Expression of metastasis-associated protein 1 (MTA1) gene correlates with the degree of invasion and metastasis in hepatocellular carcinoma (HCC). Expression of MTA1 is induced by hepatitis B virus X protein (HBx); however, little is known about the transcriptional regulation of MTA1 gene expression. Here, we report that the 5′-flanking region of the human MTA1 promoter contains two CpG islands. Transient expression of HBx in Chang liver cells increased the methylation of the CpG island1 from 18 to 49% when measured by bisulfite-modified direct sequencing. Chromatin immunoprecipitation showed that HBx recruited DNA methyltransferase 3a (DNMT3a) and DNMT3b to the CpG island1. In silico analysis of CpG island1 predicted the existence of putative p53-binding sequences. p53 was pulled down by a DNA probe encoding the p53-binding sequences but not by the methylated DNA probe. The mouse MTA1 promoter also contains a CpG island encoding a p53-binding sequence of which p53 binding was decreased in the presence of HBx, and the expression of MTA1 and DNMT3 was increased in the liver of HBx-transgenic mice. Comparison of MTA1 and DNMT3a expression in the human normal liver and HCC specimens produced a significant correlation coefficient >0.5 (r=0.5686, P=0.0001) for DNMT3a, and a marginally significant coefficient (r=0.3162, P=0.0103) for DNMT3b. These data show that HBx induces methylation of CpG island in the MTA1 promoter, which interferes with DNA binding of p53 in the specific DNA region. This result may explain the molecular mechanism responsible for the induction of MTA1 gene expression by HBx.
MTA1; p53; HBx; DNA methylation
Recently apoptotic cell death has been reported in differentiated skeletal muscle, where apoptosis was generally assumed not to occur. To investigate whether apoptosis may contribute to the steroid-induced myopathy, rats treated with triamcinolone acetonide (TA) for 9 days were sacrificed for detecting apoptosis by in situ end labeling (ISEL) and electron microscopy in the soleus muscles. Immunohistochemical stainings of Fas antigen and p53 protein were performed to examine whether apoptosis-related proteins were present in the myopathy. Muscle fiber necrosis and apoptotic myonuclei appeared in the soleus muscles following administration of TA, while control muscles showed no evidences for apoptosis. Fas antigen was not detected in control muscles, but expressed in the soleus muscles of steroid-induced myopathy. Some of the Fas antigen-expressing muscle fibers were positive for ISEL. p53 protein was not detected in any muscle fibers. These findings indicate that TA can induce apoptosis in differentiated skeletal muscles, and Fas antigen might be partly related to apoptotic muscle death in steroid-induced myopathy.
We report three autopsy cases of congenital cytomegalovirus (CMV) infection in fetuses with a review of literature. The clinical manifestations in these cases of congenital CMV infection include intrauterine fetal death, hydrops fetalis, and CMV pneumonia associated with cardiovascular defect. The pathological characteristics were as follows: 1) the kidney was the most frequently involved organ, followed by lung and liver, 2) CMV inclusions were found predominantly in epithelial cells and to a lesser degree in endothelial cells, 3) intrahepatic bile duct epithelial cells were frequently involved, and 4) inflammatory reaction around CMV inclusions was not prominent in the early stage of pregnancy. Diagnostic confirmation was obtained by in situ hybridization (ISH) using a biotinylated CMV-DNA probe, which demonstrated intranuclear inclusions and sometimes recognized cells that did not show intranuclear inclusion.
The cytokine pattern on viral antigen recognition is believed to exert a profound influence on the resolution of viral infections and viral clearance. This study was initiated to investigate whether a cytokine imbalance oriented toward Th2 type response plays a role in chronic hepatitis B. Cytokine profiles of peripheral blood mononuclear cells associated with chronic hepatitis B were analysed by RT-PCR. Upon HBsAg stimulation, expression of IFN-gamma, IL-2, IL-4, and IL-10 was detected in 41%, 8%, 41%, and 50% of the patients, respectively. Among these cytokines, the expression of IFN-gamma was associated with high levels of serum AST/ALT. However, we could not prove that Th2 type cytokines had a protective effect on hepatocytes. Upon HBxAg stimulation, there was no recognizable association of cytokine patterns with AST/ALT levels. In conclusion, production of a Th1 cytokine, IFN-gamma, by HBsAg-reactive cells was associated with hepatocyte damage in chronic hepatitis B, while no counteracting effect of Th2 cytokines produced by those cells was observed.
