Assessment of maxillary sinus fluid volume for postmortem diagnosis of drowning.

INTRODUCTION: Drowning is a comprehensive and exclusive diagnosis at autopsy. Autopsy findings such as pleural effusion and waterlogged lungs contribute to the diagnosis. Herein, we aim to reveal the practical usefulness and postmortem changes of the maxillary sinus fluid volume to diagnose drowning. METHODS: We evaluated 52 drowning and 59 nondrowning cases. The maxillary sinus fluid volume was measured using a computed tomography (CT) scan, and pleural effusion volume and lung weight were manually measured at autopsy. The utility of these three indices for diagnosing drowning and its postmortem changes was evaluated. RESULTS: The maxillary sinus fluid volume was significantly higher in drowning cases than in other external causes and cardiovascular death cases. Receiver operating characteristic curve analysis revealed that a total maxillary sinus fluid volume >1.04 mL more usefully indicated drowning (odds ratio, 8.19) than a total pleural effusion volume >175 mL (odds ratio, 7.23) and a total lung weight >829 g (odds ratio, 2.29). The combination of maxillary sinus fluid volume and pleural effusion volume more effectively predicted drowning than one index alone. Moreover, the maxillary sinus fluid volume was less influenced by the postmortem interval than the other two indices up to a week after death. CONCLUSION: Maxillary sinus fluid volume can be more useful than pleural effusion volume and lung weight with higher sensitivity and odds ratio for diagnosing drowning. IMPLICATIONS FOR PRACTICE: Fluid accumulation in both the maxillary sinuses strongly predicts drowning in the postmortem imaging.

Subjective Evaluation of Denture Adhesives: A Multicenter Randomized Controlled Trial.

INTRODUCTION: Many reports show that denture adhesives improve the retention and stability of dentures. However, few randomized controlled trials have examined the effects of denture adhesives. OBJECTIVE: This 10-center randomized controlled trial with parallel groups involving 200 edentulous patients wearing complete dentures aimed to evaluate the effects of short-term use of cream and powder denture adhesives. METHODS: Patients were allocated into 2 cream- and powder-type adhesive groups and 1 control group. Intervention groups were treated with the 2 adhesives (1 each), and the control group received saline solution. Adhesive or control was applied to the denture-mucosal surface for 4 d, and data at baseline and after day 4 of intervention (i.e., 8 meals) were obtained. Patient satisfaction was evaluated with a 100-mm visual analog scale. Oral health-related quality of life was measured with the Japanese version of the Oral Health Impact Profile for Edentulous Patients. Perceived chewing ability was evaluated by a questionnaire regarding ease of chewing and swallowing food. Between-group comparisons were performed with Kruskal-Wallis tests with the Mann-Whitney U test adjusted by Bonferroni correction. Within-group comparisons of pre- and postintervention measurements were performed with the Wilcoxon signed-rank test. Intention-to-treat analysis was also performed. RESULTS: Between-group comparisons showed no significant differences for general satisfaction or Oral Health Impact Profile for Edentulous Patients. However, significant differences in satisfaction with various denture functions with cream- and powder-type adhesives were seen in pre- and postintervention comparisons (P < 0.05). Significant differences were also observed for perceived chewing ability of hard foods (P < 0.05). CONCLUSION: These results suggest that although denture adhesives do not invariably improve denture function, they do affect subjective evaluations and possibly chewing of hard foods. Therefore, the effects of denture adhesive use are insufficient to resolve any fundamental dissatisfaction with dentures ( ClinicalTrials.gov NCT01712802 ). KNOWLEDGE TRANSFER STATEMENT: The results of this study suggest that denture adhesives should be applied under certain conditions; however, an appropriate diagnosis is important before application. These practice-based data provide information to establish evidence-based guidelines for applying denture adhesives.

Maximum Occlusal Force and Incident Functional Disability in Older Adults: The Tsurugaya Project.

