Liquid Biopsy in Organ Damage: small extracellular vesicle chip-based assessment of polytrauma.

Background: Despite major advances in medicine, blood-borne biomarkers are urgently needed to support decision-making, including polytrauma. Here, we assessed serum-derived extracellular vesicles (EVs) as potential markers of decision-making in polytrauma. Objective: Our Liquid Biopsy in Organ Damage (LiBOD) study aimed to differentiate polytrauma with organ injury from polytrauma without organ injury. We analysed of blood-borne small EVs at the individual level using a combination of immunocapture and high-resolution imaging. Methods: To this end, we isolated, purified, and characterized small EVs according to the latest Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines from human blood collected within 24 h post-trauma and validated our results using a porcine polytrauma model. Results: We found that small EVs derived from monocytes CD14+ and CD14+CD61+ were significantly elevated in polytrauma with organ damage. To be precise, our findings revealed that CD9+CD14+ and CD14+CD61+ small EVs exhibited superior performance compared to CD9+CD61+ small EVs in accurately indicating polytrauma with organ damage, reaching a sensitivity and a specificity of 0.81% and 0.97%, respectively. The results in humans were confirmed in an independent porcine model of polytrauma. Conclusion: These findings suggest that these specific types of small EVs may serve as valuable, non-invasive, and objective biomarkers for assessing and monitoring the severity of polytrauma and associated organ damage.

[Diagnosis and Treatment of Severe Fournier's Gangrene: Introduction of a Surgical Approach, Evaluation of Risk Factors, Microbiological Characteristics and Review of the Literature]. / Diagnose und zielgerichtete Therapie der Fournier-Gangrän mit septischem Verlauf: Vorstellung eines Behandlungsalgorithmus, Identifikation von Risikofaktoren, Betrachtung des Mikrobioms und Abgleich mit der Literatur.

BACKGROUND: Fournier's gangrene is a necrotising fasciitis type I occurring in the perineal and genital region. The disease expands progressively and still has poor outcome, especially in critical ill patients. This study's focus was placed on the evaluation of risk factors and comorbidities, on the bacteriological spectrum, laboratory analyses, mortality and the course according to the algorithmic approach. METHOD: The medical records were reviewed of 10 patients with severe Fournier's gangrene from 2010 to 2019 who underwent intensive care therapy for at least 48 hours. Ten patients with Fournier's gangrene and sepsis and intensive therapy lasting at least 48 hours were recorded and analysed descriptively. RESULTS: The patient cohort consisted of six men and four women with a median age of 62 years (range 42 - 78 years). The median time between the onset of symptoms and hospitalisation was four days (range 3 - 5 days). The commonest aetiological event was a minor trauma. Predisposing factors included diabetes mellitus (30%) and overweight (80% BMI > 25). 90% of patients exhibited polymicrobial infection. All patients had systemic sepsis with SOFA-Score between 3 and 17 points. The mortality rate was 40%. Significant differences between surviving and deceased patients were shown by PCT, INR and aPTT (p < 0.05). CONCLUSION: Severe Fournier's gangrene continues to be a major challenge with a high mortality rate. Only rapid diagnosis, urgent extensive surgical debridement and intensive care therapy can lead to a favourable outcome in these critically ill patients. Vacuum-assisted closure can be successfully used in the management of soft-tissue defects.

LAMP-Seq enables sensitive, multiplexed COVID-19 diagnostics using molecular barcoding.

Frequent testing of large population groups combined with contact tracing and isolation measures will be crucial for containing Coronavirus Disease 2019 outbreaks. Here we present LAMP-Seq, a modified, highly scalable reverse transcription loop-mediated isothermal amplification (RT-LAMP) method. Unpurified biosamples are barcoded and amplified in a single heat step, and pooled products are analyzed en masse by sequencing. Using commercial reagents, LAMP-Seq has a limit of detection of ~2.2 molecules per µl at 95% confidence and near-perfect specificity for severe acute respiratory syndrome coronavirus 2 given its sequence readout. Clinical validation of an open-source protocol with 676 swab samples, 98 of which were deemed positive by standard RT-qPCR, demonstrated 100% sensitivity in individuals with cycle threshold values of up to 33 and a specificity of 99.7%, at a very low material cost. With a time-to-result of fewer than 24 h, low cost and little new infrastructure requirement, LAMP-Seq can be readily deployed for frequent testing as part of an integrated public health surveillance program.

Synergistic effects of extracellular vesicle phenotyping and AFP in hepatobiliary cancer differentiation.