Multiple symmetric lipomatosis (MSL) is an extremely uncommon disorder. In the medical literatures about 200 cases have been reported. MSL is not associated with other generalized lipomatous disorders, nor are these patient to be necessarily obese. The cause of MSL is unknown. The disorder usually occurs in middle-aged males and there is frequently a history of alcoholism. Some instances of familial occurrence have been reported, but the majority of cases are sporadic. Two cases of MSL are presented.
Automated data processing and quality control of radioimmunoassays offer not only increased speed but also a more thorough and statistically rigorous analysis of results. An external quality assessment scheme for serum thyroxine, triiodothyronine and thyroid stimulating hormone (TSH) assays was performed in five nuclear medicine laboratories in Korea to compare with the assay performances of the World Health Organization Radioimmunoassay Program. The required radioimmunoassay kits were supplied through the International Atomic Energy Agency (IAEA). We have determined the weighted root mean squared error, and variance ratio as the indices of standard curve and also the average batch coefficient of variation (ABCV) as the parameters of response error relationship curve and precision profile. There was a good fit for the triiodothyronine assay, but 3 of 5 laboratories showed possible bad fit in the T4 and TSH assay systems. The ABCV was less than 5 percent for the T3 and T4 assay system, however for the TSH system, only 1 laboratory showed the ABCV value of less than 5 percent. We have also calculated the within batch variation (drift) and between laboratory variations.
Altered metabolism in cancer cells is suspected to contribute to chemoresistance but the precise mechanisms are unclear. Here we show that intracellular ATP levels are a core determinant in the development of acquired cross-drug resistance of human colon cancer cells that harbor different genetic backgrounds. Drug-resistant cells were characterized by defective mitochondrial ATP production, elevated aerobic glycolysis, higher absolute levels of intracellular ATP and enhanced HIF-1α-mediated signaling. Interestingly, direct delivery of ATP into cross-chemoresistant cells destabilized HIF-1α and inhibited glycolysis. Thus, drug-resistant cells exhibit a greater “ATP debt” defined as the extra amount of ATP needed to maintain homeostasis of survival pathways under genotoxic stress. Direct delivery of ATP was sufficient to render drug-sensitive cells drug resistant. Conversely, depleting ATP by cell treatment with an inhibitor of glycolysis, 3-bromopyruvate, was sufficient to sensitize cells cross-resistant to multiple chemotherapeutic drugs. In revealing intracellular ATP levels are a core determinant of chemoresistance in colon cancer cells, our findings may offer a foundation for new improvements to colon cancer treatment.
chemoresistance; ATP; aerobic glycolysis
Thalidomide has potent anti-inflammatory and anti-angiogenic properties. It was evaluated in combination with chemotherapy in two randomised placebo-controlled trials in patients with small cell lung cancer (SCLC, n=724) and advanced non-small cell lung cancer (NSCLC, n=722). Neither study demonstrated an improvement in overall survival with the addition of thalidomide to chemotherapy. This study investigated circulating angiogenic biomarkers in a subset of these patients.
Serial plasma samples were collected in a cohort of patients enrolled in these two trials (n=95). Vascular endothelial growth factor (VEGF), soluble truncated form of VEGF receptor-2 (sVEGFR-2), interleukin-8 (IL-8), tumour necrosis factor-α (TNF-α), basic fibroblast growth factor (bFGF) and soluble intercellular adhesion molecule-1 (sICAM-1) levels were measured by enzyme-linked immunosorbent assays. Results were correlated with patient clinical data including stage, response rate and progression-free survival (PFS).
