Interprofessional socialization of first-year medical and midwifery students: effects of an ultra-brief anatomy training.

BACKGROUND: Interprofessionalism is considered a key component in modern health profession education. Nevertheless, there remains ongoing debate about when and where to introduce interprofessional trainings in the curriculum. We identified anatomy, a subject commonly shared among health professionals, as a practical choice for initiating early intergroup-contact between first-year medical and midwifery students. Our study examined the effects of a four-hour block course in anatomy on interprofessional socialization and valuing, as well as long-term effects on intergroup contact. METHODS: Based on different concepts and theories of learning, we implemented 12 interprofessional learning stations. Several measures were taken to foster group cohesion: (1) self-directed working in interprofessional tandems on authentic obstetric tasks, (2) competing with other tandems, (3) creating positive interdependencies during task completion, and (4) allowing room for networking. In a pre-post design with a three-month follow-up, we assessed the outcomes of this ultra-brief training with qualitative essays and quantitative scales. RESULTS: After training, both groups improved in interprofessionalism scores with strong effect sizes, mean difference in ISVS-21 = 0.303 [95% CI: 0.120, 0.487], P < .001, η² = 0.171, while the scales measuring uniprofessional identity were unaffected, mean difference in MCPIS = 0.033 [95% CI: -0.236, 0.249], P = .789. A follow-up indicated that these positive short-term effects on the ISVS-21 scale diminished after 12 weeks to baseline levels, yet, positive intergroup contact was still reported. The qualitative findings revealed that, at this initial stage of their professional identity development, both medical and midwifery students considered interprofessionalism, teamwork and social competencies to be of importance for their future careers. CONCLUSION: This study advocates for an early implementation of interprofessional learning objectives in anatomical curricula. Young health profession students are receptive to interprofessional collaboration at this initial stage of their professional identity and derive strong advantages from a concise training approach. Yet, maintaining these gains over time may require ongoing support and reinforcement, such as through longitudinal curricula. We believe that an interprofessional socialization at an early stage can help break down barriers, and help to avoid conflicts that may arise during traditional monoprofessional curricula.

Evaluation of CIN2/3 Lesion Regression in GynTect® DNA Methylation-Marker-Negative Patients in a Longitudinal Study.

Cervical intraepithelial neoplasia (CIN) grade 2/3 has a high spontaneous regression rate, especially among women ≤29 years of age. To reduce overtreatment, reliable prognostic biomarkers would be helpful. The main aim of this study was to analyze the negative predictive value of the methylation marker panel GynTect® for lesion regression. In this prospective, multicenter, longitudinal observational proof-of-concept study, women aged ≤29 years with histologically confirmed CIN2 (n = 24) or CIN3 (n = 36) were closely monitored without treatment for up to 24 or 12 months, respectively. The outcome was either regression, persistence, or progression of the lesion. For each patient, a single baseline sample (V0) for cytology, hrHPV detection and methylation analysis was taken. In a primary analysis, the negative predictive value (NPV) of a GynTect®-negative test result at V0 for regression was determined. We tested the null hypothesis NPV ≤ 70% against the alternative hypothesis NPV ≥ 90%. Twelve of the eighteen GynTect®-negative CIN2 patients showed regression (NPV = 67%, 90% CI 44-85%, p = 0.53). Of the 27 GynTect®-negative CIN3 lesions, 15 regressed (NPV = 56%, 90% CI 38-72%, p = 0.92). Although the majority of GynTect®-negative lesions regressed, the postulated NPV of ≥90% was not observed. Thus, the clinical relevance for an implementation of the GynTect® assay for patients undergoing watchful waiting remains questionable. Further studies with longer observation periods should be undertaken.

Medical imaging training with eye movement modeling examples: A randomized controlled study.

PURPOSE: To determine whether ultrasound training in which the expert's eye movements are superimposed to the underlying ultrasound video (eye movement modeling examples; EMMEs) leads to better learner outcomes than traditional eye movement-free instructions. MATERIALS AND METHODS: 106 undergraduate medical students were randomized in two groups; 51 students in the EMME group watched 5-min ultrasound examination videos combined with the eye movements of an expert performing the task. The identical videos without the eye movements were shown to 55 students in the control group. Performance and behavioral parameters were compared prepost interventional using ANOVAs. Additionally, cognitive load, and prior knowledge in anatomy were surveyed. RESULTS: After training, the EMME group identified more sonoanatomical structures correctly, and completed the tasks faster than the control group. This effect was partly mediated by a reduction of extraneous cognitive load. Participants with greater prior anatomical knowledge benefited the most from the EMME training. CONCLUSION: Displaying experts' eye movements in medical imaging training appears to be an effective way to foster medical interpretation skills of undergraduate medical students. One underlying mechanism might be that practicing with eye movements reduces cognitive load and helps learners activate their prior knowledge.