The purpose of the current study was to investigate the association between maximum occlusal force, which is an objective predictor of masticatory performance, and incident functional disability in an elderly Japanese population. A prospective cohort study was conducted targeting 815 (51.7% female) community-dwelling older adults aged ≥70 y residing in the Tsurugaya district, Sendai, Japan. The outcome measurement was incident functional disability, defined as a first certification of long-term care insurance in Japan, which is determined on the basis of a strictly established, uniform, nationwide standard. During a median follow-up of 7.9 y (interquartile range, 4.8-7.9 y), information on long-term care insurance was obtained from the Sendai Municipal Authority. Bilateral maximum occlusal forces of the participants were measured using a horseshoe-shaped pressure-indicating film, and the participants were categorized into quartiles based on occlusal force. Adjusted hazard ratios for functional disability were estimated with Cox proportional hazard models, adjusted for age, sex, body mass index, medical history, smoking status, alcohol consumption, duration of education, depressive symptoms, cognitive impairment, physical functioning, marital status, history of falls, and number of remaining teeth. The multiple-adjusted hazard ratios and 95% confidence intervals (CIs) for incident functional disability compared to the greatest occlusal force quartile were 1.53 (95% CI, 1.02-2.33), 1.64 (95% CI, 1.06-2.55), and 1.64 (95% CI, 1.01-2.68) for the third, second, and first quartiles, respectively ( P for trend = 0.011). A lower maximum occlusal force was significantly associated with an increased risk of functional disability independently of possible confounders, including the number of remaining teeth. Occlusal force may be a useful indicator of the relationship between oral function and geriatric health. Knowledge Transfer Statement:This prospective cohort study demonstrated that lower maximum occlusal force was associated with an increased risk of functional disability in older adults, even after adjustment for possible confounding factors, including the number of remaining teeth. This strengthens the rationale regarding the association between oral function and geriatric health. Particularly in older adults, occlusal force is reduced by several factors other than tooth loss, such as the absence of a dental prostheses, sarcopenia in the masticatory muscle, poor periodontal condition, and orofacial pain. Our findings suggest that maximum occlusal force may be a useful biomarker associated with diverse parameters aside from the number of remaining teeth.

Deficiency in WT1-targeting microRNA-125a leads to myeloid malignancies and urogenital abnormalities.

The Wilms' tumor gene WT1 is overexpressed in leukemia and solid tumors and has an oncogenic role in leukemogenesis and tumorigenesis. However, precise regulatory mechanisms of WT1 overexpression remain undetermined. In the present study, microRNA-125a (miR-125a) was identified as a miRNA that suppressed WT1 expression via binding to the WT1-3'UTR. MiR-125a knockout mice overexpressed WT1, developed myeloproliferative disorder (MPD) characterized by expansion of myeloid cells in bone marrow (BM), spleen and peripheral blood, and displayed urogenital abnormalities. Silencing of WT1 expression in hematopoietic stem/progenitor cells of miR-125a knockout MPD mice by short-hairpin RNA inhibited myeloid colony formation in vitro. Furthermore, the incidence and severity of MPD were lower in miR-125a (-/-) mice than in miR-125a (+/-) mice, indicating the operation of compensatory mechanisms for the complete loss of miR-125a. To elucidate the compensatory mechanisms, miRNA array was performed. MiR-486 was occasionally induced in compete loss of miR-125a and inhibited WT1 expression instead of miR-125a, resulting in the cancellation of MPD occurrence. These results showed for the first time the post-transcriptional regulatory mechanisms of WT1 by both miR-125a and miR-486 and should contribute to the elucidation of mechanisms of normal hematopoiesis and kidney development.

In vivo eradication of MLL/ENL leukemia cells by NK cells in the absence of adaptive immunity.

It remains unclear how the immune system affects leukemia development. To clarify the significance of the presence of immune systems in leukemia development, we transferred MLL/ENL leukemia cells into immune-competent or immune-deficient mice without any preconditioning including irradiation. The wild-type mice did not develop leukemia, whereas all the Rag2(-/-)γc(-/-) mice lacking both adaptive immune cells and natural killer (NK) cells developed leukemia, indicating that leukemia cells were immunologically rejected. Interestingly, leukemia cells were also rejected in 60% of the Rag2(-/-) mice that lacked adaptive immune cells but possessed NK cells, suggesting that NK cells play a substantial role in the rejection of leukemia. Moreover, engraftment of leukemia cells was enhanced by NK cell depletion in Rag2(-/-) recipients and inhibited by transfer of NK cells into Rag2(-/-)γc(-/-) recipients. Upregulation of NKG2D (NK group 2, member D) ligands in MLL/ENL leukemia cells caused elimination of leukemia cells by NK cells. Finally, we found that leukemia cells resistant to elimination by NK cells had been selected during leukemia development in Rag2(-/-) recipients. These results demonstrate that NK cells can eradicate MLL/ENL leukemia cells in vivo in the absence of adaptive immunity, thus suggesting that NK cells can play a potent role in immunosurveillance against leukemia.

