Cytokine patterns in critically ill patients undergoing percutaneous tracheostomy.

The inflammatory response to acute injury among humans has proved difficult to study due to the significant heterogeneity encountered in actual patients. We set out to characterize the immune response to a model injury with reduced heterogeneity, a tracheostomy, among stable critical care patients, using a broad cytokine panel and clinical data. Twenty-three critical care patients undergoing percutaneous bedside tracheostomies were recruited in a medical intensive care unit. Blood samples were collected at five intervals during 24-h peri-procedure. Patients were followed-up for 28 days for clinical outcomes. There were no statistically significant changes in any of the cytokines between the five time-points when studied as a whole cohort. Longitudinal analysis of the cytokine patterns at the individual patient level with a clustering algorithm showed that, notwithstanding the significant heterogeneity observed, the patients' cytokine responses can be classified into three broad patterns that show increasing, decreasing or no major changes from the baseline. This analytical approach also showed statistically significant associations between cytokines, with those most likely to be associated being interleukin (IL)-6, granulocyte colony-stimulating factor (GCSF) and ferritin, as well as a strong tri-way correlation between GCSF, monocyte chemoattractant protein 1 (MCP1) and macrophage inflammatory protein-1ß (MIP1ß). In conclusion, in this standard human model of soft tissue injury, by applying longitudinal analysis at the individual level, we have been able to identify the cytokine patterns underlying the seemingly random, heterogeneous patient responses. We have also identified consistent cytokine interactions suggesting that IL-6, GCSF, MCP1 and MIP1ß are the cytokines most probably driving the immune response to this injury.

Complement C5a-Induced Changes in Neutrophil Morphology During Inflammation.

The complement and neutrophil defence systems, as major components of innate immunity, are activated during inflammation and infection. For neutrophil migration to the inflamed region, we hypothesized that the complement activation product C5a induces significant changes in cellular morphology before chemotaxis. Exposure of human neutrophils to C5a dose- and time-dependently resulted in a rapid C5a receptor-1 (C5aR1)-dependent shape change, indicated by enhanced flow cytometric forward-scatter area values. Similar changes were observed after incubation with zymosan-activated serum and in blood neutrophils during murine sepsis, but not in mice lacking the C5aR1. In human neutrophils, Amnis high-resolution digital imaging revealed a C5a-induced decrease in circularity and increase in the cellular length/width ratio. Biomechanically, microfluidic optical stretching experiments indicated significantly increased neutrophil deformability early after C5a stimulation. The C5a-induced shape changes were inhibited by pharmacological blockade of either the Cl-/HCO3--exchanger or the Cl- -channel. Furthermore, actin polymerization assays revealed that C5a exposure resulted in a significant polarization of the neutrophils. The functional polarization process triggered by ATP-P2X/Y-purinoceptor interaction was also involved in the C5a-induced shape changes, because pretreatment with suramin blocked not only the shape changes but also the subsequent C5a-dependent chemotactic activity. In conclusion, the data suggest that the anaphylatoxin C5a regulates basic neutrophil cell processes by increasing the membrane elasticity and cell size as a consequence of actin-cytoskeleton polymerization and reorganization, transforming the neutrophil into a migratory cell able to invade the inflammatory site and subsequently clear pathogens and molecular debris.

White matter lesions reduce number of brain metastases in different cancers: a high-resolution MRI study.

Brain metastases are major complications of common cancers. Tumor type and proneness to the CNS are thought to define the number and size of brain metastases. It is not known if intrinsic vascular factors can also have an effect. Restricted perfusion due to cerebral small vessel disease is frequent in elderly patients and causes white matter lesions (WML). The aim of this analysis was to evaluate a possible negative effect of WML and patient age on the number and size of brain metastases (BM) of different tumor entities. Pre-therapeutic 3 T brain magnetic resonance imaging (MRI) of 200 patients with BM were analyzed. Location, size and number of BM (NoM) were determined. T2 hyperintensive WML were scored according to Fazekas-Score (grade I-III). Patients with WML grade 1 (NoM: 5.59; p = 0.009) and grade 2 (NoM: 3.68; p = 0.002) had significantly less BM than patients without WML (NoM: 6.99). This effect was present in subgroups of different tumors: NSCLC (p = 0.05), other tumors than NSCLC (p = 0.048). Age (≤65 or >65 years) was positively correlated with the degree of WML but not with number (pNoM = 0.832) or mean diameter (pmDM = 0.662) of brain metastases. While patient age did not appear to be relevant, increasing WML were associated with lower number of brain metastases in different tumor types.

Both monovalent cations and plectin are potent modulators of mechanical properties of keratin K8/K18 networks.

