Magnetically localized and wash-free fluorescent immuno-assay: From a research platform (MLFIA) to a multiplexed POC system (MagIA).

Sexually transmitted infections (STI) remain one of the world's public health priorities: Nearly 400 million people are infected not only in emerging, but also in western countries. HIV, HBV and HCV share common infection pathways; thus these 3 diseases are recommended to be tested at the same time. However, this combined approach is currently mainly available in laboratories, and seldomly at the Point-of-care (POC). Consequently, there is a need for a STI screening POC platform with laboratory-like performance. Such a platform should be autonomous and portable and enable multiplexed screening from capillary blood. The previously developed and introduced MLFIA (Magnetically Localized and wash-free Fluorescent Immuno-Assay) technology has the potential to address these needs, as the MLFIA 18-chamber microfluidic cartridge and the MLFIA Analyzer were previously characterized and evaluated with plasma and serum from patients infected with HIV, Hepatitis B (Hep B) or C (Hep C). Here, we present the efforts to transfer this research platform (MLFIA) to a fully integrated multi-analysis solution (MagIA). First, we present the design changes of the consumable enabling to perform multiple assays in parallel, a fast filling of the cartridge with patient samples, and a homogeneous reagent/sample incubation. Second, we describe the development a piezoelectric actuator integrated into the Analyzer: this mixing module allows for an automated, fully integrated and portable workflow, with homogeneous in-situ mixing capabilities. The obtained MagIA platform was further characterized and validated for immunoassays (LOD, cartridge stability over time), using various biological models including OVA and IgG. We discuss the performances of the MLFIA and MagIA platforms for the detection of HIV / Hep B / Hep C using results from 102 patient plasma samples. Lastly, we assessed the compatibility of the MagIA platform with veinous and capillary blood samples as a final step towards its POC validation.

Magnetically localized and wash-free fluorescence immunoassay (MLFIA): proof of concept and clinical applications.

Immunoassays are used for many applications in various markets, from clinical diagnostics to the food industry, generally relying on gold-standard ELISAs that are sensitive, robust, and cheap but also time-consuming and labour intensive. As an alternative, we propose here the magnetically localized and wash-free fluorescence immunoassay (MLFIA): a no-wash assay to directly measure a biomolecule concentration, without mixing nor washing steps. To do so, a fluorescence no-wash measurement is performed to generate a detectable signal. It consists of a differential measurement between the fluorescence of fluorophores bound to magnetic nanoparticles specifically captured by micro-magnets against the residual background fluorescence of unbound fluorophores. Targeted biomolecules (antibodies or antigens) are locally concentrated on micro-magnet lines, with the number of captured biomolecules quantitatively measured without any washing step. The performance of the MLFIA platform is assessed and its use is demonstrated with several biological models as well as clinical blood samples for HIV, HCV and HBV detection, with benchmarking to standard analyzers of healthcare laboratories. Thus, we demonstrated for the first time the versatility of the innovative MLFIA platform. We highlighted promising performances with the successful quantitative detection of various targets (antigens and antibodies), in different biological samples (serum and plasma), for different clinical tests (HCV, HBV, HIV).

Knowledge and skills of emergency physicians in managing traumatic dental injuries.

PURPOSE: Emergency departments are frequently confronted with traumatic dental injuries (TDIs). The prognosis of the injured tooth is related to early dental trauma management. For this reason, physicians must be familiar with the appropriate management of TDI. This study aimed to investigate the knowledge and skills of German emergency physicians regarding TDI. METHODS: An electronic questionnaire was sent to 438 emergency departments throughout Germany. Four hundred and twenty seven questionnaires were evaluated and included in the analysis. The survey contained questions about physician characteristics and assessed their knowledge and skills of managing dental trauma. For statistical analysis, the Kruskal-Wallis, Mann-Whitney U test or ANOVA test was used as appropriate. Rank correlations were performed with the Spearman's rank correlation. RESULTS: Out of 427 participants, 256 (59.95%) stated they had no or insufficient knowledge, and 266 (71.12%) stated they had no skills in dental trauma management. Almost 76% of the participants had no previous knowledge of dentistry. Only 7.28% knew the right procedure for replanting an avulsed tooth. Just 26.06% would choose the right medium for temporary tooth storage. Having a dentist in the family (p = 0.0074) or clinical exposure to patients with dental trauma (p = 0.0384) influenced the results of the knowledge score. CONCLUSION: The knowledge and skills in dental trauma management among German emergency physicians are generally inadequate. Targeted training courses are necessary to ensure early and adequate TDI treatment to reduce the resulting medical and societal costs as much as possible.