BACKGROUND: Biliary cancer, comprising cholangio- and gallbladder carcinomas, is associated with high mortality due to asymptomatic disease onset and resulting late diagnosis. Currently, no robust diagnostic biomarker is clinically available. Therefore, we explored the feasibility of extracellular vesicles (EVs) as a liquid biopsy tool for biliary cancer screening and hepatobiliary cancer differentiation. METHODS: Serum EVs of biliary cancer, hepatocellular carcinoma, colorectal cancer and non-small cell lung cancer patients, as well as from healthy individuals, were isolated by sequential two-step centrifugation and presence of indicated EVs was evaluated by fluorescence activated cell sorting (FACS) analysis. RESULTS: Two directly tumour-related antigen combinations (AnnV+ CD44v6+ and AnnV+ CD44v6+ CD133+ ) and two combinations related to progenitor cells from the tumour microenvironment (AnnV+ CD133+ gp38+ and AnnV+ EpCAM+ CD133+ gp38+ ) were associated with good diagnostic performances that could potentially be used for clinical assessment of biliary cancer and differentiation from other cancer entities. With 91% sensitivity and 69% specificity AnnV+ CD44v6+ EVs showed the most promising results for differentiating biliary cancers from HCC. Moreover using a combined approach of EV levels of the four populations with serum AFP values, we obtained a perfect separation of biliary cancer and HCC with sensitivity, specificity, positive and negative predictive value all reaching 100% respectively. CONCLUSIONS: EV phenotyping, especially if combined with serum AFP, represents a minimally invasive, accurate liquid biopsy tool that could improve cancer screening and differential diagnosis of hepatobiliary malignancies.

Numbers of Axons in Spared Neural Tissue Bridges But Not Their Widths or Areas Correlate With Functional Recovery in Spinal Cord-Injured Rats.

The relationships between various parameters of tissue damage and subsequent functional recovery after spinal cord injury (SCI) are not well understood. Patients may regain micturition control and walking despite large postinjury medullar cavities. The objective of this study was to establish possible correlations between morphological findings and degree of functional recovery after spinal cord compression at vertebra Th8 in rats. Recovery of motor (Basso, Beattie, Bresnahan, foot-stepping angle, rump-height index, and ladder climbing), sensory (withdrawal latency), and bladder functions was analyzed at 1, 3, 6, 9, and 12 weeks post-SCI. Following perfusion fixation, spinal cord tissue encompassing the injury site was cut in longitudinal frontal sections. Lesion lengths, lesion volumes, and areas of perilesional neural tissue bridges were determined after staining with cresyl violet. The numbers of axons in these bridges were quantified after staining for class III ß-tubulin. We found that it was not the area of the spared tissue bridges, which is routinely determined by magnetic resonance imaging (MRI), but the numbers of axons in them that correlated with functional recovery after SCI (Spearman's ρ > 0.8; p < 0.001). We conclude that prognostic statements based only on MRI measurements should be considered with caution.

Liquid Biopsies in Hepatocellular Carcinoma: Are We Winning?

Hepatocellular carcinoma (HCC) represents the sixth most common cancer worldwide and the third most common cause of cancer-related death. One of the major problems faced by researchers and clinicians in this area is the lack of reliable disease biomarkers, which would allow for an earlier diagnosis, follow-up or prediction of treatment response, among others. In this regard, the "HCC circulome", defined as the pool of circulating molecules in the bloodstream derived from the primary tumor, represents an appealing target, the so called liquid biopsy. Such molecules encompass circulating tumor proteins, circulating tumor cells (CTCs), extracellular vesicles (EVs), tumor-educated platelets (TEPs), and circulating tumor nucleic acids, namely circulating tumor DNA (ctDNA) and circulating tumor RNA (ctRNA). In this article, we summarize recent findings highlighting the promising role of liquid biopsies as novel potential biomarkers in HCC, emphasizing on its clinical performance.

Anatomic conditions for bypass surgery between rostral (T7-T9) and caudal (L2, L4, S1) ventral roots to treat paralysis after spinal cord injury.

Severe spinal cord injuries cause permanent neurological deficits and are still considered as inaccessible to efficient therapy. Injured spinal cord axons are unable to spontaneously regenerate. Re-establishing functional activity especially in the lower limbs by reinnervation of the caudal infra-lesional territories might represent an effective therapeutic strategy. Numerous surgical neurotizations have been developed to bridge the spinal cord lesion site and connect the intact supra-lesional portions of the spinal cord to peripheral nerves (spinal nerves, intercostal nerves) and muscles. The major disadvantage of these techniques is the increased hypersensitivity, spasticity and pathologic pain in the spinal cord injured patients, which occur due to the vigorous sprouting of injured afferent sensory fibers after reconstructive surgery. Using micro-surgical instruments and an operation microscope we performed detailed anatomical preparation of the vertebral canal and its content in five human cadavers. Our observations allow us to put forward the possibility to develop a more precise surgical approach, the so called "ventral root bypass" that avoids lesion of the dorsal roots and eliminates sensitivity complications. The proposed kind of neurotization has been neither used, nor put forward. The general opinion is that radix ventralis and radix dorsalis unite to form the spinal nerve inside the dural sac. This assumption is not accurate, because both radices leave the dural sac separately. This neglected anatomical feature allows a reliable intravertebral exposure of the dura-mater ensheathed ventral roots and their damage-preventing end-to-side neurorrhaphy by interpositional nerve grafts.