Intermediate filament (IF) networks are a major contributor to cell rigidity and thus serve as vital elements to preserve the integrity of entire cell layers. Keratin K8 and K18 IFs are the basic constituents of the cytoskeleton of epithelial cells. The mechanical properties of K8/K18 networks depend on the structural arrangements of individual filaments within the network. This paper investigates the architecture of these networks in vitro under the influence of the monovalent cation potassium and that of the cytolinker protein plectin. Whereas increasing amounts of potassium ions lead to filament bundling, plectin interlinks filaments at filament intersection points but does not lead to bundle formation. The mechanics of the resulting networks are investigated by microrheology with assembled K8/K18 networks. It is shown that bundling induced by potassium ions significantly stiffens the network. Furthermore, our measurements reveal an increase in plectin-mediated keratin network rigidity as soon as an amount corresponding to more than 20% of the plectin present in cells is added to the keratin IF networks. In parallel, we investigated the influence of plectin on cell rigidity in detergent-extracted epithelial vulva carcinoma derived A431 cells in situ. These cytoskeletons, containing mostly IFs, actin filaments and associated proteins, exhibit a significantly decreased stiffness, when plectin is downregulated to ≈10% of the normal value. Therefore, we assume that plectin, via the formation of IF-IF connections and crosslinking of IFs to actin filaments, is an important contributor to cell stiffness.

Microrheology of keratin networks in cancer cells.

Microrheology is a valuable tool to determine viscoelastic properties of polymer networks. For this purpose measurements with embedded tracer beads inside the extracted network of pancreatic cancer cells were performed. Observing the beads motion with a CCD-high-speed-camera leads to the dynamic shear modulus. The complex shear modulus is divided into real and imaginary parts which give insight into the mechanical properties of the cell. The dependency on the distance of the embedded beads to the rim of the nucleus shows a tendency for a decreasing storage modulus. We draw conclusions on the network topology of the keratin network types based on the mechanical behavior.

Late manifestation of subclinical hyperthyroidism after goitrogenesis in an index patient with a N670S TSH receptor germline mutation masquerading as TSH receptor antibody negative Graves' disease.

In 27 families with familial non-autoimmune hyperthyroidism (FNAH) reported up to date, the onset of hyperthyroidism varies from 18 months to 60 years. Also the manifestation of goitres is variable in these families. A 74-year-old woman first presented at the age of 69 years with tachyarrhythmia and hypertension. After initial treatment of her hypertension and oral anticoagulation for her intermittent atrial fibrillation, a thyroid workup revealed a suppressed TSH and normal fT3 and fT4. TPO, TSH receptor (TSHR), and thyroglobulin antibodies were negative. Thyroid ultrasound revealed a thyroid volume of 102 ml with several nodules with diameters of up to 2.6 cm right and up to 1.8 cm left. Scintigraphy showed a homogeneous Technetium-99 m ((99 m)Tc) uptake of 1.27%. She was subsequently treated with 1 GBq radioiodine ((131)I). At the age of 74, her thyroid function was normal and her thyroid volume decreased to 90 ml. Because of the diffuse (99 m)Tc uptake and the negative TPO, TSHR, and thyroglobulin antibodies, genetic analysis of her TSHR gene was performed, in spite of her negative family history for hyperthyroidism. Sequencing revealed a N670S TSHR germline mutation. Previous in vitro characterisation of this TSHR mutation suggests a weak constitutive activity, yet the experimental data are ambiguous. This case illustrates the necessity to analyse patients with hyperthyroidism accompanied by diffuse (99 m)Tc uptake and negative TPO, TSHR, and thyroglobulin antibodies for TSHR germline mutations. Moreover, it demonstrates that TSHR germline mutations may first lead to longstanding nodular goitrogenesis before the late manifestation of subclinical hyperthyroidism.

An electromyographic analysis of the hip abductor muscles during a standing work task.

Research data strongly suggest that osteoarthritis of the hip occurs statistically more often on the right side. A possible contributing factor to this right-sided bias in frequency may be that the articular cartilage on the right hip is subjected to relatively higher muscular-based forces throughout a lifetime. As an initial attempt to study this possibility, this research examined healthy persons to determine the existence of a "dominant" hip similar to that expressed for handedness. Electromyographic (EMG) analysis was used to compare the electrical activity between the right and left hip abductor muscles during a standardized standing work task using 40 right-handed and 40 left-handed healthy subjects. Analysis of the data showed that the hip muscle on the side of the subject's handedness produced higher normalized EMG activity than did the opposite hip; however, the differences were not all statistically significant. The trend of this data set, however, warrants further research into a possible association between hip dominance, asymmetrical muscle use, and the development of hip osteoarthritis.