Baseline biomarker levels were not significantly different between SCLC and NSCLC. For pooled treatment groups, limited stage SCLC was associated with lower baseline VEGF (P=0.046), sICAM-1 (P=0.008) and IL-8 (P=0.070) than extensive stage disease. Low baseline IL-8 was associated with a significantly improved PFS in both SCLC and NSCLC (P=0.028), and a greater reduction in IL-8 was associated with a significantly improved tumour response (P=0.035). Baseline angiogenic factor levels, however, did not predict response to thalidomide.
Circulating angiogenic biomarkers did not identify patients who benefited from thalidomide treatment.
lung cancer; thalidomide; angiogenesis; biomarker
When speech is interrupted by noise, listeners often perceptually “fill-in” the degraded signal, giving an illusion of continuity and improving intelligibility. This phenomenon involves a neural process in which the auditory cortex (AC) response to onsets and offsets of acoustic interruptions is suppressed. Since meaningful visual cues behaviorally enhance this illusory filling-in, we hypothesized that during the illusion, lip movements congruent with acoustic speech should elicit a weaker AC response to interruptions relative to static (no movements) or incongruent visual speech. AC response to interruptions was measured as the power and inter-trial phase consistency of the auditory evoked theta band (4-8 Hz) activity of the electroencephalogram (EEG) and the N1 and P2 auditory evoked potentials (AEPs). A reduction in the N1 and P2 amplitudes and in theta phase-consistency reflected the perceptual illusion at the onset and/or offset of interruptions regardless of visual condition. These results suggest that the brain engages filling-in mechanisms throughout the interruption, which repairs degraded speech lasting up to ~250 ms following the onset of the degradation. Behaviorally, participants perceived greater speech continuity over longer interruptions for congruent compared to incongruent or static audiovisual streams. However, this specific behavioral profile was not mirrored in the neural markers of interest. We conclude that lip-reading enhances illusory perception of degraded speech not by altering the quality of the AC response, but by delaying it during degradations so that longer interruptions can be tolerated.
Audiovisual integration; Auditory Evoked Potentials; EEG; Illusory filling-in; phase-locking; Theta band
Although both MHC class II/CD8α double knock out and CD8β null mice show a defect in the development of MHC class I-restricted CD8+ T cells in the thymus, they possess low numbers of high avidity peripheral CTL with limited clonality and are able to contain acute and chronic infections. This in vivo data suggests that the CD8 coreceptor is not absolutely necessary for the generation of antigen-specific CTL. Lack of CD8 association causes partial TCR signaling because of the absence of CD8/Lck recruitment to the proximity of the MHC/TCR complex resulting in suboptimal MAPK activation. Therefore, there should exist a signaling mechanism that can supplement partial TCR activation caused by the lack of CD8 association. In this human study, we have shown that CD8-independent stimulation of antigen-specific CTL previously primed in the presence of CD8 coligation, either in vivo or in vitro, induced severely impaired in vitro proliferation. When naïve CD8+ T cells were primed in the absence of CD8 binding and subsequently restimulated in the presence of CD8 coligation, the proliferation of antigen-specific CTL was also severely hampered. However, when CD8-independent T-cell priming and restimulation was supplemented with IL-21, antigen-specific CD8+ CTL expanded in 2 out of 6 individuals tested. We found that IL-21 rescued partial MAPK activation in a STAT3- but not STAT1-dependent manner. These results suggest that CD8 coligation is critical for the expansion of post-thymic peripheral antigen-specific CTL in humans. However, STAT3-mediated IL-21 signaling can supplement partial TCR signaling caused by the lack of CD8 association.
Toxicity data from cancer trials are summarized into a single outcome, dose-limiting toxicity (DLT), which does not account for multiple lower grade toxic effects nor differentiates between toxicity types and gradations within DLT.
Toxicity data were summarized into a toxicity burden score (TBS) using a weighted sum. The severity weights were estimated via regression using historical data. We demonstrated the method using historical data from a bortezomib trial and illustrated the advantages of defining DLT based on TBS in a simulated dose-finding trial.
The estimated weights were 0.17, 0.40 and 0.85 for grade 1/2, grade 3 and grade 4 platelets, respectively; 0.19, 0.64, 1.03 and 2.53 for grade 1, 2, 3 and 4 neuropathy, respectively and 0.17 for each grade 3 or higher nonhematologic toxic effects unrelated to treatment. In the simulated trial, the probability of selecting doses above the maximum tolerated dose decreased when using the DLT defined based on TBS.