Digital transfer in radiation oncology education for medical students-single-center data and systemic review of the literature.

PURPOSE: Modern medical education demands innovative, competence-orientated concepts. The forced digital transfer of teaching due to the coronavirus pandemic also affected radiation oncology (RO). The following analysis investigates whether the attractivity of RO teaching at our faculty could be maintained during the pandemic and which possibilities exist to involve students (in active learning). The latter aspect is further elaborated on a broader scale by a systemic review of the literature on competence-orientated digital education. METHODS: Evaluation results and participation rates of clinical lectures in radiation oncology (RO) were analyzed between the winter semester 2018/2019 and the summer semester 2021. A systemic review of the literature on digital education in RO for medical students was conducted. RESULTS: Concerning evaluation results, a significant improvement for the 7th and 9th semesters was observed in comparison between the pre-pandemic and pandemic semesters (p = 0.046 and p = 0.05, respectively). Overall participation rates did not differ. However, the number of students attending > 75% of classes in the respective semester increased significantly between the pre-pandemic and pandemic period (median values: 38 vs. 79%, p = 0.046; 44 vs. 73%, p = 0.05; 45 vs. 64%, p = 0.05; 41 vs. 77%, p = 0.05; 41 vs. 71%, p = 0.05, for the 6th to 10th semester, respectively). CONCLUSION: The analysis demonstrates the possibility of efficient digital transfer of a core curriculum in RO to the digital era, with a more continuous participation of students. This transfer may enable amelioration of teaching quality and the introduction of innovative and interactive concepts in accordance with the literature.

"Fun slipping into the doctor's role"-The relationship between sonoanatomy teaching and professional identity formation before and during the Covid-19 pandemic.

The various psychological dimensions of professional identity formation (PIF) are an important aspect of the study course for undergraduate medical students. Anatomical learning environments have been repeatedly shown to play a critical role in forming such an identity; however, relevance of PIF during sonoanatomical training remains underexplored. At the end of their basic anatomy studies, third-semester medical students took part in a four-day block course on anatomy and imaging. Anatomical content was revised in small groups using peer teaching and imaging methods, including one hour of hands-on sonoanatomy sessions each day. On-site sonoanatomy was identified as an excellent format to support students' transition from the pre-clinical to clinical phase as medical experts-to-be. Students enjoyed practical exercises and the clinical input, which increased their interest in the medical profession and their academic studies. This study further examined the effects of the transition into an online-only format, necessitated by the current Covid-19 pandemic. A comparison was made between the quantitative and qualitative evaluation data, and the written results of examinations of several on-site (n = 1096, mean age = 22.4 years ± 2.18), and online-only cohorts (n = 230, mean age = 22.6 years ± 2.21). The online-only transition led to a reduction of all PIF-related variables measured, losing identity-related variables, increasing students' stress levels, and reducing their long-term academic performance. Together, this study demonstrates presence of PIF in undergraduate sonoanatomy teaching, and cautions against the uncritical online-only substitution of hands-on learning environments.

Performance of a six-methylation-marker assay on self-collected cervical samples - A feasibility study.

