Mycosis fungoides-derived exosomes promote cell motility and are enriched with microRNA-155 and microRNA-1246, and their plasma-cell-free expression may serve as a potential biomarker for disease burden.

BACKGROUND: Literature regarding exosomes as mediators in intercellular communication to promote progression in mycosis fungoides (MF) is lacking. OBJECTIVES: To characterize MF-derived exosomes and their involvement in the disease. METHODS: Exosomes were isolated by ultracentrifugation from cutaneous T-cell lymphoma (CTCL) cell lines, and from plasma of patients with MF and controls (healthy individuals). Exosomes were confirmed by electron microscopy, NanoSight and CD81 staining. Cell-line exosomes were profiled for microRNA array. Exosomal microRNA (exomiRNA) expression and uptake, and plasma-cell-free microRNA (cfmiRNA) were analysed by reverse-transcriptase quantitative polymerase chain reaction. Exosome uptake was monitored by fluorescent labelling and CD81 immunostaining. Migration was analysed by transwell migration assay. RESULTS: MyLa- and MJ-derived exosomes had a distinctive microRNA signature with abundant microRNA (miR)-155 and miR-1246. Both microRNAs were delivered into target cells, but only exomiR-155 was tested, demonstrating a migratory effect on target cells. Plasma levels of cfmiR-1246 were significantly highest in combined plaque/tumour MF, followed by patch MF, and were lowest in controls (plaque/tumour > patch > healthy), while cfmiR-155 was upregulated only in plaque/tumour MF vs. controls. Specifically, exomiR-1246 (and not exomiR-155) was higher in plasma of plaque/tumour MF than in healthy controls. Plasma exosomes from MF but not from controls increased cell migration. CONCLUSIONS: Our findings show that MF-derived exosomes promote cell motility and are enriched with miR-155, a well-known microRNA in MF, and miR-1246, not previously reported in MF. Based on their plasma expression we suggest that they may serve as potential biomarkers for tumour burden.

Three-dimensional concentration mapping of gases using a portable mass spectrometer system.

The visualization of hazardous gaseous emissions at volcanoes using in-situ mass spectrometry (MS) is a key step towards a better comprehension of the geophysical phenomena surrounding eruptive activity. In-situ data consisting of helium, carbon dioxide, sulfur dioxide, and other gas species, were acquired with a quadrupole based MS system. Global position systems (GPS) and MS data were plotted on ground imagery, topography, and remote sensing data collected by a host of instruments during the second Costa Rica Airborne Research and Technology Applications (CARTA) mission. This combination of gas and imaging data allowed three-dimensional (3D) visualization of the volcanic plume and the mapping of gas concentration at several volcanic structures and urban areas. This combined set of data has demonstrated a better tool to assess hazardous conditions by visualizing and modeling of possible scenarios of volcanic activity. The MS system is used for in-situ measurement of 3D gas concentrations at different volcanic locations with three different transportation platforms: aircraft, auto, and hand-carried. The demonstration for urban contamination mapping is also presented as another possible use for the MS system.

Collision induced ion ejection in an FTICR trapped-ion cell.

A collision algorithm was used with SimIon to evaluate collision-mediated ion ejection mechanisms in the ICR MS experiment. These mechanisms were characterized based on kinetic energy, ion mass, applied trapping potential, and collision gas mass. It was found that there are three collision-based energy regimes for ion loss from a trapped-ion cell. The first region is characterized by low initial cyclotron kinetic energy, a radial ejection mode, and a very high collision ratio (>100 collisions per ejection). The second region is characterized by a medium to high initial cyclotron kinetic energy leading to axial ejection at low collision ratio (1 to 10 collisions per ejection). The third region is characterized by a high initial cyclotron kinetic energy, a radial ejection mode, and a collision ratio of unity. It was also determined that there is a radial cyclotron mode limit, approximately 40% of the cell radius, after which an ion is ejected after a single collision. This has important consequences on the damping of the FTICR signal, various cooling techniques, ion activation techniques, and the remeasurement experiment.

Hyperfibrinogenemia. An important risk factor for vascular complications in diabetes.