TBS is a feasible approach to summarize toxicity. It includes information from the grades and types of multiple toxic effects and can be applied in all phases of drug development. Further efforts should focus on validating the method in a large prospective study before applying it in practice.
adverse event summary; dose-limiting toxicity; toxicity; toxicity types and grades
The blood-testis barrier (BTB) is a unique ultrastructure in the testis which creates a specialized microenvironment in the seminiferous epithelium for post-meiotic germ cell development and to maintain an immunological barrier. In this report, we have demonstrated unequivocally that a functional and intact BTB is crucial for initiation of spermatogenesis in particular differentiation of spermatogonial stem cells (SSCs). It was shown that adult rats (~300 gm body weight, b.w.) treated with adjudin at 50 (low-dose) or 250 (high-dose) mg/kg b.w. by gavage led to germ cell depletion from the seminiferous tubules and >98% of the tubules were devoid of germ cells by ~2-week and rats became infertile in both groups after the sperm reserve in the epididymis was exhausted. While the population of SSC/spermatogonia in the seminiferous tubules from both groups was similar to normal rats, only rats from the low-dose group were capable of re-initiating spermatogenesis by 20-week and by 30-week, greater than 75% of the tubules displayed normal spermatogenesis and the fertility of these rats rebounded. Detailed analysis by dual-labeled immunofluorescence analysis and a functional BTB integrity assay revealed that in both treatment groups, the BTB was disrupted from 6- to 12-week. However, the disrupted BTB “resealed” in the low, but not in the high, dose group. Our findings illustrate that that SSC/spermatogonia failed to differentiate into spermatocytes beyond Aaligned spermatogonia in the high-dose group with a disrupted BTB. In short, these findings illustrate the critical significance of BTB for re-initiation of spermatogenesis besides SSC and spermatogonia.
testis; blood-testis barrier; seminiferous epithelial cycle; spermatogonia; adjudin; spermatogonial stem cells
This study aimed to show that the horizontal relationship between the mandibular canal and the alveolar crest can influence the available bone height (ABH) measurement on panoramic radiographs.
92 mandibular edentulous sites of panoramic computed radiographs and reformatted CT images of 77 patients were used. Selected CT images were categorized into four types according to the relative location of the peak of the alveolar crest to the mandibular canal. One oral and maxillofacial radiologist measured the ABH twice on both imaging modalities with an interval of 7 days and compared the measurement differences according to the type.
The absolute average value of the differences in measurement between the values of ABHs on panoramic images and CT images was 0.97 mm. Significant difference was found only between the mean values of ABHs for Type 1 (0.60 mm), where the alveolar crest is located in the buccal side or central area with respect to the mandibular canal, and Type 4 (1.46 mm), where the alveolar crest is in the lingual side to the mandibular canal (p < 0.05).
The relative horizontal location of the alveolar crest with respect to the mandibular canal affected the ABH measurement on panoramic radiographs. In particular, ABH is overestimated when there has been resorption of the buccal aspect of the ridge, moving the alveolar crest lingually.
panoramic radiography; inferior alveolar nerve; alveolar ridge; X-ray computed tomography
In vivo anti-polysaccharide (PS) Ig responses to isolated PS are T cell-independent, rapid, and fail to generate memory. However, little is known regarding PS-specific Ig responses to intact Gram-positive (GP) and Gram-negative (GN) extracellular bacteria. We previously demonstrated that intact heat-killed Streptococcus pneumoniae (Pn), a GP bacterium, elicited a rapid primary IgG anti-capsular PS (PPS) response in mice that was dependent on CD4+ T cells, B7-dependent costimulation, and CD40-CD40L interactions. However, this response was ICOS-independent and failed to generate a boosted PPS-specific secondary IgG response. In the present study we analyzed the murine anti-capsular PS (MCPS) Ig response to i.p.-injected intact, heat-killed Neisseria meningitidis serogroup C (MenC), a GN bacterium. In contrast to Pn, the IgG anti-MCPS response to MenC exhibited delayed primary kinetics and was highly boosted after secondary immunization, whereas the IgG anti-MCPS response to isolated MCPS was rapid, without secondary boosting, and consisted of only IgG1 and IgG3, as opposed to all four IgG isotypes in response to intact MenC. The secondary, but not primary, IgG anti-MCPS response to MenC was dependent on CD4+ T cells, CD40L, CD28 and ICOS. The primary and secondary IgG anti-MCPS responses were lower in TLR4-defective (C3H/HeJ), although not TLR2−/− or MyD88−/− mice, but secondary boosting was still observed. Of interest, co-immunization of Pn and MenC, resulted in a boosted secondary IgG anti-PPS response to Pn. Our data demonstrate that the nature of the in vivo anti-PS response is markedly influenced by the composition and/or architecture of the bacterial subcapsular domain.
