined expression of E-cadherin and N-cadherin in oral SCCs (n = 63) and its implications for the disease progression. The E-cadherin-positive carcinoma cells were rapidly decreased at the invasive front. The percentage of carcinoma cells stained E-cadherin at the cell membrane was reduced in parallel with tumor dedifferentiation (P<0.01) and enhanced invasion (P<0.01). In contrast, N-cadherin-positive cells were very limited and did not correlate with the clinicopathological parameters. Mouse tongue tumors xenotransplantated oral SCC cell lines expressing both cadherins in vitro reproduced the reduction of E-cadherin-positive carcinoma cells at the invasive front and the negligible expression of N-cadherin. These results demonstrate that the reduction of E-cadherin-mediated carcinoma cell-cell adhesion at the invasive front, but not the cadherin switch, is an important determinant for oral SCC progression, and suggest that the environments surrounding carcinoma cells largely affect the cadherin expression.

Pericyte depletion results in hypoxia-associated epithelial-to-mesenchymal transition and metastasis mediated by met signaling pathway.

The functional role of pericytes in cancer progression remains unknown. Clinical studies suggest that low numbers of vessel-associated pericytes correlated with a drop in overall survival of patients with invasive breast cancer. Using genetic mouse models or pharmacological inhibitors, pericyte depletion suppressed tumor growth but enhanced metastasis. Pericyte depletion was further associated with increased hypoxia, epithelial-to-mesenchymal transition (EMT), and Met receptor activation. Silencing of Twist or use of a Met inhibitor suppressed hypoxia and EMT/Met-driven metastasis. In addition, poor pericyte coverage coupled with high Met expression in cancer cells speculates the worst prognosis for patients with invasive breast cancer. Collectively, our study suggests that pericytes within the primary tumor microenvironment likely serve as important gatekeepers against cancer progression and metastasis.

Mutational analysis of HRAS and KRAS genes in oral carcinoma cell lines.

RAS overexpression and its active mutations are involved in malignant tumorigenesis. However, the mutation rates in oral carcinoma cells differ between populations. In the present study, genomic DNA of oral carcinoma cells (HOC313, TSU, HSC2, HSC3, KOSC2, KOSC3, SCCKN, OSC19, Ca9.22, and Ho1u1 cells) or normal gingival fibroblasts (GF12 cells) derived from a Japanese population were amplified by polymerase chain reaction using primer sets, spanning HRAS and KRAS exons. Nucleotide substitutions were analyzed by single strand conformation polymorphism. In contrast to no substitutions in KRAS, nine different substitutions were detected in HRAS. Of the nine, six substitutions were located at intron 1 (HSC2 and HSC3 cells) or intron 2 (HSC3, SCCKN and Ca9.22 cells), and one each of exon 1 (all cells), exon 2 (HOC313, TSU, HSC2 and HSC3 cells) and the 5' upstream region (all cells). Substitutions at exons 1 and 2 did not affect the amino acid sequence; the exon 1 substitution was positioned at the 5' untranslated region, which may be a single nucleotide polymorphism (SNP) sequence because all the cells were isolated from a Japanese population, and the mutations at exon 2 was a silent mutation. A substitution at the 5' upstream region was an SNP. These data demonstrate that SNPs and point mutations observed in HRAS do not change the amino acid sequence, and suggest that the mutations affecting the amino acid sequence may be a rare event in oral carcinomas of the Japanese population.

Loss of p53 and acquisition of angiogenic microRNA profile are insufficient to facilitate progression of bladder urothelial carcinoma in situ to invasive carcinoma.