Recovery after spinal cord injury by modulation of the proteoglycan receptor PTPσ.

SCI is followed by dramatic upregulation of chondroitin sulfate proteoglycans (CSPGs) which limit axonal regeneration, oligodendrocyte replacement and remyelination. The recent discovery of the specific CSPGs signaling receptor protein tyrosine phosphatase sigma (RPTPσ) provided an opportunity to refine the therapeutic approach to overcome CSPGs inhibitory actions. In previously published work, subcutaneous (s.c.) delivery of 44 µg/day of a peptide mimetic of PTPσ called intracellular sigma peptide (ISP), which binds to PTPσ and blocks CSPG-mediated inhibition, facilitated recovery after contusive SCI. Since this result could be of great interest for clinical trials, we independently repeated this study, but modified the method of injury as well as peptide application and the dosage. Following SCI at the Th10-segment, 40 rats were distributed in 3 groups. Animals in group 1 (20 rats) were subjected to SCI, but received no treatment. Rats in group 2 were treated with intraperitoneal (i.p.) injections of 44 µg/day ISP (SCI + ISP44) and animals of group 3 with s.c. injections of 500 µg/day ISP (SCI + ISP500) for 7 weeks after lesioning. Recovery was analyzed at 1, 3, 6, 9 and 12 weeks after SCI by determining (i) BBB-score, (ii) foot-stepping angle, (iii) rump-height index, (iv) number of correct ladder steps, (v) bladder score and (vi) sensitivity (withdrawal latency after thermal stimulus). Finally, we determined the amount of serotonergic fibers in the preserved neural tissue bridges (PNTB) around the lesion site. Our results show that, systemic therapy with ISP improved locomotor, sensory and vegetative recovery which correlated with more spared serotonergic fibers in PNTB.

Anatomy of the human orbital muscle (OM): Features of its detailed topography, syntopy and morphology.

The human orbital muscle (OM) is not readily accessible during ordinary anatomical teaching because of insufficient time and difficulties encountered in the preparation. Accordingly, its few anatomical descriptions are supported only by drawings, but not by photographs. The aim of this study was to present OM in dissected anatomic specimens in more detail. Following microscope-assisted dissection, its location, syntopy and morphology were analyzed in 88 orbits of 51 cadavers. Together with the periorbital connective tissue OM filled the infraorbital fissure (IOF) and extended back to the cavernous sinus. As a new finding, we here report that in 34% of the orbits we observed OM-fibers, which proceeded from IOF caudally to the facies infratemporalis of the maxilla. OM had a mean width of 4±1mm, a mean length of 22±5mm and its mean mass was 0.22±0.19g. The subsequent histological analysis of all specimens showed features of smooth muscle tissue: long, spindle-like cells with a centrally located cell nucleus (hematoxylin-eosin staining) which were innervated by tyrosine-hydroxylase immunopositive adrenergic fibers. We conclude that precise knowledge on OM might be very helpful not only to students in medicine and dentistry during anatomical dissection courses, but also to head and neck surgeons, ear-nose-throat specialists and neurosurgeons working in this field.

Microsurgical anatomy of the human carotid body (glomus caroticum): Features of its detailed topography, syntopy and morphology.

The human glomus caroticum (GC) is not readily accessible during ordinary anatomical teaching courses because of insufficient time and difficulties encountered in the preparation. Accordingly, most anatomical descriptions of its location, relationship to neighboring structures, size and shape are supported only by drawings, but not by photographs. The aim of this study is to present the GC with all associated roots and branches. Following microscope-assisted dissection and precise photo-documentation, a detailed analysis of location, syntopy and morphology was performed. We carried out this study on 46 bifurcations of the common carotid artery (CCA) into the external (ECA) and internal (ICA) carotid arteries and identified the GC in 40 (91%) of them. We found significant variations regarding the location of the GC and its syntopy: GC was associated with CCA (42%), ECA (28%) and ICA (30%) lying on the medial or lateral surface (82% or 13%, respectively) or exactly in the middle (5%) of the bifurcation. The short and long diameter of its oval form varied from 1.0 × 2.0 to 5.0 × 5.0mm. Connections with the sympathetic trunk (100%), glossopharyngeal (93%), vagus (79%) and hypoglossal nerve (90%) could be established in 29 cadavers. We conclude that precise knowledge of this enormous variety might be very helpful not only to students in medicine and dentistry during anatomical dissection courses, but also to surgeons working in this field.