OBJECTIVE: To evaluate the determinants of elevated fibrinogen levels and the impact of hyperfibrinogenemia on vascular complications in diabetes. RESEARCH DESIGN AND METHODS: Plasma fibrinogen, glucose, HbA1, and lipids were measured in 116 ambulatory type I and type II diabetic patients with (n = 59) or without (n = 57) clinical evidence of micro- or macrovascular complications. In 56 of these patients, factor VII activity and CRP also were measured. Univariate and multivariate data analyses were conducted. RESULTS: Overall mean +/- SE fibrinogen levels in patients (339 +/- 7.3 mg/dl) were elevated markedly compared with control subjects (248 +/- 9.1 mg/dl). Fibrinogen levels were elevated disproportionately in patients with type II diabetes (P less than 0.0001), hypertension (P = 0.0001), obesity (P less than 0.0001), and vascular complications (P less than 0.0001). Fibrinogen was correlated significantly with age (P less than 0.001), cholesterol (P = 0.002), CRP (P less than 0.001), and factor VII activity (P = 0.032), but not with plasma glucose, triglycerides, HDL cholesterol, or disease duration. Stepwise multiple regression analyses revealed that type II diabetes and presence of vascular complications were major determinants of fibrinogen. For vascular complications, fibrinogen emerged as one of only three independent predictors, the other two being diabetes duration and hypertension.

Unmanned aerial mass spectrometer systems for in-situ volcanic plume analysis.

Technology advances in the field of small, unmanned aerial vehicles and their integration with a variety of sensor packages and instruments, such as miniature mass spectrometers, have enhanced the possibilities and applications of what are now called unmanned aerial systems (UAS). With such technology, in situ and proximal remote sensing measurements of volcanic plumes are now possible without risking the lives of scientists and personnel in charge of close monitoring of volcanic activity. These methods provide unprecedented, and otherwise unobtainable, data very close in space and time to eruptions, to better understand the role of gas volatiles in magma and subsequent eruption products. Small mass spectrometers, together with the world's smallest turbo molecular pump, have being integrated into NASA and University of Costa Rica UAS platforms to be field-tested for in situ volcanic plume analysis, and in support of the calibration and validation of satellite-based remote sensing data. These new UAS-MS systems are combined with existing UAS flight-tested payloads and assets, such as temperature, pressure, relative humidity, SO2, H2S, CO2, GPS sensors, on-board data storage, and telemetry. Such payloads are capable of generating real time 3D concentration maps of the Turrialba volcano active plume in Costa Rica, while remote sensing data are simultaneously collected from the ASTER and OMI space-borne instruments for comparison. The primary goal is to improve the understanding of the chemical and physical properties of emissions for mitigation of local volcanic hazards, for the validation of species detection and abundance of retrievals based on remote sensing, and to validate transport models.

Coadministration of naproxen and low-dose methotrexate in patients with rheumatoid arthritis.

Fifteen patients (30 to 78 years of age) with diagnoses of rheumatoid arthritis were administered oral and intravenous methotrexate (15 mg), alone or with concomitant naproxen (1000 mg/day). Serial blood samples and urine were collected for 24 hours after the dose of methotrexate and were assayed for methotrexate by a specific radioenzymatic method. In twelve patients who completed the study, methotrexate systemic clearance was not statistically different with naproxen (103.3 +/- 35.0 ml/min) versus without naproxen (113.4 +/- 48.3 ml/min; p = 0.37). Oral clearance of methotrexate was not statistically different with naproxen (161.7 +/- 55.0 ml/min) versus without naproxen (176.7 +/- 68.3 ml/min; p = 0.14). Likewise, there was not a significant difference in methotrexate renal clearance or plasma protein binding with or without naproxen. No toxicity was observed when patients received methotrexate alone or with naproxen. This study indicates that concomitant naproxen does not abruptly alter the disposition of low-dose methotrexate in patients with rheumatoid arthritis who have normal renal function.

Central ring electrode for trapping and excitation/detection in Fourier transform ion cyclotron resonance mass spectrometry.