CD138-negative clonogenic cells are plasma cells but not B cells in some multiple myeloma patients.

Clonogenic multiple myeloma (MM) cells reportedly lacked expression of plasma cell marker CD138. It was also shown that CD19(+) clonotypic B cells can serve as MM progenitor cells in some patients. However, it is unclear whether CD138-negative clonogenic MM plasma cells are identical to clonotypic CD19(+) B cells. We found that in vitro MM colony-forming cells were enriched in CD138(-)CD19(-)CD38(++) plasma cells, while CD19(+) B cells never formed MM colonies in 16 samples examined in this study. We next used the SCID-rab model, which enables engraftment of human MM in vivo. CD138(-)CD19(-)CD38(++) plasma cells engrafted in this model rapidly propagated MM in 3 out of 9 cases, while no engraftment of CD19(+) B cells was detected. In 4 out of 9 cases, CD138(+) plasma cells propagated MM, although more slowly than CD138(-) cells. Finally, we transplanted CD19(+) B cells from 13 MM patients into NOD/SCID IL2Rγc(-/-) mice, but MM did not develop. These results suggest that at least in some MM patients CD138-negative clonogenic cells are plasma cells rather than B cells, and that MM plasma cells including CD138(-) and CD138(+) cells have the potential to propagate MM clones in vivo in the absence of CD19(+) B cells.

Quantitative analysis of masticatory activity during unilateral mastication using muscle fMRI.

OBJECTIVE: Quantitative analysis of the activities of all masticatory muscles is required to elucidate the mechanism of stomatognathic dysfunction. Electromyography can be used to record the activity of masticatory muscles, but quantification of the overall activity of every masticatory muscle has not been accomplished because of methodological limitations. In this study, we used muscle functional magnetic resonance imaging for simultaneous quantification of the overall activities of the masseter, medial pterygoid and lateral pterygoid muscles during unilateral gum chewing. METHODS: Seven healthy male volunteers participated in the study. We evaluated changes in the mean proton transverse relaxation time in the bilateral masseter, medial pterygoid and lateral pterygoid muscles before and after unilateral gum chewing, and to quantify the overall activity of these muscles simultaneously during unilateral gum chewing. RESULTS: After 5 min of chewing, the activity of the ipsilateral masseter was highest among the six muscles, followed by the ipsilateral medial pterygoid, contralateral lateral pterygoid and contralateral masseter muscles. CONCLUSION: These results affirm the importance of the ipsilateral masseter muscle and quantitatively demonstrate the important contribution of the ipsilateral medial pterygoid and contralateral lateral pterygoid muscles during unilateral mastication.

Response properties of periodontal mechanosensitive neurons in the trigeminal ganglion of rabbit and neuronal activities during grinding-like jaw movement induced by cortical stimulation.

OBJECTIVE: To examine the response properties of incisor- and molar-sensitive periodontal mechanosensitive (PM) neurons in the trigeminal ganglion of rabbit and the activities of the molar-sensitive PM neurons during the grinding-like jaw movement. DESIGN: Discharges of PM units were recorded from the trigeminal ganglion with a microelectrode. The grinding-like jaw movement was induced by repetitive electrical stimulation of the cortical masticatory area. RESULTS: Upper-incisor (UI) and upper-molar (UM) units were recorded from the rostromedial area of the trigeminal ganglion, and lower-incisor (LI) and lower-molar (LM) units were distributed in the caudolateral area. Most PM units were responsive to only one tooth, slowly adapting ones and responded to tooth stimulation of a force of less than 0.05 N. The optimal stimulus direction for most UI units was labio-lingual, axial or linguo-labial, and that for most LI units was linguo-labial or axial. The optimal stimulus direction of anterior UM and LM units was oriented predominantly mesio-distal or axial. The maximum frequency of spike discharges for UM units for which the optimal stimulus direction was axial or bucco-lingual was in the middle period of the grinding phase. However, UM units for which the optimal stimulus direction was mesio-distal or linguo-buccal were fired mostly in the early period. CONCLUSIONS: Periodontal sensory information in the grinding phase of jaw movement is transmitted by PM neurons with various response properties encoding the magnitude and direction of a force at least, in a weaker range of force than a saturating response level.

