When Palliative Care May Be the Only Option in the Management of Severe Burns: A Case Report Written With the Help of ChatGPT.

We present a case of 100% third-degree burns. The patient received full resuscitative measures, but the family was prepared for a poor outcome based on the severe extent of the injuries. After several days of treatment, it became apparent that the patient indeed could not survive the injuries and palliative care was instituted, including mechanical ventilation, fluid therapy, and analgesia. Surgery was not possible without causing major disfigurement, including enucleation of both eyes and amputation of all limbs.

Longitudinal patterns of cytokine expression at the individual level in humans after laparoscopic sleeve gastrectomy.

The study of the human response to injury has been hampered by the inherent heterogeneity in the models and methods used. By studying a standard injury longitudinally, using individual patient-level analysis, we endeavoured to better describe its dynamics. We analysed clinical variables, clinical laboratory and plasma cytokines from 20 patients at five time points. Clustering analysis showed two prototype patterns of cytokine behaviour: a concordant type, where cytokines behave the same way for all patients (notably IL-0 and TNFα), and a variable type, where different patterns of expression are seen for different patients (notably IL-8, IL-6 and IL-1RA). Analysis of the cytokines at the individual patient-level showed a strong four-way correlation between IL-1RA, GCSF, MIP-1ß and MCP-1. As it holds for most patients and not just on average, this suggests that they form a network which may play a central role in the response to gastro-intestinal injuries in humans. In conclusion, the longitudinal analysis of cytokines in a standard model allowed the identification of their underlying patterns of expression. We propose that the two prototype patterns shown may reflect the mechanism that separates the common and individual aspects of the injury response.

Alleviation of exhaustion-induced immunosuppression and sepsis by immune checkpoint blockers sequentially administered with antibiotics-analysis of a new mathematical model.

BACKGROUND: Sepsis-associated immune dysregulation, involving hyper-inflammation and immunosuppression, is common in intensive care patients, often leading to multiple organ dysfunction and death. The aim of this study was to identify the main driving force underlying immunosuppression in sepsis, and to suggest new therapeutic avenues for controlling this immune impairment and alleviating excessive pathogen load. METHODS: We developed two minimalistic (skeletal) mathematical models of pathogen-associated inflammation, which focus on the dynamics of myeloid, lymphocyte, and pathogen numbers in blood. Both models rely on the assumption that the presence of the pathogen causes a bias in hematopoietic stem cell differentiation toward the myeloid developmental line. Also in one of the models, we assumed that continuous exposure to pathogens induces lymphocyte exhaustion. In addition, we also created therapy models, both by antibiotics and by immunotherapy with PD-1/PD-L1 checkpoint inhibitors. Assuming realistic parameter ranges, we simulated the pathogen-associated inflammation models in silico with or without various antibiotic and immunotherapy schedules. RESULTS: Computer simulations of the two models show that the assumption of lymphocyte exhaustion is a prerequisite for attaining sepsis-associated immunosuppression, and that the ability of the innate and adaptive immune systems to control infections depends on the pathogen's replication rate. Simulation results further show that combining antibiotics with immune checkpoint blockers can suffice for defeating even an aggressive pathogen within a relatively short period. This is so as long as the drugs are administered soon after diagnosis. In contrast, when applied as monotherapies, antibiotics or immune checkpoint blockers fall short of eliminating aggressive pathogens in reasonable time. CONCLUSIONS: Our results suggest that lymphocyte exhaustion crucially drives immunosuppression in sepsis, and that one can efficiently resolve both immunosuppression and pathogenesis by timely coupling of antibiotics with an immune checkpoint blocker, but not by either one of these two treatment modalities alone. Following experimental validation, our model can be adapted to explore the potential of other therapeutic options in this field.

CognitionMaster: an object-based image analysis framework.

