Phenotypic and functional characterisation of synovial fluid-derived neutrophils in knee osteoarthritis and knee infection.

OBJECTIVE: An increase in the number of neutrophils (NEUs) has long been associated with infections in the knee joints; however, their impact on knee osteoarthritis (KOA) pathophysiology remains largely unexplored. DESIGN: This study compared the phenotypic and functional characteristics of synovial fluid (SF)-derived NEUs in KOA and knee infection (INF). RESULTS: KOA NEUs were characterised by a lower expression of CD11b, CD54, and CD64 and higher expression of CD62L, TLR2, and TLR4 compared with INF NEUs. Except for CCL2, lower levels of inflammatory mediators and proteases were detected in KOA SF than in INF SF. Functionally, KOA NEUs displayed increased reactive oxygen species production and phagocytic activity compared with INF NEUs. Moreover, KOA and INF NEUs differed in cell sizes, histological characteristics of the surrounding synovial tissues, and their effects on the endothelial cells assessed by human umbilical vein endothelial cells. When KOA patients were subdivided based on the SF NEU abundance, patients with high NEUs (10%-60%) were characterised by i) elevated SF protein levels of TNF-α, IL-1RA, MMP-9, sTREM-1, VILIP-1 and ii) lower CD54, CD64, TLR2 and TLR4 expression compared to patients with low NEUs (<10%). Analysis of paired SF samples suggests that low or high NEU percentages, respectively, persist throughout the course of disease. CONCLUSIONS: Our findings suggest that NEU may play a significant role in KOA pathophysiology. Further studies should explore the mechanisms that contribute to the increased number of NEUs in SF and the clinical consequences of neutrophilic phenotype in KOA.

[Ectopic duodenal varices as a cause of difficult diagnosis of bleeding in extrahepatic portal hypertension]. / Varikoznye veny dvenadtsatiperstnoi kishki kak prichina «trudno¼ diagnostiruemogo krovotecheniya pri vnepechenochnoi portal'noi gipertenzii.

Bleeding from ectopic varicose veins is a rare life-threatening cause of upper gastrointestinal hemorrhage. Alberti first described duodenal varices in 1931. According to the literature, incidence of duodenal varicose veins in patients with portal hypertension is 1-3% of all varicose veins. Bleeding from duodenal varices makes up 17% of all bleedings from other ectopic varices. Mortality in these patients may be up to 40%. The causes are delayed diagnosis, technical difficulties in endoscopic therapeutic procedures (sclerotherapy, endoscopic ligation), as well as ineffective Blackmore tube for hemorrhage in distal stomach and bowel. We report a rare case of upper gastrointestinal bleeding from ectopic duodenal varices.

[Self-expanding nitinol stents for bleeding from esophageal varices in patients with portal hypertension]. / Lechenie krovotecheniya iz varikozno rasshirennykh ven pishchevoda nitinolovym stentom u bol'nykh portal'noi gipertenziei.

Blakemore probe-obturator was previously preferable for primary hemostasis in patients with bleeding from esophageal varices. Currently, Danis self-expanding nitinol stent became an effective alternative. According to some manufacturers, Danis stent has some advantages over balloon tamponade. We report implantation of nitinol stent for hemostasis in a patient with multiple recurrent bleeding and ineffective endoscopic manipulations. A method of stent fixation for prevention of distal migration as the most common complication is described.

Gender-related differences observed among immune cells in synovial fluid in knee osteoarthritis.