Novel modified endoscopic mucosal resection of large GI lesions (> 20 mm) using an external additional working channel (AWC) may improve R0 resection rate: initial clinical experience.

BACKGROUND: En-bloc resection of large, flat dysplastic mucosal lesions of the luminal GI tract can be challenging. In order to improve the efficacy of resection for lesions ≥2 cm and to optimize R0 resection rates of lesions suspected of harboring high-grade dysplasia or early adenocarcinoma, a novel grasp and snare EMR technique utilizing a novel over the scope additional accessory channel, termed EMR Plus (EMR+), was developed. The aim of this pilot study is to describe the early safety and efficacy data from the first in human clinical cases. METHODS: A novel external over-the-scope additional working channel (AWC) (Ovesco, Tuebingen, Germany) was utilized for the EMR+ procedure, allowing a second endoscopic device to be used through the AWC while using otherwise standard endoscopic equipment. The EMR+ technique allows tissue retraction and a degree of triangulation during endoscopic resection. We performed EMR+ procedure in 6 patients between 02/2018-12/2018 for lesions in the upper and lower GI tract. RESULTS: The EMR+ technique utilizing the AWC was performed successfully in 6 resection procedures of the upper and/or lower GI tract in 6 patients in 2 endoscopy centers. All resections were performed successfully with the EMR+ technique, all achieving an R0 resection. No severe adverse events occurred in any of the procedures. CONCLUSIONS: The EMR+ technique, utilizing an additional working channel, had an acceptable safety and efficacy profile in this preliminary study demonstrating it's first use in humans. This technique may allow an additional option to providers to remove complex, large mucosal-based lesions in the GI tract using standard endoscopic equipment and a novel AWC device.

Shallow whole genome sequencing of adenoid cystic carcinomas of the salivary glands identifies specific chromosomal aberrations related to tumor progression.

BACKGROUND AND PURPOSE: Adenoid cystic carcinomas (ACC) are characterized by high rate of local recurrence and late distant metastasis. Chromosomal changes in the evolution from primary tumors to metastatic disease of ACC have not been appointed. Here we investigated the chromosomal alterations of 53 primary tumors from ACC patients with different progressive states by shallow whole genome sequencing to identify potential new markers for metastatic spread. METHODS: Illumina paired-end libraries were generated using DNA from the primary tumor of 53 ACC patients. Fragmented DNA was end-repaired, A-tailed and multiplex sequencing adapters were ligated. Sequence data were mapped to HG19 and a copy-number analysis was conducted using the QDNAseq R package (version 1.10.0). Outliers were removed and data was smoothed by applying the circular binary segmentation algorithm implemented in the R package copynumber version 1.22.0. A modified chromosomal instability (CNI) score was used to analyze deletions and amplifications. RESULTS: Cluster analysis of the whole genome sequencing revealed that the frequency of chromosomal aberrations were increased in ACC with local recurrence and distant metastases in comparison to ACC patients with no metastatic spread. Specifically, chromosome 6 and 12 and exclusively the entire chromosome 4 showed an increased frequency of chromosomal alterations with tumor progression. CONCLUSION: Our data show a molecular evolution from primary tumors to local recurrences and distant metastases and pinpoint the critical chromosomal regions involved in this process. These regions should be in the focus of the search for therapeutic targets of progressive ACC.