BACKGROUND: Automated image analysis methods are becoming more and more important to extract and quantify image features in microscopy-based biomedical studies and several commercial or open-source tools are available. However, most of the approaches rely on pixel-wise operations, a concept that has limitations when high-level object features and relationships between objects are studied and if user-interactivity on the object-level is desired. RESULTS: In this paper we present an open-source software that facilitates the analysis of content features and object relationships by using objects as basic processing unit instead of individual pixels. Our approach enables also users without programming knowledge to compose "analysis pipelines" that exploit the object-level approach. We demonstrate the design and use of example pipelines for the immunohistochemistry-based cell proliferation quantification in breast cancer and two-photon fluorescence microscopy data about bone-osteoclast interaction, which underline the advantages of the object-based concept. CONCLUSIONS: We introduce an open source software system that offers object-based image analysis. The object-based concept allows for a straight-forward development of object-related interactive or fully automated image analysis solutions. The presented software may therefore serve as a basis for various applications in the field of digital image analysis.

Properties of intermediate filament networks assembled from keratin 8 and 18 in the presence of Mg²+.

The mechanical properties of epithelial cells are modulated by structural changes in keratin intermediate filament networks. To investigate the relationship between network architecture and viscoelasticity, we assembled keratin filaments from recombinant keratin proteins 8 (K8) and 18 (K18) in the presence of divalent ions (Mg(2+)). We probed the viscoelastic modulus of the network by tracking the movement of microspheres embedded in the network during assembly, and studied the network architecture using scanning electron microscopy. Addition of Mg(2+) at physiological concentrations (<1 mM) resulted in networks whose structure was similar to that of keratin networks in epithelial cells. Moreover, the elastic moduli of networks assembled in vitro were found to be within the same magnitude as those measured in keratin networks of detergent-extracted epithelial cells. These findings suggest that Mg(2+)-induced filament cross-linking represents a valid model for studying the cytoskeletal mechanics of keratin networks.

Inflammation, regeneration, and transformation in the pancreas: results of the Collaborative Research Center 518 (SFB 518) at the University of Ulm.

The primary diseases of the pancreas include diabetes mellitus, acute and chronic pancreatitis, as well as pancreatic carcinoma. This review presents findings and emerging questions on the diseases of the pancreas obtained by the consortium of the Collaborative Research Center 518 (SFB 518), "Inflammation, Regeneration, and Transformation in the Pancreas" at the University of Ulm. During the last 12 years, the SFB 518 contributed considerably to the understanding of the cellular and molecular basis of pancreatic diseases and established the basis for the development of new strategies for prevention and causal therapy for diabetes, pancreatitis, and pancreatic cancer.

Critical review: cellular mechanobiology and amoeboid migration.

Cell motility is important for tissue homeostasis and plays a central role in various pathologies, notably inflammation and cancer. Research into the critical processes involved in cell migration has so far mostly focused on cell adhesion and proteolytic degradation of the extracellular matrix. However, pharmacological interference with these processes only partially blocks cell motility in vivo. In this review we summarize the arising evidence that the mechanical properties of the cell body have a major role to play in cell motility--especially in a low-adhesion, amoeboid-like migration mode in three-dimensional tissue structures. We summarize the processes determining cell mechanics and discuss relevant measurement technologies including their applications in medical cell biology.

Novel electron tomographic methods to study the morphology of keratin filament networks.

The three-dimensional (3D) keratin filament network of pancreatic carcinoma cells was investigated with different electron microscopical approaches. Semithin sections of high-pressure frozen and freeze substituted cells were analyzed with scanning transmission electron microscope (STEM) tomography. Preservation of subcellular structures was excellent, and keratin filaments could be observed; however, it was impossible to three-dimensionally track the individual filaments. To obtain a better signal-to-noise ratio in transmission mode, we observed ultrathin sections of high-pressure frozen and freeze substituted samples with low-voltage (30 kV) STEM. Contrast was improved compared to 300 kV, and individual filaments could be observed. The filament network of samples prepared by detergent extraction was imaged by high-resolution scanning electron microscopy (SEM) with very good signal-to-noise ratio using the secondary electron signal and the 3D structure could be elucidated by SEM tomography. In freeze-dried samples it was possible to discern between keratin filaments and actin filaments because the helical arrangement of actin subunits in the F-actin could be resolved. When comparing the network structures of the differently prepared samples, we found no obvious differences in filament length and branching, indicating that the intermediate filament network is less susceptible to preparation artifacts than the actin network.

The regulatory role of cell mechanics for migration of differentiating myeloid cells.