OBJECTIVES: In high-income countries, a high proportion of cervical cancers is diagnosed in screening non-attendees. One approach to improve screening coverage is to offer self-sampling for human papillomavirus (HPV) testing. However, especially young women are often HPV positive without having a precancerous lesion in need of treatment. To improve the rather low specificity of HPV testing additional markers such as DNA-methylation can be used. The aim of this feasibility study was to examine the performance of the methylation marker assay GynTect®, comprising six methylation markers, on dry self-collected cervico-vaginal samples compared to physician-taken samples. METHODS: We recruited 89 patients from our colposcopy clinic of whom 87 qualified for the study. The women took a self-sample with the Evalyn-Brush. Afterwards the planned colposcopy was performed and smears for cytology and reference HPV testing were taken as well as a biopsy in cases of abnormalities. Physician-taken and self-collected samples were tested for HPV DNA and were analyzed with GynTect®. RESULTS: We obtained 95.5 % valid results for the self-collected samples which was very close to the physician-taken samples. Only about half of the self-collected samples were GynTect® positive in comparison to the physician-taken samples. GynTect® scores were significantly lower for self-collected than for physician-taken samples (p = 0.001, paired t-test). The overall concordance for GynTect® results was moderate (kappa 0.394; p < 0.001). For HPV testing we obtained a good concordance (kappa 0.586; p < 0.001). The GynTect® results for the self-collected samples showed a sensitivity for the detection of cervical intraepithelial neoplasia 2 or worse (CIN2+) of 26.1 % (95 %-CI: 0.13-0.46) and a specificity of 95.6 % (95 %-CI: 0.85-0.99), in comparison to a sensitivity of 45.5 % (95 %-CI: 0.27-0.65) and a specificity of 78.3 % (95 %-CI: 0.64-0.88) for the physician-taken samples. CONCLUSIONS: GynTect® methylation marker testing has a satisfactory amount of valid results on self-collected samples. However, the results of the self-collected samples differed clearly in comparison to the reference samples. To justify an application in screening, a larger study with more cases of high-grade cervical dysplasia and HPV positive patients will be needed.

Triage of hrHPV-positive women: comparison of two commercial methylation-specific PCR assays.

AIM: High-risk human papillomavirus (hrHPV)-based screening is becoming increasingly important, either by supplementing or replacing the traditional cytology-based cervical Pap smear. However, hrHPV screening lacks specificity, because it cannot differentiate between transient virus infection and clinically relevant hrHPV-induced disease. Therefore, reliable triage methods are needed for the identification of HPV-positive women with cervical intraepithelial neoplasia (CIN) in need of treatment. Promising tools discussed for the triage of these patients are molecular diagnostic tests based on epigenetic markers. Here, we compare the performance of two commercially available DNA methylation-based diagnostic assays-GynTect® and the QIAsure Methylation Test-in physician-taken cervical scrapes from 195 subjects. FINDINGS: Both GynTect® and the QIAsure Methylation Test detected all cervical carcinoma and carcinoma in situ (CIS). The differences observed in the detection rates between both assays for the different grades of cervical lesions (QIAsure Methylation Test: CIN1 26.7%, CIN2 27.8% and CIN3 74.3%; GynTect®: CIN1 13.3%, CIN2 33.3% and CIN3 60%) were not significant. Concerning the false-positive rates, significant differences were evident. For the healthy (NILM) hrHPV-positive group, the false-positive rates were 5.7% for GynTect® and 26.4% for QIAsure Methylation Test (p = 0.003) and for the NILM hrHPV-negative group 2.2% vs. 23.9% (p = 0.006), respectively. When considering hrHPV-positive samples only for comparison (n = 149), GynTect® delivered significantly higher specificity compared to the QIAsure Methylation Test for CIN2 + (87.6% vs. 67.4% (p < 0.001)) and CIN3 + (84.1% vs. 68.2% (p = 0.002)). Overall our findings suggest that DNA methylation-based tests are suitable for the triage of hrHPV-positive women. With the goal to provide a triage test that complements the limited specificity of HPV testing in HPV-based screening, GynTect® may be preferable, due to its higher specificity for CIN2+ or CIN3+ .

Combined assessment of 3q26 amplification and promoter methylation in patients with high grade cervical lesions show age specific differences.

A considerable proportion of high grade cervical intraepithelial lesions (CIN2/3) are known to resolve on their own especially among young women. However, since reliable prognostic markers are still lacking, the diagnosis "CIN3" is still an indication for surgery which may result in overtreatment. It is conceivable that a combination of different, ideally independent molecular markers may provide more reliable results. In the present cross-sectional study two established triage markers, 3q26 amplification and a methylation signature, were evaluated in an age-dependent manner. The patient cohort comprised 60 patients with histologically confirmed CIN2/3 in two equally sized age groups (<30 years, ≥30 years). Cervical scrapes were analyzed by interphase fluorescence in situ hybridization for 3q26 amplification and methylation specific PCR (GynTect®) for six different genome regions. Both assays showed a significantly different pattern of test outcome independent of age (P = .001). Moreover, the combination of both assays differed significantly for double positive and double negative cases when comparing the two age groups: In patients <30 years there were clearly less cases with positive methylation signature and amplification of 3q26 as in women ≥30 years (23% vs 63%, Bonferroni adjusted P = .016). Of particular interest is the finding that double negative results were exclusive for the young age group (0% vs 27%, Bonferroni adjusted P = .020). Since regression of CIN2/3 characteristically occurs among young women it is tempting to speculate that a double negative test result could be prognostic for regression of CIN2/3. This will have to be investigated further in a prospective longitudinal intervention study.