The use of a central trapping ring electrode for Fourier transform ion cyclotron resonance (FTICR) mass spectrometry is demonstrated. Ions are trapped with an oppositely biased static potential superimposed on both the excite and detect electrodes and maintained throughout the experiment, including the application of a dipolar rf excite waveform and the image current ion detection event. The use of a central trapping electrode for FTICR coupled with an open cell design retains the advantages of high ion throughput and gas conductance, while simplifying the electrode geometry and reducing the overall dimensions of the cell. This allows the central trapping electrode to be of utility in volume-limited vacuum chambers including FTICR instrument miniaturization. Presented here are the preliminary experimental results using the central trapping electrode as an FTICR cell in which the excitation and detection electrodes also create a trapping depression to constrain the z-axis motion of the ions. The cell overcomes the principle limitation of an earlier single trapping electrode design by producing a 91% effective potential well depth compared to 19% for the single trapping electrode and 33% for standard open cells. This allows the central trapping electrode configuration to achieve an order of magnitude improvement in ion capacity compared to more conventional open cell designs.

Evaluation of small mass spectrometer systems for permanent gas analysis.

This work is aimed at understanding the aspects of designing a miniature mass spectrometer (MS) system. Several types of small MS systems are evaluated and discussed, including linear quadrupole, quadrupole ion trap, time of flight, and sector. Analysis of hydrogen, helium, oxygen, and argon in a nitrogen background with the concentrations of the components of interest ranging from 0 to 5000 parts per million (ppm). The performance of each system in terms of accuracy, precision, limits of detection, response time, recovery time, scan rate, size, and weight is assessed. The relative accuracies of the systems varied from <1% to approximately 40% with an average below 10%. Relative precisions varied from 1% to 20%, with an average below 5%. The detection limits had a large distribution, ranging from 0.2 to 170 ppm. The systems had a diverse response time ranging from 4 to 210 s, as did the recovery time with a 6-to-210-s distribution. Most instruments had scan times near 1 s; however, one instrument exceeded 13 s. System weights varied from 9 to 52 kg and sizes ranged from 15 x 10(3) cm3 to 110 x 10(3) cm3. A performance scale is set up to rank each system, and an overall performance score is given to each system.

Second messenger regulation of mouse gonadotropin-releasing hormone gene expression in immortalized mouse hypothalamic GT1-3 cells.

Using a transgenic mouse derived GnRH expressing neuronal cell line, GT1-3, we studied the effects of activation of cAMP, Ca2+ and protein kinase C pathways by forskolin, ionomycin and the phorbol ester phorbol 12-myristate 13-acetate (PMA), respectively, upon gonadotropin-releasing hormone (GnRH) secretion, cellular peptide content, mRNA and RNA primary transcript levels. Forskolin, ionomycin and phorbol ester all caused an increase in GnRH secretion in GT1-3 cells in a time and dose-dependent manner during a short-term (1 h) static incubation. Prolonged treatment with forskolin (10 microM), ionomycin (1 microM) and PMA (10 nM) for 12 or 24 h resulted in significant decreases in GnRH mRNA levels. Time-course studies showed that the increases in GnRH secretion stimulated by forskolin, ionomycin and PMA were gradually attenuated over time in parallel with the decreases in mRNA expression. In contrast, there were only small and variable changes in the GnRH cellular content. Studies using a GnRH antagonist (100 microM) suggested that the released GnRH has a negative feedback effect on its own secretion. However, co-incubation with the GnRH antagonist did not alter the inhibitory effects on GnRH mRNA levels by the secretagogues. Further studies on the transcriptional effects of forskolin, ionomycin and PMA on GnRH gene expression in GT1-3 cells revealed that all three secretagogues suppressed GnRH RNA primary transcript levels, with forskolin having a slower time course of action. Thus, the inhibition of cytoplasmic GnRH mRNA, and presumably its synthesis, after 12-24 h of secretagogue treatment may be due at least in part to a suppression of GnRH gene transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Relapsing polychondritis: review of current status and case report,.

This communication has attempted to review the present state of published knowledge on the syndrome of relapsing polychondritis. Basic anatomic, physiologic, and biochemical changes in this disorder are summarized and the role of metabolic and immunologic alterations in the pathogenesis discussed. An additional case of relapsing polychondritis is reported, and the clinical features of this case, plus those of 131 previously reported, are reviewed with discussion of present day therapeutic experience and prognosis.