Rodent; B cells; T cells; bacterial; antibodies; antigens; vaccination; memory; costimulation; transgenic/knockout mice
In this video we demonstrate a protocol to establish mouse thymic lymphoma cell lines. By following this protocol, we have successfully established several T-cell lines from Atm−/− and p53−/− mice with thymic lymphoma.
Established cell lines are a critical research tool that can reduce the use of laboratory animals in research. Certain strains of genetically modified mice, such as Atm−/− and p53−/− consistently develop thymic lymphoma early in life (1,2), and thus, can serve as a reliable source for derivation of murine T-cell lines. Here we present a detailed protocol for the development of established murine thymic lymphoma T-cell lines without the need to add interleukins as described in previous protocols (1,3). Tumors were harvested from mice aged three to six months, at the earliest indication of visible tumors based on the observation of hunched posture, labored breathing, poor grooming and wasting in a susceptible strain (1,4). We have successfully established several T-cell lines using this protocol and inbred strains of Atm−/− [FVB/N-Atmtm1Led/J] (2) and p53−/− [129/S6-Trp53tm1Tyj/J] (5) mice. We further demonstrate that more than 90% of the established T-cell population expresses CD3, CD4 and CD8. Consistent with stably established cell lines, the T-cells generated by using the present protocol have been passaged for over a year.
Multimarker quantitative real-time polymerase chain reaction (qRT-PCR) represents an effective method for detecting circulating tumour cells in the peripheral blood of patients with melanoma.
To investigate whether the phenotype of circulating melanoma cells represents a useful indicator of disease stage, recurrence and treatment efficacy.
Peripheral blood was collected from 230 patients with melanoma and 152 healthy controls over a period of 3 years and 9 months. Clinical data and blood samples were collected from patients with primary melanoma (early stages, 0–II, n = 154) and metastatic melanoma (late stages, III–IV, n = 76). Each specimen was examined by qRT-PCR analysis for the expression of five markers: MLANA, ABCB5, TGFβ2, PAX3d and MCAM.
In total, 212 of the patients with melanoma (92%) expressed markers in their peripheral blood. Two markers, MLANA and ABCB5, had the greatest prognostic value, and were identified as statistically significant among patients who experienced disease recurrence within our study period, being expressed in 45% (MLANA) and 49% (ABCB5) of patients with recurrence (P = 0·001 and P = 0·031, respectively). For patients administered nonsurgical treatments, MCAM expression correlated with poor treatment outcome.
Circulating tumour cells were detectable at all stages of disease and long after surgical treatment, even when patients were considered disease free. Specifically, expression of ABCB5 and MLANA had significant prognostic value in inferring disease recurrence, while MCAM expression was associated with poor patient outcome after treatment, confirming multimarker qRT-PCR as a potential technique for monitoring disease status.