Differences in Stability of Viral and Viral-Cellular Fusion Transcripts in HPV-Induced Cervical Cancers.

HPV-DNA integration results in dysregulation of viral oncogene expression. Because viral-cellular fusion transcripts inherently lack the viral AU-rich elements of the 3'UTR, they are considered to be more stable than episome-derived transcripts. The aim of this study is to provide formal proof for this assumption by comparing the stability of viral early transcripts derived from episomal and integrated HPV16 DNA, respectively. Full-length cDNA of three fusion transcripts comprising viral and cellular sequences in sense orientation were amplified and cloned into the adeno-viral-vector pAd/CMV/V5-DEST. The most abundant HPV16 oncogene transcript E6*I-E7-E1vE4-E5 with and without 3'UTR, served as reference and control, respectively. Human primary keratinocytes were transduced using high titer virus stocks. qRT-PCR was performed to determine mRNA stability in relation to GAPDH in the presence of actinomycin-D. In four independent transduction experiments, all three viral-cellular fusion transcripts were significantly more stable compared to the episome-derived reference. Among the three viral-cellular fusion transcripts the most stable transcript was devoid of the instability core motif "AUUUA". Unexpectedly, there was no significant difference in the stability between the episome-derived transcripts either with or without 3'UTR, indicating that the AU-rich elements of the 3'UTR are not contributing to RNA stability. Instead, the three "AUUUA" motifs located in the untranslated region between the viral E4 and E5 genes may be responsible for the instability. This is the first report showing that authentic viral-cellular fusion transcripts are more stable than episome-derived transcripts. The longer half-life of the fusion transcripts may result in increased levels of viral oncoproteins and thereby drive the carcinogenic process.

Successful integration of radiation oncology in preclinical medical education : Experiences with an interdisciplinary training project.

PURPOSE: Modern impartation of both anatomic and radiation oncology (RO) knowledge in medical education enables a transfer of preclinical knowledge to clinical practice, which may be addressed by multidisciplinary concepts. The faculty's "Anatomy and imaging" course attempts to integrate RO, radiology and nuclear medicine into the preclinical curriculum. The present analysis focuses on the description of the course concept and discusses the potential didactic impact of the implementation of RO. METHODS: In total 5 semester cohorts have undertaken the course since the introduction of RO in the winter semester of 2015/2016 with 682 students participating. It is designed as a small group circuit training with a teaching content of 8 h daily. Course evaluation was performed on a 100-item Likert scale. RESULTS: General evaluation showed an average of 9.3-12.7 on a Likert scale (0 being the best, 100 being the worst grade). Use of media, relevance for medical training, gain of interest in medicine in general and overall satisfaction with the course received excellent mean values. For RO, there was a high degree of consent with the following statements: "the course was well organized", "subjects and presentation were well-structured", "topics were well chosen", "the time for exercises was sufficient" and "teaching by student tutors and physicians was adequate". CONCLUSION: The present evaluation demonstrates the feasibility of introducing RO in the preclinical part of medical education. The course concept shows excellent results in evaluation and may help in broadening RO knowledge and in recruiting new doctoral candidates and residents.

Performance of a DNA methylation marker panel using liquid-based cervical scrapes to detect cervical cancer and its precancerous stages.

BACKGROUND: A change of cervical cancer screening algorithms to an HPV-based screening setting is discussed in many countries, due to higher sensitivity of HPV testing compared to cytology. Reliable triage methods are, however, an essential prerequisite in such a setting to avoid overtreatment and higher screening costs. RESULTS: In this study, a series of cervical scrapes collected in PreservCyt liquid-based cytology (LBC) medium from women with cervical cancer (n = 5), cervical intraepithelial neoplasia grade 1-3 (n = 74), and normal cytology (n = 201; further n = 352 collected in SureThin®) were assessed for methylation of the marker regions ASTN1, DLX1, ITGA4, RXFP3, SOX17, and ZNF671 using the GynTect assay and compared to cobas® HPV and CINtec Plus® biomarker results. All samples from women with cervical cancer, 61.2% of CIN3, 44.4% of CIN2 and 20.0% of CIN1 cases were scored positive for the GynTect methylation assay. In contrast, all CIN, irrespective of severity grade, and carcinomas were positive by both, CINtec Plus and cobas HPV. The specificity of GynTect for CIN3+ was 94.6% compared to 69.9% for CINtec Plus and 82.6% for cobas HPV (all HPV types) and 90.6% for cobas HPV 16/18. DNA methylation analysis of this methylation marker panel (GynTect assay) in cervical scrapes consistently detects cervical cancer and the majority of CIN3 as well as a subset of CIN1/2 lesions. The detection rate among cytologically normal samples is extraordinarily low (1.5%). CONCLUSION: GynTect shows excellent performance when using cervical scrape material collected in liquid-based cytology media, a prerequisite for employing such a test as a triage in screening programs. Compared to the other test systems used in this work, GynTect showed higher specificity while still detecting all cancer cases.