OBJECTIVE: There is no existing comprehensive report on the cellular composition of synovial fluids (SFs) from knee osteoarthritis (OA). We therefore aimed to characterise the immune cell composition in SFs from knee OA (KOA) and in subgroups according to gender. DESIGN: The immunophenotyping of monocyte/macrophage lineage cells, T and B cells, NK cells, neutrophils, dendritic and mast cells (MC) present in SFs from 53 patients (24 males/29 females) with KOA was performed using 6-colour flow cytometry. RESULTS: SFs from patients with OA contained 90% hematopoietic cells. Lymphocytes were the predominant cell population (44.8%) in the SFs of OA patients, with CD4+ T lymphocytes being more prevalent than CD8+ T cells (CD4+/CD8+ ratio = 1.3). Within the monocyte/macrophage lineage gating, monocytes accounted for 33.9%, macrophages 14.8%, myeloid dendritic cells 16.4%. The rest of the hematopoietic cells were comprised of neutrophils (8%), NK cells (3.8%), T regulatory cells (1.2%), plasmacytoid dendritic cells (1.1%), mast cells (0.3%). In OA females, a higher percentage of CD4+ T cells (P = 0.023), macrophages (P = 0.012), and a lower percentage of monocytes (P = 0.008) and CD8+ T cells (P = 0.002) were detected in comparison to OA males. CONCLUSIONS: Based on the immune cell composition of SFs, data mining analysis revealed distinct phenotypes (monocyte- and lymphocyte-predominant) within each gender group. This first study on the cellular complexity of SFs in KOA showed marked differences between male and female patients. The findings give a rational starting point for patient stratification according to their phenotypes, as is required for phenotype-specific treatment strategies.

ANTIPHOSPHOLIPID SYNDROME AND MONOCYTES: NEW ASPECTS.

After discovery of antiphospholipid syndrome (APS) our understanding of molecular mechanisms of living matter has become more sophisticated and on this way monocytes has become crucial player, particularly in pathogenesis of APS. Thrombotic and non-thrombotic complications of APS could be explained by monocytes' activation too. But mechanisms underlying their activation are poorly investigated. So we aimed to determine transcriptional activity of monocytes after exposing them to low concentrations of lipopolysaccharide (LPS) and LPS+ATP using comparative of RT-PCR. Our study included eleven women suffering from recurrent miscarriages and APS (mean age 30±5,6 years). Nine healthy women (mean age of 29±8,5 years) without a positive family history of APS, autoimmune diseases and thrombosis were chosen as a control group. The results showed increasing levels of TLR2, IL-23, CCL2, CXCL10, IL-1ß and IL-6 in APS cells, while in healthy cells LPS resulted in IL-6 and STAT3 elevated mRNAs. Double stimulation of APS cells resulted in decreased mRNA levels of CCL-2, IL-1ß, and mRNA NLRP3 in healthy cells. At the same time TLR2 mRNAs were elevated in both groups after double stimulation. Thus increased sensitivity of APS cells to LPS may contribute to thrombus formation. Low concentration of ATP diminishes LPS-induced inflammatory state of APS monocytes, which might be one of potential regulatory mechanisms.

Isolation of Induced Pluripotent Cells from Stromal Liver Cells of Patients with Alcoholic Cirrhosis.

Stromal liver cells obtained from liver biopsy specimens of a patient with alcoholic cirrhosis can proliferate for a long time in culture passing more than 30 passages. In the course of culturing from early to late passages, acceleration of cell proliferation, decrease of the expression of some markers, and loss of hepatogenic differentiation potential were observed. On passage 30, induced pluripotent stem cells were obtained from these cells and comparative analysis of adipogenic and hepatic differentiation potencies of these cells and original liver stromal cells was performed. Induced pluripotent stem cells differentiated into both directions more efficiently and more rapidly than initial cells. Under conditions of hepatic differentiation, liver stromal cells started to express markers of definitive endoderm, but not markers of immature/mature hepatocytes, whereas induced pluripotent stem cells consistently expressed markers of definitive endoderm, immature/mature hepatocytes.

[The hepatic differentiation of adult and fetal liver stromal cells in vitro]. / Gepatogennaia differentsirovka stromal'nykh kletok vzrosloi i fetal'noi pecheni in vitro.

The liver has a marked capacity for regeneration. In most cases the liver regeneration is determined by hepatocytes. The regenerative capacity of hepatocytes is significantly reduced in acute or chronic damage. In particular, repair mechanisms are not activated in patients with alcoholic cirrhosis. Organ transplantation or advanced methods of regenerative medicine can help such patients. The promising results were obtained in clinical trials involving patients with various forms of liver disease who received transplantation of autologous bone marrow stem cells. However, to improve the effectiveness of such treatment it is necessary to search for more optimal sources of progenitor cells, as well as to evaluate the possibility of using descendants of these cells differentiated in vitro. In this study we isolated stromal cells from the liver biopsies of three patients with alcoholic cirrhosis, conducted their morphological and phenotypic analysis, and evaluated the hepatic potential of these cells in vitro. The stromal cells isolated from fetal liver were used for comparison. The results of this can serve as a basis for the development of a new method for the treatment of end-stage liver disease. The stromal cells isolated from the liver biopsies for a long time proliferate in a culture and this which makes it possible to expand them to large amounts for subsequent differentiation into hepatocyte-like cells and autologous transplantation.