Migration of cells is important for tissue maintenance, immune response, and often altered in disease. While biochemical aspects, including cell adhesion, have been studied in detail, much less is known about the role of the mechanical properties of cells. Previous measurement methods rely on contact with artificial surfaces, which can convolute the results. Here, we used a non-contact, microfluidic optical stretcher to study cell mechanics, isolated from other parameters, in the context of tissue infiltration by acute promyelocytic leukemia (APL) cells, which occurs during differentiation therapy with retinoic acid. Compliance measurements of APL cells reveal a significant softening during differentiation, with the mechanical properties of differentiated cells resembling those of normal neutrophils. To interfere with the migratory ability acquired with the softening, differentiated APL cells were exposed to paclitaxel, which stabilizes microtubules. This treatment does not alter compliance but reduces cell relaxation after cessation of mechanical stress six-fold, congruent with a significant reduction of motility. Our observations imply that the dynamical remodeling of cell shape required for tissue infiltration can be frustrated by stiffening the microtubular system. This link between the cytoskeleton, cell mechanics, and motility suggests treatment options for pathologies relying on migration of cells, notably cancer metastasis.

Integration and acceleration of virtual microscopy as the key to successful implementation into the routine diagnostic process.

BACKGROUND: The virtual microscopy is widely accepted in Pathology for educational purposes and teleconsultation but is far from the routine use in surgical pathology due to the technical requirements and some limitations. A technical problem is the limited bandwidth of a usual network and the delayed transmission rate and presentation time on the screen. METHODS: In this study the process of secondary diagnostic was evaluated using the "T.Konsult Pathologie" service of the Professional Association of German Pathologists within the German breast cancer screening program. The characteristics of the access to the WSI (Whole Slide Images) have been analyzed to explore the possibilities of prefetching and caching to reduce the presentation and transfer time with the goal to increase user acceptance. The log files of the web server were analyzed to reconstruct the movements of the pathologist on the WSI and to create the observation path. Using a specialized tool the observation paths were extracted automatically from the log files. The attributes linearity, 3-point-linearity, changes per request, and number of consecutive requests were calculated to design, develop and evaluate different caching and prefetching strategies. RESULTS: The analysis of the observation paths showed that a complete accordance of two image requests is a very rare event. But more frequently a partial covering of two requested image areas can be found. In total 257 diagnostic paths from 131 WSI have been extracted and analysed. On average a diagnostic path consists of 16 image requests and takes 189 seconds between first and last image request. The mean linearity was 0,41 and the mean 3-point-linearity 0,85. Three different caching algorithms have been compared with respect to hit rate and additional image requests on the WSI server. Tests demonstrated that 95% of the diagnostic paths could be loaded without any deletion of entries in the cache (cache size 12,2 Megapixel). If the image parts are stored after JPEG compression this complies with less than 2 MB. DISCUSSION: WSI telepathology is a technology which offers the possibility to break the limitations of conventional static telepathology. The complete histological slide may be investigated instead of sets of images of lesions sampled by the presenting pathologist. The benefit is demonstrated by the high diagnostic security of 95% accordance between first and second diagnosis.

Simulating the formation of keratin filament networks by a piecewise-deterministic Markov process.

Keratin intermediate filament networks are part of the cytoskeleton in epithelial cells. They were found to regulate viscoelastic properties and motility of cancer cells. Due to unique biochemical properties of keratin polymers, the knowledge of the mechanisms controlling keratin network formation is incomplete. A combination of deterministic and stochastic modeling techniques can be a valuable source of information since they can describe known mechanisms of network evolution while reflecting the uncertainty with respect to a variety of molecular events. We applied the concept of piecewise-deterministic Markov processes to the modeling of keratin network formation with high spatiotemporal resolution. The deterministic component describes the diffusion-driven evolution of a pool of soluble keratin filament precursors fueling various network formation processes. Instants of network formation events are determined by a stochastic point process on the time axis. A probability distribution controlled by model parameters exercises control over the frequency of different mechanisms of network formation to be triggered. Locations of the network formation events are assigned dependent on the spatial distribution of the soluble pool of filament precursors. Based on this modeling approach, simulation studies revealed that the architecture of keratin networks mostly depends on the balance between filament elongation and branching processes. The spatial distribution of network mesh size, which strongly influences the mechanical characteristics of filament networks, is modulated by lateral annealing processes. This mechanism which is a specific feature of intermediate filament networks appears to be a major and fast regulator of cell mechanics.