Performance of a methylation specific real-time PCR assay as a triage test for HPV-positive women.

BACKGROUND: HPV DNA testing as a primary screening marker is being implemented in several countries. Due to the high HPV prevalence in the screening population, effective triage strategies for HPV-positive cases are required. The aim of this study was to evaluate the performance of a methylation-specific real-time PCR  assay (GynTect®) comprising six marker regions as a triage test. RESULTS: An analytical sensitivity of 0.1 ng genomic DNA corresponding to 15 SiHa cells was achieved. Absolute specificity was observed in the presence of 20 ng unmethylated genomic DNA. In a clinical setting, cervical scrapes of 306 women showing abnormal colposcopy were tested for cytology, HPV positivity, and the GynTect markers ASTN1, DLX1, ITGA4, RXFP3, SOX17, and ZNF671. Of all women, histopathological data were available. The overall sensitivity for GynTect to detect CIN3+ was 67.7% (95% CI 57.3%-77.1%) whereas sensitivity was significantly higher for women of age ≥ 30 years (p = 0.04). All cancer cases (n = 5) were detected by GynTect. The overall false positive rate (= 1-specificity) for women with no CIN was 17.4% (95% CI 12.5-23.1%), with a higher proportion among HPV-positive women (24.0%, 95% CI 16.0-33.6%). In a triage screening setting, where all women underwent HPV testing and the HPV positives in addition GynTect testing, the overall sensitivity would slightly decline but specificity would reach the maximum value of 88.7% (95% CI 83.7-92.6%). CONCLUSION: The GynTect® assay is a robust easy to use assay with high analytical sensitivity and specificity. Moreover, the performance of the assay based on cervical scrapes provides further evidence for the usefulness of methylation markers to detect HPV-positive women with clinically relevant disease.

Hepatic Overexpression of Soluble Urokinase Receptor (uPAR) Suppresses Diet-Induced Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient (LDLR-/-) Mice.

OBJECTIVE: Atherosclerosis, a chronic inflammatory disease, arises from metabolic disorders and is driven by inappropriate recruitment and proliferation of monocytes / macrophages and vascular smooth-muscle-cells. The receptor for the urokinase-type plasminogen activator (uPAR, Plaur) regulates the proteolytic activation of plasminogen. It is also a coactivator of integrins and facilitates leukocyte-endothelial interactions and vascular smooth-muscle-cell migration. The role of uPAR in atherogenesis remains elusive. METHODS AND RESULTS: We generated C57Bl6/J low-density lipoprotein receptor (LDL) and uPAR double knockout (uPAR-/-/LDLR-/-) mice to test the role of uPAR in two distinct atherosclerosis models. In LDLR-/- mice, hepatic overexpression following hydrodynamic transfection of soluble uPAR that competes with endogenous membrane-bound uPAR was performed as an interventional strategy. Aortic root atherosclerotic lesions induced by feeding a high-fat diet were smaller and comprised less macrophages and vascular smooth-muscle-cells in double knockout mice and animals overexpressing soluble uPAR when compared to controls. In contrast, lesion size, lipid-, macrophage-, and vascular smooth muscle cell content of guide-wire-induced intima lesions in the carotid artery were not affected by uPAR deficiency. Adhesion of uPAR-/--macrophages to TNFα-stimulated endothelial cells was decreased in vitro accompanied by reduced VCAM-1 expression on primary endothelial cells. Hepatic overexpression of soluble full-length murine uPAR in LDLR-/- mice led to a reduction of diet-induced atherosclerotic lesion formation and monocyte recruitment into plaques. Ex vivo incubation with soluble uPAR protein also inhibited adhesion of macrophages to TNFα-stimulated endothelial cells in vitro. CONCLUSION: uPAR-deficiency as well as competitive soluble uPAR reduced diet-promoted but not guide-wire induced atherosclerotic lesions in mice by preventing monocyte recruitment and vascular smooth-muscle-cell infiltration. Soluble uPAR may represent a therapeutic tool for the modulation of hyperlipidemia-associated atherosclerotic lesion formation.