Mesenchymal-Epithelial Transition in Culture of Stromal Progenitor Cells Isolated from the Liver of a Patient with Alcoholic Cirrhosis.

The cells isolated from biopsy specimen of a patient with alcoholic liver cirrhosis and cultured under standard conditions for obtaining stromal cell culture clearly diverged during early passages into two morphologically and phenotypically different subtypes: epithelial and mesenchymal. Mesenchymal cells expressed CD90 and CD44 and epithelial cells expressed CD166, CD227, and hepatocyte growth factor receptor Met. Starting from passage 6, the culture underwent spontaneous morphological changes and by passages 8-10 contained only epithelium-like cells. CD90 and CD44 expression disappeared, CD166 and CD227 expression remained unchanged, and Met expression increased. A small fraction of cells expressed GATA-4, HNF3ß, HNF1α, and HNF4α. After addition of inducers of hepatogeneic differentiation, the cells started producing albumin.

Transcriptional activity of neutrophils exposed to high doses of colchicine: short communication.

Colchicine is an antimitotic drug which binds to tubulin and at high doses results in cytoskeleton disruption. Colchicine is believed to be an anti-inflammatory agent, though its modulatory effects on the level and transcriptional activity of genes is still a matter of debate. There is growing evidence that alterations in the cytoskeleton exert specific effects on the expression of various genes. This study was undertaken to analyze whether disrupting the microtubule cytoskeleton by colchicine modulates transcriptional levels of MEFV, NF-κB p65, NLRP3, HMGB1, and caspase-3 in neutrophils from patients with familial Mediterranean fever (FMF) and healthy subjects. In the present study, colchicine caused increased expression of NLRP3 (p=0.007) and MEFV (p=0.03), but had no effect on caspase-3, NF-κB p65 and HMGB1 genes in healthy neutrophils. FMF neutrophils were less responsive to the drug treatment. This study supports the hypothesis that, being an anti-inflammatory agent, colchicine at relatively high concentrations might lead to the activation of pro-inflammatory signalling pathways in neutrophils.

Colchicine modulates expression of pro-inflammatory genes in neutrophils from patients with familial Mediterranean fever and healthy subjects.

Colchicine (Col) is a microtubule depolymerizing drug, widely used for treatment of familial Mediterranean fever (FMF). Mechanisms by which Col exerts its beneficial effects are not yet completely understood, especially with respect to gene expression in polymorphonuclear neutrophils (PMNs), the main effector cells in acute inflammatory attacks of FMF. This study was, therefore, designed to elucidate possible modulatory effect of Col on expression of inflammation-related genes in circulating PMNs from 16 FMF patients in the remission period and 11 healthy subjects. In vitro effect of Col exposure (1 microg/ml) on expression of 8 selected genes was examined using quantitative real-time RT-PCR. Col up-regulated expression of IL-8 and IL-1beta genes in FMF (13-fold and 2.7-fold, p less than 0.05, respectively) and healthy (3-fold and 6.5-fold, p less than 0.05, respectively) PMNs, and down-regulated caspase-1 in FMF neutrophils (3-fold, p less than 0.05). In FMF PMNs treated with Col mRNAs of IL-8 (51-fold, p less than 0.01) and c-FOS (7-fold, p less than 0.05) transcripts were elevated compared to those from healthy subjects. By contrast, caspase-1 mRNA was decreased in FMF neutrophils compared to healthy cells (1.6-fold, p less than 0.05). Hereby, we provide evidence that, at least in vitro, Col displays pro-inflammatory potential in respect to IL-1beta and IL-8 genes. At the same time, our findings implicate suppression of caspase-1 expression by Col as a potential mechanism for its effects in FMF treatment.

Wetting of a drop on a sphere.