It is becoming increasingly clear that many diseases are the result of infection from multiple genetically distinct strains of a pathogen. Such multi-strain infections have the capacity to alter both disease and pathogen dynamics. Infection with multiple strains of human cytomegalovirus (HCMV) is common and has been linked to enhanced disease. Suggestions that disease enhancement in multi-strain infected patients is due to complementation have been supported by trans-complementation studies in mice during co-infection of wild type and gene knockout strains of murine CMV (MCMV). Complementation between naturally circulating strains of CMV has, however, not been assessed. In addition, many models of multi-strain infection predict that co-infecting strains will compete with each other and that this competition may contribute to selective transmission of more virulent pathogen strains. To assess the outcome of multi-strain infection, C57BL/6 mice were infected with up to four naturally circulating strains of MCMV. In this study, profound within-host competition was observed between co-infecting strains of MCMV. This competition was MCMV strain specific and resulted in the complete exclusion of certain strains of MCMV from the salivary glands of multi-strain infected mice. Competition was dependent on Ly49H+ natural killer (NK) cells as well as the expression of the ligand for Ly49H, the MCMV encoded product, m157. Strains of MCMV which expressed an m157 gene product capable of ligating Ly49H were outcompeted by strains of MCMV expressing variant m157 genes. Importantly, within-host competition prevented the shedding of the less virulent strains of MCMV, those recognized by Ly49H, into the saliva of multi-strain infected mice. These data demonstrate that NK cells have the strain specific recognition capacity required to meditate within-host competition between strains of MCMV. Furthermore, this within-host competition has the capacity to shape the dynamics of viral shedding and potentially select for the transmission of more virulent virus strains.
Infection of the host with multiple strains of a pathogen is common and occurs with the herpesvirus, human cytomegalovirus (HCMV). However the effects of multi-strain infection on the host and the pathogen remain poorly studied. Here we show, in a mouse model, that infection of C57BL/6 mice with multiple strains of murine CMV (MCMV) results in profound within-host competition. Competition between the strains of MCMV is dependent on Ly49H+ natural killer (NK) cells. The NK cell activation receptor Ly49H receptor targets certain genotypes of the viral protein, m157. During multi-strain infection, strains of MCMV encoding an m157 capable of binding Ly49H are excluded from the salivary gland and the saliva of C57BL/6 mice, allowing for the shedding of only non-Ly49H binding strains of MCMV in the saliva. This within-host competition could therefore have significant impacts on the circulation of MCMV strains, as only the most virulent MCMV strains were present in the saliva.
To reduce stimulus transduction artifacts in EEG while using insert earphones.
Reference Equivalent Threshold SPLs (RETSPLs) were assessed for Etymotic ER-4B earphones in fifteen volunteers. Auditory brainstem responses (ABRs) and middle latency responses (MLRs) – as well as long-duration complex ABRs – to click and /dɑ/ speech stimuli were recorded in a single-case design.
Transduction artifacts occurred in raw EEG responses, but they were eliminated by shielding, counter-phasing (averaging across stimuli 180° out of phase) or re-referencing.
Clinical-grade ABRs, MLRs, and cABRs can be recorded with a standard digital EEG system and high-fidelity insert earphones, provided one or more techniques are used to remove the stimulus transduction artifact.
ABR; cABR; ER-4B; headphones; RETSPL
To assess the health literacy and numeracy skills of Spanish-speaking parents of young children and to validate a new Spanish language health literacy assessment for parents, the Spanish Parental Health Literacy Activities Test (PHLAT-10 Spanish).
Cross-sectional study of Spanish-speaking caregivers of young children (<30 months) enrolled at primary care clinics in 4 academic medical centers. Caregivers were administered the 10-item PHLAT in addition to validated tests of health literacy (S-TOFHLA) and numeracy (WRAT-3 Arithmetic). Psychometric analysis was used to examine item characteristics of the PHLAT-10 Spanish, to assess its correlation with sociodemographics and performance on literacy/numeracy assessments, and to generate a shorter 8-item scale (PHLAT-8).
Of 176 caregivers, 77% had adequate health literacy (S-TOFHLA), while only 0.6% had 9th grade or higher numeracy skills. Mean PHLAT-10 score was 41.6% (SD 21.1). Fewer than half (45.5%) were able to read a liquid antibiotic prescription label and demonstrate how much medication to administer within an oral syringe. Less than a third (31.8%) were able to interpret a food label to determine whether it met WIC guidelines. Higher PHLAT-10 score was associated with higher years of education (r=0.49), S-TOFHLA (r=0.53) and WRAT-3 (r=0.55) scores (p<0.001). Internal reliability was good (KR-20=0.61). An 8-item scale was highly correlated with the full 10-item scale (r=0.97, p<0.001), with comparable internal reliability (KR-20= 0.64).