Measuring the nanomechanical properties of cancer cells by digital pulsed force mode imaging.

In this paper, we demonstrate that the digital pulsed force mode data can distinguish two cancer cell lines (HeLa, Panc) by their mechanical properties. The live cells were imaged in buffer solution. The digital pulsed force mode measured 175 force-distance curves per second which, due to the speed of the measurement, were distorted by the viscous drag in the buffer. We show that this drag force causes a sinusoidal addition to the force-distance curves. By subtracting the viscous drag effect one obtains standard force-distance curves. The force-distance curves are then evaluated to extract key data on the curves, such as adhesion energies, local stiffness or the width of the hysteresis loop. These data are then correlated to classify the force-distance curves. We show examples based on the width of the hysteresis loop and the adhesion energies. Outliers in this classification scheme are points where, potentially, interesting new physics or different physics might happen. Based on classification schemes adapted to experimental settings, we propose that the digital pulsed force mode is a tool to evaluate the time evolution of the mechanical response of cells.

The bioactive lipid sphingosylphosphorylcholine induces differentiation of mouse embryonic stem cells and human promyelocytic leukaemia cells.

Sphingosylphosphorylcholine (SPC) is the major component of high-density lipoproteins (HDL) in blood plasma. The bioactive lipid acts mainly via G protein coupled receptors (GPCRs). Similar to ligands of other GPCRs, SPC has multiple biological roles including the regulation of proliferation, migration, angiogenesis, wound healing and heart rate. Lysophospholipids and their receptors have also been implicated in cell differentiation. A potential role of SPC in stem cell or tumour cell differentiation has been elusive so far. Here we examined the effect of SPC on the differentiation of mouse embryonic stem (ES) cells and of human NB4 promyelocytic leukemia cells, a well established tumour differentiation model. Our data show that mouse embryonic stem cells and NB4 cells express the relevant GPCRs for SPC. We demonstrate both at the level of morphology and of gene expression that SPC induces neuronal and cardiac differentiation of mouse ES cells. Furthermore, SPC induces differentiation of NB4 cells by a mechanism which is critically dependent on the activity of the MEK-ERK cascade. Thus, the bioactive lipid SPC is a novel differentiation inducing agent both for mouse ES cells, but also of certain human tumour cells.

Fitting of random tessellation models to keratin filament networks.

The role of specific structural patterns in keratin filament networks for regulating biophysical properties of epithelial cells is poorly understood. This is at least partially due to a lack of methods for the analysis of filament network morphology. We have previously developed a statistical approach to the analysis of keratin filament networks imaged by scanning electron microscopy. The segmentation of images in this study resulted in graph structures, i.e. tessellations, whose structural characteristics are now further investigated by iteratively fitting geometrical statistical models. An optimal model as well as corresponding optimal parameters are detected from a given set of possible random tessellation models, i.e. Poisson-Line tessellations (PLT), Poisson-Voronoi tessellations (PVT) and Poisson-Delaunay tessellations (PDT). Using this method, we investigated the remodeling of keratin filament networks in pancreatic cancer cells in response to transforming growth factor alpha (TGFalpha), which is involved in pancreatic cancer progression. The results indicate that the fitting of random tessellation models represents a suitable method for the description of complex filament networks.

The tyrosine kinase Yes regulates actin structure and secretion during pancreatic acinar cell damage in rats.

Acinar cells require a functional apical actin web for secretion. During stimulation with supraphysiological concentrations of cholecystokinin (CCK), a condition that mimics acute pancreatitis, the actin filaments disintegrate. This leads to retention of secretory enzymes and, together with their premature activation, results in cell injury. Actin filaments are anchored through membrane-associated protein complexes that can be regulated through Src-family kinases in some model systems. Here we show that the Src-family kinases Yes and Lyn, but not Src and Fyn, are expressed in isolated pancreatic acini of Wistar rats. Upon stimulation with supramaximal secretory CCK (10(-8) M), Yes became reversibly tyrosine-phosphorylated and activated within 2 min. Immunocytochemical and subcellular fractionation studies showed reversible redistribution of Yes to the apical actin web and to the membrane fraction within 5 min. Coimmunoprecipitation demonstrated that Yes forms a complex with the focal adhesion protein Pyk2, which increased with CCK stimulation. In functional studies, inhibition of Src-kinase activity with PP2 partially reversed actin disintegration and also restored amylase secretion. We conclude that Yes participates in the regulation of the acinar cell actin, probably by interaction with Pyk2.