In this work, the equilibrium morphology of a drop on a sphere is analyzed as a function of the contact angle and drop volume experimentally and with analytical effective interfacial energy calculations. Experimentally, a drop on a sphere geometry is realized in an oil bath by placing a water drop on a sphere coated with a dielectric, of which the radii of curvature are comparable with that of the drop. Electrowetting (EW) is used to change the contact angle of the water drop on the sphere. To validate the applicability of EW and the Lippman-Young equation on nonflat surfaces, we systematically investigate the response of the contact angle to the applied voltage (EW response) for various drop volumes and compared the results with the case of a planar surface. The effective interfacial energy of two competing morphologies, namely, the spherically symmetric "completely engulfing" and "partially engulfing" morphologies are calculated analytically. The analytical calculations are then compared to the experimental results to confirm which morphology is energetically more favored for a given contact angle and drop volume. Our findings indicate that the "partially engulfing" morphology is always the energetically more favorable morphology.

Electrical switching of wetting states on superhydrophobic surfaces: a route towards reversible Cassie-to-Wenzel transitions.

We demonstrate that the equilibrium shape of the composite interface between superhydrophobic surfaces and drops in the superhydrophobic Cassie state under electrowetting is determined by the balance of the Maxwell stress and the Laplace pressure. Energy barriers due to pinning of contact lines at the edges of the hydrophobic pillars control the transition from the Cassie to the Wenzel state. Barriers due to the narrow gap between adjacent pillars control the lateral propagation of the Wenzel state. We demonstrate how reversible switching between the two wetting states can be achieved locally using suitable surface and electrode geometries.

Laparoscopic intracorporal colorectal sutured anastomosis using the Radius Surgical System in a phantom model.

BACKGROUND: The Radius Surgical System (RSS) is a manipulator with additional degrees of freedom to enhance the dexterity of laparoscopic suturing. Our aim was to determine the feasibility and potentially added value of laparoscopic intracorporal sutured colorectal anastomosis (RSS) compared with suturing with conventional laparoscopic instruments (CLI). METHODS: A total of 72 colorectal anastomoses and 30 single sutures using RSS and CLI were performed in the study. The experiment was divided as follows: One surgeon performed 40 colorectal anastomoses using RSS to assess the learning curve and the feasibility of the technique; The same surgeon performed 10 additional colorectal anastomoses with CLI which were then compared to the last 10 cases of the 40 anastomoses with RSS; Fifteen single sutures in the horizontal plane with RSS and 15 with CLI between two segments of colon were performed to compare the traction force to disrupt the suture; Twelve anastomoses were performed by the other three participants to evaluate ergonomy. RESULTS: Three leakages (7.5%) were found in the 40 anastomoses with RSS but none after the eighth case. There was no stenosis. The mean time for the anastomoses once the learning curve was achieved was 32.7 min. After 21 anastomoses with RSS there was no improvement in the operating time. The quality of the suture was superior with RSS, with a larger anastomosis diameter, higher bursting pressure, and fewer suturing failures being found. The RSS suture withstood a higher traction force. The participants showed more discomfort suturing with CLI. CONCLUSION: This study demonstrated the feasibility of laparoscopic colorectal anastomosis using RSS. Anastomosis with RSS was shown to be safer. The three participants evaluating ergonomy reflected less discomfort in hand/wrist using RSS. Others ergonomic problems were comparable to CLI.

Direct observation of a nonequilibrium electro-osmotic instability.

We present a visualization of the predicted instability in ionic conduction from a binary electrolyte into a charge selective solid. This instability develops when a voltage greater than critical is applied to a thin layer of copper sulfate flanked by a copper anode and a cation selective membrane. The current-voltage dependence exhibits a saturation at the limiting current. With a further increase of voltage, the current increases, marking the transition to the overlimiting conductance. This transition is mediated by the appearing vortical flow that increases with the applied voltage.

Cytokine profile of Armenian patients with Familial Mediterranean fever.

OBJECTIVES: Despite the well-defined genetics of FMF, limited information is available regarding the regulation of inflammation by cytokines. DESIGN AND METHODS: The levels of systemic cytokines and other markers of inflammation in FMF patients and control were measured by ELISA and Cytometric Bead Array (CBA). RESULTS: In FMF attack the levels of IL-6, IL-10, IL-17, TGF-beta, CRP, and sIL-2R were significantly different from the norm and FMF remission. CONCLUSIONS: Inflammation in FMF involves Treg and Th17 lineages.