Activation of oncogenes or inactivation of tumor suppressors in urothelium is considered critical for development of urothelial cancer. Here we report cloning of the urothelium-specific promoter uroplakin-II (UPK II) and generation of transgenic mice in which expression of SV40 large T antigen is driven by UPK II promoter. Inactivation of tumor suppressor p53 and pRb in urothelium by SV40 T antigen resulted in urothelial carcinoma, resembling human high-grade carcinoma in situ. Specific deletion of p53 in urothelial cells using the newly generated UPK II-Cre mice results in normal bladders without any evidence of cancer. The high-grade carcinoma in situ in the UPK II-SV40 mice is associated with significant activation of angiogenic signals consisting of hypoxia-inducible factor-1α (HIF-1α) and VEGF and a down-regulation of thrombospondin-1. Interestingly, such pro-angiogenic activity was not associated with progression to invasive cancer. Analysis of bladder-associated microRNAs in carcinoma in situ lesions reveals a pro-angiogenic profile, with specific overexpression of miR-18a and miR-19a and down-regulation of miR-107. A group of microRNAs (miRs) identified as associated with invasive human urothelial cancer remained unchanged in this mouse model. Collectively, our results support the notion that activation of angiogenesis and loss of p53 are not sufficient for progression to invasive cancer. Our studies identify a new mouse model for bladder cancer that can be used to study factors that determine progression to an invasive phenotype of bladder cancer.

Epigenetic loss of mucosa-associated lymphoid tissue 1 expression in patients with oral carcinomas.

Mucosa-associated lymphoid tissue 1 (MALT1), which is located in a genomic region that encodes unknown tumor suppressor gene(s), activates nuclear factor-kappaB in lymphocyte lineages. However, its expression and role in the pathology of malignant tumors of epithelial origin is not known. In the present study, we examined MALT1 expression and its implications for the pathology of oral carcinomas. Immunostaining localized MALT1 in the nucleus of normal oral epithelial cells, but the expression was absent in 45.0% of carcinomas (49 of 109 cases) especially at the invasive front. The loss of expression was correlated with tumor recurrence (P = 0.007) and poor patient survival (P < 0.001), and it was an independent prognostic determinant (P < 0.001). MALT1-negative carcinomas exhibited microsatellite instability at the MALT1 locus and a specific cytosine methylation positioned at -256 from the gene, and the expression was recovered by demethylation treatment. In contrast to lymphocyte lineages, carcinoma cells showed MALT1 located at the nucleus independent of its domain structures, and its loss of expression induced the epithelial-mesenchymal transition. These results show that MALT1 is expressed in the nucleus of oral epithelial cells and that its expression is epigenetically inactivated during tumor progression, suggesting that the detection of MALT1 expression is a useful predictive and prognostic determinant in the clinical management of oral carcinomas.

Identification of GATA3 binding sites in Jurkat cells.

Determining binding sites of transcription factors is important for understanding the transcriptional control of target genes. Although a transcription factor GATA3 plays a pivotal role in Th2 lymphocyte development, its physiological role is not clearly defined because the target genes remain largely unknown. In this study, we modified chromatin immunoprecipitation (ChIP), and isolated 121 GATA3 binding sites and 83 different annotated target genes. Re-ChIP analysis using anti-GATA3 and anti-RNA polymerase II mAbs and chromosome conformation capture assay demonstrate that GATA3-bound fragments interact with basal transcriptional units of target genes. GATA3 regulation of target genes under the control of binding fragments was confirmed by reporter assay and quantification of target gene mRNA expression in the presence of GATA inhibitor or short interfering RNA against GATA3. These data demonstrate that GATA3 binds to regulatory elements and controls target gene expression through physical interaction with core promoter regions.

The role of WNT in rheumatoid arthritis and its therapeutic implication.

Rheumatoid arthritis (RA) is a systematic inflammatory and intractable disease, which progressively affects multiple joints. Recent findings strongly suggest a key role of WNT signaling in the disease initiation and progression. In this review, we discuss the role and possibility of treatment by targeting WNT signaling.

Epigenetic inactivation of IkappaB Kinase-alpha in oral carcinomas and tumor progression.