Transcriptionally active regions are the preferred targets for chromosomal HPV integration in cervical carcinogenesis.

Integration of human papillomavirus (HPV) into the host genome is regarded as a determining event in cervical carcinogenesis. However, the exact mechanism for integration, and the role of integration in stimulating cancer progression, is not fully characterized. Although integration sites are reported to appear randomly distributed over all chromosomes, fragile sites, translocation break points and transcriptionally active regions have all been suggested as being preferred sites for integration. In addition, more recent studies have reported integration events occurring within or surrounding essential cancer-related genes, raising the question whether these may reflect key events in the molecular genesis of HPV induced carcinomas. In a search for possible common denominators of the integration sites, we utilized the chromosomal coordinates of 121 viral-cellular fusion transcripts, and examined for statistical overrepresentation of integration sites with various features of ENCODE chromatin information data, using the Genomic HyperBrowser. We find that integration sites coincide with DNA that is transcriptionally active in mucosal epithelium, as judged by the relationship of integration sites to DNase hypersensitivity and H3K4me3 methylation data. Finding an association between integration and transcription is highly informative with regard to the spatio-temporal characteristics of the integration process. These results suggest that integration is an early event in carcinogenesis, more than a late product of chromosomal instability. If the viral integrations were more likely to occur in destabilized regions of the DNA, a completely random distribution of the integration sites would be expected. As a by-product of integration in actively transcribing DNA, a tendency of integration in or close to genes is likely to be observed. This increases the possibility of viral signals to modulate the expression of these genes, potentially contributing to the progression towards cancer.

A promising DNA methylation signature for the triage of high-risk human papillomavirus DNA-positive women.

High-risk human papillomavirus (hrHPV)-DNA testing is frequently performed parallel to cytology for the detection of high-grade dysplasia and cervical cancer particularly in women above 30 years of age. Although highly sensitive, hrHPV testing cannot distinguish between HPV-positive women with or without clinically relevant lesions. However, in principle discrimination is possible on the basis of DNA methylation markers. In order to identify novel DNA regions which allow an effective triage of hrHPV-positive cases, hypermethylated DNA enriched from cervical cancers was compared with that from cervical scrapes of HPV16-positive cases with no evidence for disease by CpG island microarray hybridization. The most promising marker regions were validated by quantitative methylation-specific PCR (qMSP) using DNA from archived cervical tissues and cervical scrapes. The performance of these markers was then determined in an independent set of 217 hrHPV-positive cervical scrapes from outpatients with histopathological verification. A methylation signature comprising the 5' regions of the genes DLX1, ITGA4, RXFP3, SOX17 and ZNF671 specific for CIN3 and cervical cancer (termed CIN3+) was identified and validated. A high detection rate of CIN3+ was obtained if at least 2 of the 5 markers were methylated. In the subsequent cross-sectional study all cervical carcinomas (n = 19) and 56% (13/23) of CIN3 were identified by this algorithm. Only 10% (11/105) of hrHPV-positive women without histological evidence of cervical disease were scored positive by the methylation assay. Of note is that the detection rate of CIN3 differed between age groups. Eight of nine CIN3 were detected among women ≥30 years of age but only five of fourteen among <30 year old group (p = 0.03). The specificity for CIN3+ in the older age group was 76.6% (95% CI 65.6-85.5%). Clinical validation studies are required to determine the usefulness of these novel markers for triage after primary hrHPV testing in a cervical cancer screening setting.

Thrombomodulin's lectin-like domain reduces myocardial damage by interfering with HMGB1-mediated TLR2 signalling.