Many Spanish-speaking parents have difficulty carrying out health-related literacy and numeracy tasks. The Spanish PHLAT demonstrates good psychometric characteristics and may be useful for identifying parents who would benefit from receiving low-literacy child health information.
anticipatory guidance; parenting skills; infants; health literacy
To evaluate the effect of gadoxetic acid enhancement on the detection and characterisation of focal hepatic lesions on T2 weighted and diffusion weighted (DW) images.
A total of 63 consecutive patients underwent T2 weighted and DW imaging before and after gadoxetic acid enhancement. Two blinded readers independently identified all of the focal lesions using a five-point confidence scale and characterised each lesion using a three-point scale: 1, non-solid; 2, indeterminate; and 3, solid. For both T2 weighted and DW imaging, the accuracies for detecting focal lesions were compared using the free-response receiver operating characteristic analysis; the accuracies for lesion characterisation were compared using the McNemar test between non-enhanced and gadoxetic acid-enhanced image sets. For hepatic lesions ≥1 cm, the lesion-to-liver contrast-to-noise ratio (CNR) and the apparent diffusion coefficient (ADC) were compared in the non-enhanced and enhanced image sets using the generalised estimating equations.
For both T2 weighted and DW images, the accuracies for detecting focal lesions (p≥0.52) and those for lesion characterisation (p≥0.63) did not differ significantly between the non-enhanced and enhanced image sets. The lesion-to-liver CNR was significantly higher on enhanced DW images than on non-enhanced DW images (p=0.02), although the difference was not significant for T2 weighted imaging (p=0.65). The mean ADC values of lesions did not differ significantly on enhanced and non-enhanced DW imaging (p=0.75).
The acquisition of T2 weighted and DW images after administration of gadoxetic acid has no significant effect on the detection or characterisation of focal hepatic lesions, although it improves the lesion-to-liver CNR on DW images.
Surveys reveal limited screening and counseling for alcohol misuse by primary care physicians despite evidence-based recommendations. We developed and evaluated an alcohol screening and misuse counseling tool designed to assist clinicians at the point-of-care (POC).
Mixed methods, prospective cohort study conducted in a practice-based research network with licensed clinicians. A software tool was designed to guide clinicians through evidence-based alcohol misuse assessment and interventions.
Participants (N=12) used the tool an average of 3 sessions and 71% were satisfied with the tool. Participants increased their ability to differentiate between patients who are “at risk” drinkers vs. those with alcohol use disorders including dependence/abuse (21%; t=2.4, p=.04). Thematic analysis of interviews suggest that barriers to overall use included perceptions of alcohol use; clinical need to intervene; time; and issues with use of technology generally at the POC. However, the tool added confidence and a valuable framework for interventions and was valued as an educational tool. Users felt that increased training and practice could increase comfort and impact future POC use. Increased POC usability may also be achieved through tool simplification and additional flexibility in POC use options.
A computer-assisted counseling tool for alcohol misuse and abuse can be implemented in primary care settings and shows promise for improving physician screening and interventions for alcohol misuse. To enhance utility in daily clinical practice we recommend design enhancements and strategies to enhance usage as described in this research.