EMG study for perioral facial muscles function during mastication.

This study aimed to clarify the temporal and quantitative modulation in the orbicularis oris (OO) and buccinator (BUC) muscle activities during mastication. Ten healthy males (26.9 +/- 1.0 years) participated. Electromyograms (EMGs) of the facial muscles were recorded with fine wire electrodes when chewing the chewing gum (one to four sticks) and peanuts (one to five pieces). Surface EMGs of the masseter (MAS) and digastric muscles were recorded simultaneously. EMGs of the OO and BUC showed rhythmic single-peaked bursts corresponding to the jaw-opening phase of chewing cycles. The total cycle lengths were constant regardless of the food amount. Integrated EMGs of the OO changed significantly when the amount of both foods changed (anova: P < 0.05). Those of the BUC changed significantly with the amount of gum changed (P < 0.05), but did not change with the amount of peanuts changed. The burst duration of OO changed significantly when the amount of gum changed during ipsilateral chewing (P < 0.05). When the amount of peanuts changed during ipsilateral chewing, the onset of OO and the peak of BUC based on the onset of MAS activity changed significantly (P < 0.05). However, the onset, peak and offset of the OO and BUC based on the offset of MAS did not change regardless of the amounts chewed. The changes of the OO and BUC activities may derive from chewing-generated sensory inputs in accordance with the physical property of food in part, which would relate to the function of these muscles during mastication.

The Wilms' tumor gene WT1-GFP knock-in mouse reveals the dynamic regulation of WT1 expression in normal and leukemic hematopoiesis.

The Wilms' tumor gene WT1 is overexpressed in most of human leukemias regardless of disease subtypes. To characterize the expression pattern of WT1 during normal and neoplastic hematopoiesis, we generated a knock-in reporter green fluorescent protein (GFP) mouse (WT1(GFP/+)) and assayed for WT1 expression in normal and leukemic hematopoietic cells. In normal hematopoietic cells, WT1 was expressed in none of the long-term (LT) hematopoietic stem cells (HSC) and very few (<1%) of the multipotent progenitor cells. In contrast, in murine leukemias induced by acute myeloid leukemia 1 (AML1)/ETO+TEL/PDGFbetaR or BCR/ABL, WT1 was expressed in 40.5 or 38.9% of immature c-kit(+)lin(-)Sca-1(+) (KLS) cells, which contained a subset, but not all, of transplantable leukemic stem cells (LSCs). WT1 expression was minimal in normal fetal liver HSCs and mobilized HSCs, both of which are stimulated for proliferation. In addition, overexpression of WT1 in HSCs did not result in proliferation or expansion of HSCs and their progeny in vivo. Thus, the mechanism by which expansion of WT1-expressing cells occurs in leukemia remains unclear. Nevertheless, our results demonstrate that the WT1(GFP/+) mouse is a powerful tool for analyzing WT1-expressing cells, and they highlight the potential of WT1, as a specific therapeutic target that is expressed in LSCs but not in normal HSCs.

Development of WT1 peptide cancer vaccine against hematopoietic malignancies and solid cancers.