AIMS: Thrombomodulin (TM), via its lectin-like domain (LLD), exhibits anti-inflammatory properties partly by sequestering the pro-inflammatory cytokine, high-mobility group box 1 (HMGB1). Since myocardial damage after ischaemia and reperfusion is mediated by inflammation, we evaluated the cardioprotective effects of the LLD of TM. Using an in vivo mouse model of transient ischaemia and in vitro models of cardiomyocyte hypoxia, we assessed the ability of the LLD to suppress HMGB1-mediated activation of the receptors, receptor for advanced glycation endproducts (RAGEs) and Toll-like receptors (TLRs) 2 and 4. METHODS AND RESULTS: Thirty-minute myocardial ischaemia was induced in isoflurane-anaesthetized mice followed by 24 h of reperfusion in wild-type (WT) mice, in mice lacking the LLD of TM (TM(LeD/LeD) mice), and in WT with systemic overexpression of the LLD of TM induced by hydrodynamic transfection. Infarct size, HMGB1 protein, and apoptotic cells were significantly increased in TM(LeD/LeD) mice when compared with WT. Neonatal rat cardiomyocytes transfected with TLR2-, TLR4-, and RAGE-siRNA were exposed to hypoxia (0.8% O2) and reoxygenation (21% O2). HMGB1 augmented hypoxia-induced apoptosis in TLR2- but not in RAGE- or TLR4-suppressed cells. Administration of HMGB1- and TLR2-blocking antibodies in TM(LeD/LeD) mice prior to myocardial ischaemia diminished apoptosis. Therapeutic systemic gene therapy using the LLD reduced the infarct size and HMGB1 protein levels 24 h after reperfusion. CONCLUSION: The LLD of TM suppresses HMGB1-induced and TLR2-mediated myocardial reperfusion injury and apoptosis in vitro and in vivo.

Multiplex Identification of Human Papillomavirus 16 DNA Integration Sites in Cervical Carcinomas.

Cervical cancer is caused by high-risk human papillomaviruses (HPV), in more than half of the worldwide cases by HPV16. Viral DNA integration into the host genome is a frequent mutation in cervical carcinogenesis. Because integration occurs into different genomic locations, it creates unique viral-cellular DNA junctions in every single case. This singularity complicates the precise identification of HPV integration sites enormously. We report here the development of a novel multiplex strategy for sequence determination of HPV16 DNA integration sites. It includes DNA fragmentation and adapter tagging, PCR enrichment of the HPV16 early region, Illumina next-generation sequencing, data processing, and validation of candidate integration sites by junction-PCR. This strategy was performed with 51 cervical cancer samples (47 primary tumors and 4 cell lines). Altogether 75 HPV16 integration sites (3'-junctions) were identified and assigned to the individual samples. By comparing the DNA junctions with the presence of viral oncogene fusion transcripts, 44 tumors could be classified into four groups: Tumors with one transcriptionally active HPV16 integrate (n = 12), tumors with transcribed and silent DNA junctions (n = 8), tumors carrying episomal HPV16 DNA (n = 10), and tumors with one to six DNA junctions, but without fusion transcripts (n = 14). The 3'-breakpoints of integrated HPV16 DNA show a statistically significant (p<0.05) preferential distribution within the early region segment upstream of the major splice acceptor underscoring the importance of deregulated viral oncogene expression for carcinogenesis. Half of the mapped HPV16 integration sites target cellular genes pointing to a direct influence of HPV integration on host genes (insertional mutagenesis). In summary, the multiplex strategy for HPV16 integration site determination worked very efficiently. It will open new avenues for comprehensive mapping of HPV integration sites and for the possible use of HPV integration sites as individualized biomarkers after cancer treatment of patients for the early diagnosis of residual and recurrent disease.

The receptor for activated complement factor 5 (C5aR) conveys myocardial ischemic damage by mediating neutrophil transmigration.

Tissue loss after myocardial ischemia with reperfusion (MI/R) is in part conveyed by neutrophil recruitment to post-ischemic myocardium. Strategies to prevent reperfusion injury would help to limit myocardial damage. The receptor for activated complement factor 5 (C5aR) plays a prominent role in inflammation. We examine the effects of C5aR-deficiency on reperfusion injury after MI/R. C5aR(-/-)-mice and their C57BL/6- (WT) littermates underwent transient myocardial ischemia followed by different time points of reperfusion. Infarct size and leukocyte infiltration were determined. Expression of C5aR, inflammatory cytokines and adhesion molecules were analyzed by real-time RT-PCR. Leukocyte-endothelial interactions were assessed by low-shear adhesion- and transmigration-assays in vitro. Myocardial C5aR mRNA expression was 2.8-fold increased by ischemia. Infarct size per area-at-risk and leukocyte recruitment into infarctions were reduced in C5aR(-/-)-compared to WT-mice as well as in WT mice treated with the C5aR-antagonist JPE1375. IL-6, IL-1ß, ICAM-1 and VCAM-1 expression were not different, while TNFα expression was reduced in C5aR(-/-)-mice after MI/R. In vitro, C5aR on leukocytes is required for effective transendothelial migration but not adhesion. Expression of MMP9 and JAM-A, molecules that are involved in leukocyte transmigration, were reduced in C5aR(-/-) mice in vivo. Genetic C5aR deficiency blunts the inflammatory response in murine MI/R resulting in reduced inflammatory cell recruitment, which is due to a C5aR-dependent effect on leukocyte transmigration across inflamed endothelium into the ischemic myocardium. This effect could be related to MMP9- and JAM-A expression in response to ischemia and reperfusion.