CGS 35601 and its orally active prodrug CGS 37808 as triple inhibitors of endothelin-converting enzyme-1, neutral endopeptidase 24.11, and angiotensin-converting enzyme.

CGS 35601 is a potent triple inhibitor of endothelin-converting enzyme-1, neutral endopeptidase 24.11, and angiotensin-converting enzyme. It inhibited the activities of these three enzymes with IC50 values of 55, 2 and 22 nM, respectively. In conscious rats, CGS 35601 suppressed the big endothelin-1-induced pressor response by 82% and 72% at 30 and 120 minutes, respectively, following injection at a dose of 10 mg/kg, intravenously. At the same dose, CGS 35601 increased plasma atrial natriuretic peptide (ANP) immunoreactivity by 170% for up to 4 hours in conscious rats infused with ANP, and it inhibited the angiotensin I-induced pressor response by 74-94% within the first 2 hours after dosing. Similar in vivo activities were also observed with its orally active prodrug, CGS 37808. This compound blocked the big endothelin-1- induced pressor response by 71% and 67% at 30 and 120 minutes, respectively, after an oral dose of 10 mgEq/kg in conscious rats. It also increased plasma ANP immunoreactivity by 103% for up to 4 hours and inhibited the angiotensin I-induced pressor response by an average of 49% within the first 4 hours after the same dosing regimen. By suppressing the biosyntheses of endothelin-1 and angiotensin II, two potent vasoconstrictors, while simultaneously potentiating the circulating levels of ANP, a vasorelaxant and diuretic, CGS 35601 and CGS 37808 may represent novel agents for the treatment of cardiovascular and renal diseases.

Sphingosylphosphorylcholine regulates keratin network architecture and visco-elastic properties of human cancer cells.

Sphingosylphosphorylcholine (SPC) is a naturally occurring bioactive lipid that is present in high density lipoproteins (HDL) particles and found at increased levels in blood and malignant ascites of patients with ovarian cancer. Here, we show that incubation of human epithelial tumour cells with SPC induces a perinuclear reorganization of intact keratin 8-18 filaments. This effect is specific for SPC, largely independent of F-actin and microtubules, and is accompanied by keratin phosphorylation. In vivo visco-elastic probing of single cancer cells demonstrates that SPC increases cellular elasticity. Accordingly, SPC stimulates migration of cells through size-limited pores in a more potent manner than lysophosphatidic acid (LPA). LPA induces actin stress fibre formation, but does not reorganize keratins in cancer cells and hence increases cellular stiffness. We propose that reorganization of keratin by SPC may facilitate biological phenomena that require a high degree of elasticity, such as squeezing of cells through membranous pores during metastasis.

Transgenic overexpression of the oncofetal RNA binding protein KOC leads to remodeling of the exocrine pancreas.

BACKGROUND & AIMS: To elucidate the function of the oncofetal RNA-binding protein, K-homologous (KH) domain containing protein overexpressed in cancer (KOC), we studied the effect of a constitutive reexpression of KOC in transgenic mice. METHODS: Transgenic mouse lines expressing KOC under the control of the mouse metallothionein promoter were generated and were shown to express the 69-kilodalton protein. Two mouse lines with moderate to strong gene expression of the transgene were further analyzed. RESULTS: The pancreas of KOC-transgenic mice showed progressive morphologic alterations, including an increased proliferation of acinar cells, acinar-ductal metaplasia, net loss of acinar tissue, and the appearance of numerous interstitial cells. Acinar-ductal metaplasia led to the development of duct-like structures exhibiting the characteristics of normal intralobular ducts. Interstitial cells expressed markers of endocrine or ductal differentiation. Nerve growth factor alpha (NGF-alpha) and the GTPase kir/Gem were identified as potential targets of KOC by expression profiling analyses. CONCLUSIONS: Reexpression of KOC in the transgenic model is apparently incompatible with the maintenance of a fully differentiated, adult acinar phenotype and may lead to a more fetal ductal phenotype via acinar-ductal metaplasia. This and the appearance of interstitial cells with a ductal and endocrine differentiation capacity suggest that transgenic reexpression of the oncofetal gene KOC may recapitulate a developmental program active during embryogenesis.