Wild-type Wilms' tumor gene WT1 is highly expressed not only in hematopoietic malignancies, including leukemia and myelodysplastic syndromes (MDS), but also in various kinds of solid tumors. Human cytotoxic T lymphocytes (CTLs) which could specifically lyse WT1-expressing tumor cells with HLA class I restriction were generated in vitro. We have also demonstrated that mice immunized with the WT1 peptide or WT1 cDNA rejected challenges by WT1-expressing tumor cells and survived with no signs of auto-aggression to normal organs which physiologically expressed WT1 in prophylactic and therapeutic models. Furthermore, we and others detected IgM and IgG WT1 antibodies in the patients with hematopoietic malignancies, indicating that WT1 protein was highly immunogenic, and that immunoglobulin class-switch-inducing WT1-specific cellular immune responses were elicited in the patients. CD8+ WT1-specific CTLs were also detected in peripheral blood or tumor-draining lymph nodes of cancer patients. These results provided us with the rationale for elicitation of CTL responses targeting the WT1 product for cancer immunotherapy. On the basis of the findings mentioned above, we performed a phase I clinical trial of WT1 peptide cancer vaccine for the patients with malignant neoplasms. These results strongly suggested that WT1 peptide cancer vaccine had efficacy in the clinical setting, because clinical responses, including reduction of leukemic blast cells or regression of tumor masses, were observed after the WT1 vaccination in patients with hematopoietic malignancies or solid cancers. The power of TAA-derived cancer vaccine may be enhanced by combination with stronger adjuvants, helper peptide, or conventional treatments such as molecular-target-based drugs.

Functional properties and regional differences of human masseter motor units related to three-dimensional bite force.

The aim of this study was to estimate numerically the properties of masseter motor units (MUs) in relation to bite force magnitude and direction three-dimensionally and to confirm the hypothesis that the properties differ between different parts of the muscle by means of simultaneous recording of MU activity along with the MU location and three-dimensional (3D) bite force. The MU activity of the right masseter of four healthy men was recorded using a monopolar needle electrode in combination with a surface reference electrode. The location of the needle electrode was estimated stereotactically with the aid of magnetic resonance images and a reference plate. The magnitude and direction of the bite force was recorded with a custom-made 3D bite force transducer. The recorded bite force was displayed on a signal processor, which enabled the participant to adjust the direction and magnitude of the force. The activities of 65 masseter MUs were recorded. Each MU had specific ranges of bite force magnitude and direction (firing range: FR) and an optimum direction for recruitment (minimum firing threshold: MFT). There was a significant negative correlation between MFT and FR width. There were functional differences in MU properties between the superficial and deep masseter and between the superficial layer and deep layer in the superficial masseter. These results indicate that the contribution of human masseter motor units to bite force production is heterogeneous within the muscle.

Antiapoptotic function of 17AA(+)WT1 (Wilms' tumor gene) isoforms on the intrinsic apoptosis pathway.

The WT1 gene is overexpressed in human primary leukemia and a wide variety of solid cancers. The WT1 gene is alternatively spliced at two sites, yielding four isoforms: 17AA(+)KTS(+), 17AA(+)KTS(-), 17AA(-)KTS(+), and 17AA(-)KTS(-). Here, we showed that 17AA(+)WT1-specific siRNA induced apoptosis in three WT1-expressing leukemia cell lines (K562, HL-60, and Kasumi-1), but not in WT1-non-expressing lymphoma cell line (Daudi). 17AA(+)WT1-specific siRNA activated caspase-3 and -9 in the intrinsic apoptosis pathway but not caspase-8 in the extrinsic one. On the other hand, 17AA(-)WT1-specific siRNA did not induce apoptosis in the three WT1-expressing cell lines. The apoptosis was associated with activation of proapoptotic Bax, which was activated upstream of the mitochondria. Constitutive expression of 17AA(+)WT1 isoforms inhibited apoptosis of K562 leukemia cells induced by apoptosis-inducing agents, etoposide and doxorubicin, through the protection of mitochondrial membrane damages, and DNA-binding zinc-finger region of 17AA(+)WT1 isoform was essential for the antiapoptotic functions. We further studied the gene(s) whose expression was altered by the expression of 17AA(+)WT1 isoforms and showed that the expression of proapoptotic Bak was decreased by the expression of 17AA(+)KTS(-)WT1 isoform. Taken together, these results indicated that 17AA(+)WT1 isoforms played antiapoptotic roles at some points upstream of the mitochondria in the intrinsic apoptosis pathway.

Th1-biased humoral immune responses against Wilms tumor gene WT1 product in the patients with hematopoietic malignancies.