Influence of tumour necrosis factor alpha on epithelial-mesenchymal transition of oral cancer cells in co-culture with mesenchymal stromal cells.

Tumour progression in head and neck squamous cell carcinoma (HNSCC) is influenced by the surrounding stroma and inflammatory cytokines such as tumour necrosis factor alpha (TNF-α). The aim of this study was to test the hypothesis that TNF-α modulates the interactions of HNSCC cell line PCI-13 and bone marrow mesenchymal stromal cells (BMSCs) and influences markers of epithelial-mesenchymal transition (EMT). Following induction with TNF-α, mono- and co-cultures of BMSCs and the established HNSCC cell line PCI-13 were analyzed; protein expression of E-cadherin and vimentin and qRT-PCR expression of Snail, Twist, MMP14, vimentin, E-cadherin, and ß-catenin were examined, and changes in cellular AKT signalling were analyzed. TNF-α induced a significant decrease in E-cadherin (64.5±6.0%, P=0.002) and vimentin (10.4±3.5%, P=0.04) protein expression in co-cultured PCI-13, while qRT-PCR showed a significant increase in ß-catenin (BMSCs P<0.0001; PCI-13 P=0.0005) and Snail (BMSCs P=0.009; PCI-13 P=0.01). TNF-α also resulted in a down-regulation of AKT downstream targets S6 (38.7±20.9%, P=0.01), p70S6 (16.7±12%, P=0.05), RSK1 (23.6±28.8%, P=0.02), and mTOR (27.4±17.5%, P=0.004) in BMSC co-cultures. In summary, while reducing the expression of vimentin and AKT-signalling in PCI-13 and BMSC, respectively, TNF-α introduced an inflammatory-driven tumour-stroma transition, marked by an increased expression of markers of EMT.

Multiple, Multiloculated, and Recurrent Keratocysts of the Mandible and Maxilla in Association with Gorlin-Goltz (Nevoid Basal-Cell Carcinoma) Syndrome: A Pediatric Case Report and Follow-up over 5 Years.

BACKGROUND: We report a case of multiple keratocysts first diagnosed in an 8-year-old boy. CASE REPORT: The incidental radiographic finding of a cystic lesion in an 8-year-old boy led to the surgical enucleation and further diagnosis of a keratocyst associated with a tooth crown. In the course of dental maturation from deciduous to permanent teeth, the boy presented new lesions, always associated with the crowns of teeth. Gorlin-Goltz (nevoid basal-cell carcinoma) syndrome was suspected, and the genetic analysis detected a previously undescribed germline variant in the PTCH1 gene. TREATMENT: This included a surgical removal of the cystic lesions, as well as the affected teeth. FOLLOW-UP: Due to the high recurrence rate of the keratocysts, frequent radiological checks were performed over a 5-year period.

Do we need retarded delivery of bone growth factors in facial bone repair? An experimental study in rats.

The aim of the present study was to evaluate the effect of different dosages of retarded vs. rapid release of bone morphogenic protein 2 (BMP2) at different recipient sites. Porous composite poly(D,L-lactic acid) (PDLLA)/CaCO3 scaffolds were loaded with three different dosages of rhBMP2 (24 µg, 48 µg and 96 µg) and implanted, together with blank controls, both into non-healing defects of the mandibles and into the gluteal muscles of 24 adult male Wistar rats. After 26 weeks, bone formation and expression of bone specific markers [alkaline phosphatase (AP) and Runx2] were evaluated by histomorphometry and immunohistochemistry. Results showed that the mode of delivery had no quantitative effect on bone formation in mandibular sites. Expression of AP and Runx2 showed significant differences among the three dosage groups. There were significant correlations between the expression of both AP and Runx2 as well as the extent of bone formation, with both retarded and rapid release of rhBMP2. In ectopic sites, retarded release significantly enhanced bone formation in the low and medium dosage groups, compared to rapid release. Expression of AP was significantly higher and Runx2 significantly lower in ectopic sites, compared to mandibular sites. Significant correlations between the expression of bone specific markers and bone formation occurred only in the retarded delivery groups, but not in the rapid release groups. Within the limitations of the experimental model, it was concluded that retarded delivery of BMP2 was effective, preferably in sites with low or non-existing pristine osteogenic activity. Expression of bone specific markers indicated that osteogenic pathways might be different in mandibular vs. ectopic sites.