CGS 34226, a thiol-based dual inhibitor of endothelin converting enzyme-1 and neutral endopeptidase 24.11.

Endothelins (ETs) are potent vasoconstrictors and have been implicated in the pathogenesis of various cardiovascular and renal diseases. In contrast, atrial natriuretic peptide (ANP) is a potent vasorelaxant and diuretic agent, which is mainly degraded by neutral endopeptidase 24.11 (NEP) in vivo. Thus, compounds that can suppress the biosynthesis of ETs by inhibiting endothelin converting enzymes (ECEs), which catalyse the final step of post-translational processing of the vasoconstrictors, while simultaneously potentiating the levels of ANP by inhibiting NEP may have novel therapeutic utility. Through targeted screening of our compound library and subsequent optimization, CGS 34226 was identified as a potent, dual inhibitor of ECE-1 and NEP, inhibiting the enzymes with respective IC(50) values of 11 and 4.6 nM. In vivo, CGS 34226 suppressed the big endothelin-1 (big ET-1)-induced pressor response dose-dependently. At 15 and 90 min after an intravenous dose of 30 mg/kg in anaesthetized rats, this compound inhibited the big ET-1-induced effect by 79% and 65% respectively. In addition, CGS 34226 increased plasma ANP immunoreactivity by 120% up to 4 h after an intravenous dose of 10 mg/kg in conscious rats infused with ANP at a rate of 450 ng/kg per min, intravenously. These results show that CGS 34226 is a potent dual inhibitor of ECE-1 and NEP in vitro and in vivo and that the compound may represent a novel agent for the treatment of cardiovascular and renal dysfunction.

Effects of the ECE/NEP inhibitor CGS 34225 on the big ET-1-induced pressor response and plasma atrial natriuretic peptide concentration in conscious rats.

CGS 34226 is a thiol-containing, potent dual inhibitor of endothelin converting enzyme-1 (ECE-1) and neutral endopeptidase 24.11 (NEP) with IC(50) values of 11 and 5 nM respectively. The purpose of the present study was to characterize the inhibitory effects of CGS 34225, an orally active prodrug of CGS 34226, on ECE-1 and NEP in vivo. The effects on ECE-1 and NEP were assessed by determining the inhibition of big endothelin-1 (big ET-1)-induced increases in mean arterial pressure (MAP) and increases in plasma atrial natriuretic peptide (ANP) concentrations respectively, in conscious rats. Thirty and 120 min after the administration of vehicle, big ET-1 (0.3 nmol/kg, intravenously; i.v.) produced pressor responses of approximately 800 mmHg.min (area under the curve for change in MAPxtime). Treatment with CGS 34225 at 1 mgEq/kg, per os (p.o.), decreased the pressor effect of big ET-1 by 39 and 53% at 30 and 120 min respectively (P<0.05, both times). Increasing the dose of CGS 34255 to 30 mgEq/kg, p.o., resulted in greater inhibition, 84 and 92% (P<0.05) at 30 and 120 min respectively. Furthermore, at this higher dose, the inhibitory effect on ECE-1 was long-lasting, averaging 86, 75 and 30% (P<0.05, all times) at 4, 8 and 24 h respectively. In rats treated with vehicle, the infusion of ANP at 450 ng/kg per min i.v. resulted in plasma ANP concentrations of 3.9-4.8 ng/ml that remained relatively constant for 4 h. Treatment with CGS 34225 at 10 mgEq/kg, p.o., increased the ANP level to 7.7+/-1.0 and 10.6+/-1.8 ng/ml at 1 and 4 h after dosing (P<0.05, both times). These data demonstrate that CGS 34225 is a potent, orally active and long-acting inhibitor of ECE-1 and NEP in vivo. It is anticipated that compounds with this dual function may be useful in the treatment of cardiovascular diseases where the ET system plays a pathogenic role and the potentiation of ANP elicits therapeutic benefits.