Porphyromonas gingivalis is a primary etiological agent of chronic periodontal disease, an infection-driven chronic inflammatory disease that leads to the resorption of tooth-supporting alveolar bone. We previously reported that TLR2 is required for P. gingivalis–induced alveolar bone loss in vivo, and our in vitro work implicated TNF as a key downstream mediator. In this study, we show that TNF-deficient (Tnf−/−) mice are resistant to alveolar bone loss following oral infection with P. gingivalis, and thus establish a central role for TNF in experimental periodontal disease. Using bone marrow–derived macrophages (BMDM) from wild-type and gene-specific knockout mice, we demonstrate that the initial inflammatory response to P. gingivalis in naive macrophages is MyD88 dependent and requires cooperative signaling of TLR2 and TLR4. The ability of P. gingivalis to activate cells via TLR2 or TLR4 was confirmed in TLR2- or TLR4-transformed human embryonic kidney cells. Additional studies using bacterial mutants demonstrated a role for fimbriae in the modulation of TLR-mediated activation of NF-κB. Whereas both TLR2 and TLR4 contributed to TNF production in naive macrophages, P. gingivalis preferentially exploited TLR2 in endotoxin-tolerant BMDM to trigger excessive TNF production. We found that TNF induced surface TLR2 expression and augmented TLR-induced cytokine production in P. gingivalis–stimulated BMDM, establishing a previously unidentified TNF-dependent feedback loop. Adoptive transfer of TLR2-expressing macrophages to TLR2-deficient mice restored the ability of P. gingivalis to induce alveolar bone loss in vivo. Collectively, our results identify a TLR2- and TNF-dependent macrophage-specific mechanism underlying pathogen-induced inflammatory bone loss in vivo.
Aberrant activation of Wnt signalling through hypermethylation of Wnt inhibitor genes is involved in several human malignancies, including acute myeloid leukaemia (AML). It remains unclear whether hypermethylation of Wnt inhibitors is associated with molecular gene mutations in the development of AML.
We investigated the association of the promoter hypermethylation of six Wnt inhibitors (Wif-1, SFRP1, SFRR2, SFRP4, SFRP5, and DKK1) with gene aberrations in the leukaemogenesis of 269 AML patients.
In total, 166 patients (61.7%) had hypermethylation of at least one Wnt inhibitor. The majority (68.5%) of patients with Wnt inhibitor hypermethylation had concurrent Class II gene mutations that affect transcription factors or cofactors. There was a close association of Wif-1 hypermethylation with t(15;17) (P=0.0005) and CEBPA mutation (P<0.0001), DKK1 hypermethylation with t(8;21) (P<0.0001) and ASXL1 mutation (P=0.0078), SFRP-1 hypermethylation with t(8;21) (P<0.0001), SFRP-2 hypermethylation with AML1/RUNX1 mutation (P=0.0012), and SFRP-5 hypermethylation with MLL/PTD (P=0.0505). On the other side, hypermethylation of Wnt inhibitors was always negatively associated with NPM1 mutation and FLT3/ITD.
There was distinct association between hypermethylation of individual Wnt inhibitors and specific gene aberrations, especially Class II mutations. The Wnt inhibitor hypermethylation might interact with genetic alterations in the leukaemogenesis.
Wnt pathway inhibitors; methylation; genetic alteration; acute myeloid leukaemia
Bisphenol A, an estrogenic environmental toxicant, has been implicated to have hazardous effects to reproductive health in humans and rodents. However, there are conflicting reports in the literature regarding its effects to male reproductive function. In this study, it was shown that in adult rats treated with acute doses of bisphenol A, a small but statistically insignificant percentage of seminiferous tubules in the testes displayed signs of germ cell loss, consistent with some earlier reports. It also failed to disrupt the blood-testis barrier in vivo. This is possibly due to the low bioavailability of free bisphenol A in the systemic circulation. However, bisphenol A disrupted the blood-testis barrier when administered to immature 20-day-old rats, consistent with earlier reports concerning the higher susceptibility of immature rats towards bisphenol A. This observation was confirmed using primary Sertoli cells cultured in vitro with established tight junction-permeability barrier that mimicked the blood-testis barrier in vivo. The reversible disruption of Sertoli cell tight junction barrier by bisphenol A was associated with an activation of ERK, and a decline in the levels of selected proteins at the tight junction, basal ectoplasmic specialization, and gap junction at the blood-testis barrier. Studies by dual-labeled immunofluorescence analysis and biotinylation techniques also illustrated declining levels of occludin, connexin 43, and N-cadherin at the cell-cell interface following bisphenol A treatment. In summary, bisphenol A reversibly perturbs the integrity of the blood-testis barrier in Sertoli cells, which can also serve as a suitable model for studying the dynamics of the blood-testis barrier.
Testis; spermatogenesis; seminiferous epithelial cycle; tight junction; anchoring junction; Sertoli cells; bisphenol A