PURPOSE: The loss of epithelial phenotypes in the process of carcinoma progression correlates with clinical outcome, and genetic/epigenetic changes accumulate aggressive clones toward uncurable disease. IkappaB kinase-alpha (IKKalpha) has a decisive role in the development of the skin and establishes keratinocyte phenotypes. We assessed clinical implications of IKKalpha expression in oral carcinomas and epigenetic aberrations for the loss of expression. EXPERIMENTAL DESIGN: We examined IKKalpha expression in oral carcinomas by immunostaining (n = 64) and genetic instability by microsatellite PCR (n = 46). Promoter methylation status was analyzed by bisulfite-modified sequence (n = 11). RESULTS: IKKalpha was expressed in the nucleus of basal cells of normal oral epithelium, but not or marginally detected in 32.8% of carcinomas. The immunoreactivity was significantly decreased in less differentiated carcinomas (P < 0.05) and correlated to long-term survival of patients (P < 0.01) with an independent prognostic value (P < 0.05). Although allelic/biallelic loss of the gene was limited to four cases, we detected microsatellite instability in 63.0% cases in which the immunoreactivities were decreased and the promoter was hypermethylated. CONCLUSION: These results showed that oral carcinomas exhibiting genetic instability and promoter hypermethylation down-regulate expression of IKK and suggest that the epigenetic loss of the expression closely associates with disease progression toward unfavorable prognosis.

Epigenetic inactivation of E-cadherin by promoter hypermethylation in oral carcinoma cells.

The loss of E-cadherin expression by epigenetic aberrations, including promoter hypermethylation and transcription repressor binding, plays a key role in the initiation of the epithelial-mesenchymal transition, which leads to the progression of oral squamous cell carcinomas. However, mutual actions and roles of the epigenetic pathways remain to be elucidated. In this study, we determined the methylation status of cytosine within CpG islands of the E-cadherin promoter region in relation to the expression level of SIP1, a major E-cadherin repressor in oral carcinoma cells. Methylation-specific polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism analyses showed that the expression of E-cadherin was downregulated in parallel with promoter hypermethylation. The use of a bisulfite-modified sequence further validated that methylation was observed in 22.6 +/- 38.7% (mean +/- 1 SD) of cytosines in carcinoma cells negligibly expressing E-cadherin, in contrast to 7.5 +/- 1.8% in E-cadherin-expressing cells. Treatment with a demethylating reagent, 5-azacytidine, induced upregulation of E-cadherin in some E-cadherin-expressing carcinoma cell lines but not in others. The finding that the unresponsive cell lines retained high expression of SIP1 supports the repressive effect of SIP1 on E-cadherin expression regardless of promoter hypermethylation. Collectively, the overall results suggest the dynamic but differential regulation of E-cadherin by epigenetic aberrations in the pathology of oral carcinomas.

Expression of SIP1 in oral squamous cell carcinomas: implications for E-cadherin expression and tumor progression.

Loss of E-cadherin expression allows carcinoma cells to liberate from the primary site and enhances invasion and metastasis. The genetic aberration of E-cadherin is a rare event in sporadic carcinomas, and transcription repressors are considered to take a central role in E-cadherin loss. However, expression of E-cadherin repressors is largely dependent on tissue and cell type. To identify the repressor expressed in oral squamous carcinomas, we compared the expression levels of E-cadherin and repressors by real-time RT-PCR. Among the repressors including SNAIL, SLUG, SIP1, E12 and E47, SIP1 was inversely correlated to E-cadherin (P < 0.05). Chromatin immunoprecipitation showed that SIP1 specifically bound to the E-cadherin promoter region. SIP1 expression was immuno-histochemically detected in 27.7% of 47 oral carcinomas, and SIP1-positive carcinomas did not express E-cadherin (P < 0.01). Thirteen patients with SIP1 staining showed a lower disease-specific survival rate (P < 0.05). Multivariate risk factor analysis demonstrated that SIP1 expression was an independent prognostic value for disease-specific overall survival (P < 0.05). These results suggest that SIP1 contributes to the loss of E-cadherin expression and that detection of SIP1 expression is a predictive and prognostic tool in clinical management of oral carcinomas.