In vivo fluorescence-mediated tomography imaging demonstrates atorvastatin-mediated reduction of lesion macrophages in ApoE-/- mice.

BACKGROUND: Macrophage recruitment into atherosclerotic plaques drives lesion progression, destabilization, and rupture. Chronic statin treatment reduces macrophage plaque content. Information on dynamics of macrophage recruitment would help assessing plaque vulnerability and guiding therapy. Techniques to image macrophage homing to vulnerable plaques in vivo are scarcely available. The authors tested if noninvasive fluorescence-mediated tomography (FMT) can assess plaque-stabilizing effects of short-term high-dosage atorvastatin. METHODS: Macrophages from green-fluorescent-protein-transgenic mice were labeled with a near-infrared fluorescent dye and were injected IV in apolipoprotein E-deficient mice (n=9) on Western diet 7 days after guidewire-injury of the carotid artery. FMT-scans, 2 and 7 days thereafter, quantified macrophage recruitment into carotid artery plaques. Atorvastatin was tested for macrophage adhesion, proliferation, and viability (n=5 to 6) in vitro. Fourteen mice received atorvastatin or vehicle for 4 days after 16 weeks on Western diet. FMT assessed macrophage recruitment into aortic and innominate artery lesions. Means (±SD)% are reported. RESULTS: Double-labeled macrophages were recruited into carotid artery lesions. FMT resolved fluorescence projecting on the injured carotid artery and detected a signal increase to 300% (±191) after guidewire injury. Atorvastatin reduced macrophage adhesion to activated endothelial cells by 36% (±19). In a clinically relevant proof-of-concept intervention, FMT-imaging detected that 4 days atorvastatin treatment reduced macrophage recruitment by 57% (±8) indicating plaque stabilization. Immunohistochemistry confirmed reduced macrophage infiltration. CONCLUSIONS: FMT optical imaging proved its high potential for clinical applicability for tracking recruitment of near-infrared fluorescent-labeled macrophages to vulnerable plaques in vivo. FMT-based quantification of macrophage recruitment demonstrated rapid plaque stabilization by 4-day atorvastatin treatment in apolipoprotein E-deficient mice.

Non-random integration of the HPV genome in cervical cancer.

HPV DNA integration into the host genome is a characteristic but not an exclusive step during cervical carcinogenesis. It is still a matter of debate whether viral integration contributes to the transformation process beyond ensuring the constitutive expression of the viral oncogenes. There is mounting evidence for a non-random distribution of integration loci and the direct involvement of cellular cancer-related genes. In this study we addressed this topic by extending the existing data set by an additional 47 HPV16 and HPV18 positive cervical carcinoma. We provide supportive evidence for previously defined integration hotspots and have revealed another cluster of integration sites within the cytogenetic band 3q28. Moreover, in the vicinity of these hotspots numerous microRNAs (miRNAs) are located and may be influenced by the integrated HPV DNA. By compiling our data and published reports 9 genes could be identified which were affected by HPV integration at least twice in independent tumors. In some tumors the viral-cellular fusion transcripts were even identical with respect to the viral donor and cellular acceptor sites used. However, the exact integration sites are likely to differ since none of the integration sites analysed thus far have shown more than a few nucleotides of homology between viral and host sequences. Therefore, DNA recombination involving large stretches of homology at the integration site can be ruled out. It is however intriguing that by sequence alignment several regions of the HPV16 genome were found to have highly homologous stretches of up to 50 nucleotides to the aforementioned genes and the integration hotspots. One common region of homologies with cellular sequences is between the viral gene E5 and L2 (nucleotides positions 4100 to 4240). We speculate that this and other regions of homology are involved in the integration process. Our observations suggest that targeted disruption, possibly also of critical cellular genes, by HPV integration remains an issue to be fully resolved.