The Wilms' tumor gene WT1 is highly expressed in leukemias and myelodysplastic syndrome (MDS), and WT1 expression levels increase along with the disease progression in chronic myeloid leukemia and MDS. We previously reported that IgM and IgG WT1 antibodies were detected with significantly higher detection rate and antibody titers in leukemias and MDS compared to those in healthy volunteers. In this study, whether IgG humoral immune responses against WT1 protein were Th1- or Th2-type were determined by measurement of four subclasses of IgG WT1 antibody, IgG1, IgG2, IgG3, and IgG4. In leukemias and MDS, Th1-type WT1 antibodies such as IgG1, IgG2, and IgG3 were significantly increased in both detection rate and antibody titers compared to those in healthy volunteers, whereas Th2-type WT1 antibody such as IgG4 did not increase. These results showed that Th1-biased humoral immune responses against WT1 protein were generated in leukemias and MDS. These results should allow us to consider that Th1-biased cellular immune responses against WT1 protein, which was essentially needed for cancer immunotherapy targeting WT1, should be elicited in patients with hematopoietic malignancies.

Preferential expression of the vasoactive intestinal peptide (VIP) receptor VPAC1 in human cord blood-derived CD34+CD38- cells: possible role of VIP as a growth-promoting factor for hematopoietic stem/progenitor cells.

Primitive hematopoietic progenitor cells such as severe combined immunodeficiency- repopulating cells and long-term culture-initiating cells are enriched in CD34+CD38- cells derived from various stem cell sources. In this study, to elucidate the features of such primitive cells at the molecular level, we tried to isolate genes that were preferentially expressed in umbilical cord blood (CB)-derived CD34+CD38- cells by subtractive hybridization. The gene for VPAC1 receptor, a receptor for the neuropeptide vasoactive intestinal peptide (VIP), was thereby isolated and it was shown that this gene was expressed in both CD34+CD38- and CD34+CD38+ CB cells and that the expression levels were higher in CD34+CD38- CB cells. Next, we assessed the effects of VIP on the proliferation of CD34+ CB cells using in vitro culture systems. In serum-free single-cell suspension culture, VIP enhanced clonal growth of CD34+ CB cells in synergy with FLT3 ligand (FL), stem cell factor (SCF), and thrombopoietin (TPO). In serum-free clonogenic assays, VIP promoted myeloid (colony-forming unit-granulocyte/macrophage (CFU-GM)) and mixed (CFU-Mix) colony formations. Furthermore, in Dexter-type long-term cultures, VIP increased colony-forming cells at week 5 of culture. These results suggest that VIP functions as a growth-promoting factor of CB-derived hematopoetic progenitor cells.

Absence of mutations in the Wilms' tumor gene wt1 in de novo non-small cell lung cancers.

We recently demonstrated that the WT1 gene was overexpressed in the majority of de novo lung cancers regardless of cancer subtypes. Here, we examined WT1 genomic DNA in 38 cases of de novo non-small cell lung cancers (NSCLC) for mutations using direct sequencing. The sequencing analysis showed no mutations of WT1 genomic DNA in any of 38 de novo non-small cell lung cancers examined. These results indicated that the non-mutated, wild-type WT1 gene played an important role in de novo NSCLC.

Identification of a gene element essential for leukemia-specific expression of transgenes.

Leukemia-specific promoters and enhancers for gene therapy had never been reported. Since the Wilms' tumor gene WT1 is overexpressed in almost all types of leukemia, WT1 is an ideal target of leukemia-specific therapy. To explore the possibility of gene therapy for leukemia using WT1 promoter and enhancer, their activities in several kinds of cells were analyzed by using the enhanced green fluorescent protein (EGFP) gene as a reporter. First, we identified the best combination (654P/EGFP/int3- enh/3'-enh vector) of the 654-bp WT1 promoter and the two WT1 enhancers located in intron 3 and at the 3' end of the WT1 gene for inducing EGFP expression in K562 cells, which endogenously expressed WT1. When this was transfected into WT1-expressing leukemia cells (K562, HEL), WT1-nonexpressing hematopoietic cells (Daudi, U937), and WT1-expressing nonhematopoietic cells (TYK-nu-CPr, SW480, 293 T), 19.8, 22.9, 1.47, 1.43, 4.50, 4.16, and 1.09 times EGFP expression was induced, respectively, compared to that by the promoter-less EGFP vector. These results showed that the 654P/EGFP/int3-enh/3'-enh vector specifically induced high levels of EGFP expression in WT1-expressing leukemia cells. 654P/int3- enh/3'-enh vector containing transgenes such as suicide genes might become useful tools for leukemia-specific gene therapy.