Remodeling of vimentin cytoskeleton correlates with enhanced motility of promyelocytic leukemia cells during differentiation induced by retinoic acid.

The intermediate filament (IFs) cytoskeleton is one of the major determinants for the mechanical properties of cytoplasm. Vimentin is the major IFs protein in peripheral blood neutrophils. We investigated its expression and function during neutrophil differentiation using the promyelocytic leukemia cell line NB4. The differentiation of NB4 cells along the neutrophil lineage and the monocytic pathway was induced by all-trans retinoic acid (ATRA) and phorbol esters (PMA), respectively. We demonstrated a down-regulation of vimentin after ATRA treatment of NB4 cells by immunoblotting and immunofluorescence. The architecture of the vimentin cytoskeleton in differentiated NB4 cells resembled that observed in mature neutrophils. In contrast, we showed a slight increase of vimentin content in phorbol ester (PMA)-treated NB4 cells. The structural features of the vimentin cytoskeleton obtained by image analysis showed significant differences in network density and directionality between ATRA-treated NB4 cells and controls. The functional consequence of the cytoskeletal remodeling for the mechanical properties of NB4 cells was assessed in migration assays. After ATRA treatment, we found a 4-fold increased migration of NB4 cells across transwell membranes with a 8 microm pore size without any cell size modification. No significant differences between PMA-treated NB4 cells and control cells could be observed using similar tests. These results indicate that both vimentin expression and network architecture are tightly controlled during neutrophil differentiation to regulate the mechanical properties of these cells.

Statistical analysis of the three-dimensional structure of centromeric heterochromatin in interphase nuclei.

Translocation of genes into the pericentromeric heterochromatin occurs during cellular differentiation and leads to a long-term silencing of these genes. Consequently, a structural remodelling of this heterochromatin compartment is observed during differentiation but this remains to be defined from a topological point of view. In a previous study, we analysed the three-dimensional (3D) distribution patterns of centromere clusters (chromocentres) by confocal scanning laser microscopy and found that differentiation of the promyelocytic leukaemia cell line NB4 along the neutrophil lineage is associated with a progressive clustering of centromeres. This clustering was reflected by a decreased number of detectable chromocentres, i.e. groups of centromeres with a distance below the diffraction-limited resolution of optical microscopy. The purpose of this study was to perform a statistical analysis of the 3D distribution of chromocentres in NB4 cells. Several point field characteristics (Ripley's K-function, L-function, pair correlation function, nearest-neighbour distribution function) were investigated to describe the topology of chromocentres during differentiation of NB4 cells. The pair correlation function revealed a higher frequency of chromocentre distances between 350 nm and 800 nm in undifferentiated NB4 cells as compared with differentiated cells. The L-function and the nearest-neighbour distribution function confirmed these results. These data imply the existence of intranuclear heterochromatin zones formed by functionally related centromeric regions. In view of the observed decrease in the number of detectable chromocentres during differentiation, we hypothesize that these zones with a diameter of 350-800 nm in undifferentiated NB4 cells contract into zones with a diameter below 350 nm in differentiated cells.

Contractile hyporesponsiveness of hepatic arteries in humans with cirrhosis: evidence for a receptor-specific mechanism.

Splanchnic vasodilatation and vascular hyporesponsiveness to vasopressors are characteristic features of patients with cirrhosis. Although the vascular response to different vasopressors has been shown to be attenuated in cirrhosis, alterations on the receptor level are discussed controversially. Thus, impaired postreceptor signaling has been postulated. However, so far this has not been studied in human splanchnic vessels. Therefore, we assessed the vascular response of human hepatic arteries after activating the G-protein-dependent signal transduction pathway by stimulation with angiotensin II, the thromboxane A(2) analog U46619, or by G-protein activation with NaF/AlCl(3). After endothelium denudation, cumulative isometric concentration contraction curves were obtained for hepatic arteries from 32 cirrhotic patients undergoing liver transplantation and from 40 organ donors after stimulation with either angiotensin II (10(-11)-10(-5) mol/L), U46619 (10(-10)-10(-6) mol/L) or AlCl(3) (30 micromol/L)/NaF (10(-4)-3 x 10(-2) mol/L). Hepatic arteries from cirrhotic patients were markedly less responsive to angiotensin II (P <.0001) than those from organ donors. Both stimulation of the G-protein phospholipase C pathway via the thromboxane A(2) receptor and receptor-independent G-protein stimulation with AlCl(3)/NaF, induced an intact contractile response. In conclusion, the G-protein-dependent signal transduction system itself is unaltered in cirrhosis. Hence, the cause of the hyporesponsiveness to some vasoconstrictors in cirrhosis appears to be a receptor-specific phenomenon localized upstream from the G-protein level.