Presentation of a variation of the chorioallantoic membrane set up as a potential model for individual therapy for squamous cell carcinoma of the oropharynx.

The chorioallantoic membrane of fertilized chicken eggs in an early phase of breeding presents an approved test situation for the growth and treatment of human cancer cells.These models work due to the inoculation of cells into the membrane that stays within the egg shell during the time of investigation. In this study a modification of this model is presented. Samples of native tumors, rather than cell lines, are transplanted into the membrane and the body of the egg is taken out of the shell and placed in a plastic bowl. These modifications lead to an enhanced accessibility to the chorioallantoic membrane and the surrounding vessels thus facilitating intra venous access and application of pharmaceuticals and a focused radiotherapy. With the current modifications the embryo was kept alive and additionally, the vascularized tumor environment was preserved.

Deep neck infections: A single-center analysis of 63 cases.

BACKGROUND AND PURPOSE: With the use of antibiotic therapy, the incidence of deep neck infections has decreased in recent decades. The aim of this investigation was to review the clinical course and the management of deep neck infections in our department, compare them to the experiences of the common literature and identify predisposing factors for lethal complications. MATERIAL AND METHODS: In this single-center analysis, 63 patients with deep neck infections were treated surgically. The following clinical data were analyzed and compared: age, gender, laboratory data, spatial manifestation, therapeutic modalities, comorbidities, length of hospitalization and complications. RESULTS: There was a predominance of male patients (58.7%) and a mean age of 57.9 years. The most common symptoms at diagnosis were sore throat (96.8%) and neck swelling (92.0%). Cardio/pulmonary diseases and diabetes mellitus were the most common comorbidities. There was a significantly longer hospital stay for patients with diabetes mellitus. The most common manifestation was a parapharyngeal abscess in 24 patients (38.1%), followed by peri-/retrotonsillar infections in 19 patients (30.2%). In 29 patients, a multiple space infection was observed, with a significantly longer duration of hospitalization and a higher rate of complications. The main life-threatening complication was the development of airway obstruction in 20 patients (31.7%), who all received a tracheostomy. The duration of hospitalization for patients with complications was significantly longer. CONCLUSION: Close attention must be paid to the management of patients with deep neck infections, especially patients with diabetes mellitus and cardio/pulmonary diseases or patients with multiple space infections.

[Relevance of eosinopenia as marker of sepsis in the Emergency Department]. / Intérêt de l'éosinopénie dans le diagnostic de sepsis aux urgences.

BACKGROUND: Several studies in internal medicine departments and in intensive care units have shown the interest of eosinopenia in the diagnosis of infected patients. The aim of the present study was to test the value of this marker in the Emergency Department (ED), either alone or associated with other common sepsis markers. METHODS: We report on a retrospective and monocentric study. We reviewed the complete blood count (CBC) of all patients visiting the ED during one-week duration (in February 2014). Every element of the CBC and other inflammation markers (such as CRP) were analyzed. RESULTS: During the week of our study, 725 patients had a CBC (33 exclusions) and 692 patients were included for analysis. The median age was 59 years (IQR: 16-100). One hundred and twenty-five patients (18.1%) had a sepsis. The ROC curve demonstrated a cut off level of 10/mm3 eosinophils for which the specificity for sepsis was 91%. The association of eosinopenia (< 10/mm3) and white blood cells (WBC) or CRP elevation also showed a good specificity in patients with sepsis. CONCLUSION: In the ED, with a "simple" CBC, a profound eosinopenia appears to be very specific for sepsis, alone or in association with other markers of inflammation. Eosinopenia may become a helpful tool in our daily practice in the ED. Further studies are needed to further evaluate this marker.