Precision in stitches: Radius Surgical System.

BACKGROUND: The Radius Surgical System is a manual manipulator with two additional degrees of freedom compared with conventional laparoscopic instruments (CLIs). This study aimed to compare the performance of laparoscopic suturing tasks with the use of the Radius Surgical System and CLIs, respectively. METHODS: Five experienced laparoscopic surgeons performed laparoscopic surgical tasks in a training box. The tasks consisted of knot-tying, suturing, and needle control tasks. The needle control task was performed to evaluate the precision of the needle drive by analysis of the needle exit point on a suture pad. In the knot-tying and suturing tasks, required time and accuracy value were measured. Needle control tasks were performed on three different angulations of plane. The angles between the instrument plane and the target plane (AIT) were 30 degrees, 60 degrees, and 90 degrees. The distance of the exit point to the center of the target field, the number of actions needed to fulfill a single task, and the required time were recorded and analyzed. RESULTS: In the knot-tying and frontal suturing tasks, there were no significant differences between the two groups. In the sagittal suturing task, the required time in the Radius group was significantly shorter than in the CLI group. In the needle control tasks on 30 degree and 60 degree AIT, the distance was significantly shorter in the Radius group than in the CLI group. There were no significant differences in the number of actions or the required time. In the frontal and sagittal needle control task on 90 degree AIT, the distance was significantly shorter in the Radius group than in the CLI group. The number of actions and the required time were significantly less in the Radius group than in the CLI group. CONCLUSIONS: The two additional degrees of freedom contributed to accurate and controlled needle guidance, especially in difficult spatial situations.

Ergonomics with the use of curved versus straight laparoscopic graspers during rectosigmoid resection: results of a multiprofile comparative study.

BACKGROUND: A detailed ergonomic comparison of motions and muscular activity in the left upper extremity using a laparoscopic straight or curved grasper in rectosigmoid resection is presented. METHODS: The study had two parts: experimental and clinical. In the experiment part, 30 laparoscopic sigmoid resections were performed under animal organ phantom conditions. The operations were divided into three groups according to instrument and trocar position. Group 1 (n = 10) underwent operations performed with a curved grasper in the excentral trocar position (in relation to the telescope trocar), with the left-hand curved grasper placed in the right flank and the right hand instrument in the right lower quadrant. In group 2 (n = 10), straight forceps were used in the excentral trocar position. Group 3 (n = 10) underwent laparoscopic sigmoid resection performed with a straight grasper in the central position (in relation to the telescope trocar), with the instruments placed at both sides of the lower abdomen. To measure ergonomic aspects during rectosigmoid resection, several overview video cameras, surface electromyography (EMG), an ultrasound tracking system (UTS), and a questionnaire were used. In the clinical part of the study, laparoscopic rectosigmoid resections (n = 5) were performed using a curved instrument in the excentral trocar position. The surgeon's left-hand movement and body posture were recorded for further analysis. RESULTS: The curved grasper required the fewest contractions (group 1) of the measured muscles. A comparison of the UTS analysis in the experimental part of the study and the video analysis in the clinical part showed economy of movements in group 1. According to subjective estimation, both physical activity and mental stress remain at the lowest level when the excentral trocar position is used (groups 1 and 2). CONCLUSIONS: The combination of the curved grasper and the excentral trocar position (in relation to the telescope trocar) is, according to our examinations, the best ergonomic adjustment for laparoscopic rectosigmoid surgery.

Study of ethidium bromide interaction peculiarities with DNA.

The helix-coil transition of DNA-ethidium bromide complexes in an interval of ionic strength of 2.0 x 10(-3) M < or = muNa+ < or = 2.0 x 10(-2) M has been investigated. It has been revealed that at the certain high ligand-DNA ratios (r(b)) the transition interval of the complex--(deltaT) becomes equal to that of DNA itself (deltaTo). It has been shown that the values of r(b) at which delta deltaT=deltaT-deltaT0=0 depends on ionic strength of a solution. Further increasing of ligand concentration leads to its conversion from stabilizer into the destabilizer of the double-stranded DNA.