Histopathology of the donor gallbladder removed at orthotopic liver transplantation: correlation with graft function.

The histopathology of 50 consecutive donor gallbladders removed during orthotopic liver transplantation was reviewed and correlated with graft function. Multiple sections of the gallbladders were examined for the presence of mucosal congestion, hemorrhage, and necrosis, without prior knowledge of the clinical outcome. Each pathologic feature was graded as absent (0), involving less than 10% (1+), 10% to 50% (2+), or more than 50% (3+) of the histologically examined mucosa. Graft function was determined by two transplant surgeons, a poor diagnosis being worsening of liver function tests associated with declining mental status and resulting in immediate retransplantation or early postoperative death; all others were categorized as good. Of 39 patients with good graft function, 18 had normal donor gallbladders, 11 had congestion only, and 10 had hemorrhage and/or necrosis. Of 11 patients with poor graft function, eight had hemorrhage and/or necrosis (2+ in seven), three had congestion only, and none had a normal gallbladder mucosa. Congestion alone was found to be a poor predictor of graft damage. Presence of any grade of hemorrhage and/or necrosis in donor gallbladders as related to poor liver graft function had a sensitivity of 73%, a specificity of 74%, a positive predictive value of 44%, and a negative predictive value of 91%. When hemorrhage and/or necrosis of 2+ severity was separately grouped and correlated with poor graft function, the specificity rose to 97% and the positive predictive value to 88%, and the negative predictive value was similar at 90%. We conclude that donor gallbladders often show mucosal abnormalities consisting of varying degrees of congestion, hemorrhage, and necrosis. The finding of hemorrhage and/or necrosis affecting more than 10% of the mucosa appears to be a specific lesion of ischemic damage that correlates highly with poor liver graft function.

The T-test and clinical relevance. Is your beta error showing.

The t-test, as usually performed in the clinical laboratory for methods comparisons, does not consider the clinical relevance of the difference between the methods. Two procedures are presented which incorporate medical criteria into the t-test thereby making it a more relevant statistical tool. This is done by defining beta error in addition to alpha error. Since a positive action; i.e., adoption of the "test" methods, is taken when there is no difference between the "test" and reference methods, commission of a beta error has a potentially damaging effect. Therefore, evaluation of beta error is an appropriate part of methods comparison in the clinical laboratory.

Effect of drawing blood specimens proximal to an in-place but discontinued intravenous solution. Can blood be drawn above the site of a shut-off i.v.?

To assess the effects of drawing blood specimens from a site proximal to an intravenous (IV) infusion line, 24 volunteers were infused with approximately 30 mL of 5% (w/v) dextrose in 0.9% (w/v) NaCl solution. Specimens were drawn proximal to the IV line, while the solution was infusing, and at 1, 2, and 3 minutes after discontinuance of the infusion and assayed for glucose, sodium, chloride, and red blood cell (RBC) count. Control samples were obtained simultaneously from the opposite arm to determine any residual or dilutional effects from the infusing solution. Statistical analysis using the paired sample t-test showed no significant difference of RBCs between arms at or beyond 1 minute. Statistical power analysis reveals that there is a 95% level of certainty that there is less than 1% dilution of the test arm specimen. Analysis of sodium and chloride levels showed no contamination of the test arm specimen at 1 minute, but glucose concentrations still showed an average elevation over control of 5% at 3 minutes. The authors concluded that the drawing of blood specimens proximal to an IV infusion, 3 minutes after its discontinuance, has a clinically negligable dilutional effect but substances present at relatively high levels in the infused solution may still be detected.

Acute B-cell leukemia occurring with Hodgkin's disease.

The association of lymphoid neoplasms with Hodgkin's disease is being recognized with increasing frequency. These tumors often occur concurrently with Hodgkin's disease and are not as clearly related to therapy as are other secondary tumors. Presumably, the immune deficiency state found in Hodgkin's disease plays an important role. A patient with Hodgkin's disease and simultaneous acute lymphocytic leukemia is reported. The leukemia was B cell in type, a rare form that most likely is identical to acute lymphosarcoma cell leukemia of immature cell type. This case expands the spectrum of lymphoid neoplasms known to occur with Hodgkin's disease. The fact that the leukemia responded to therapy for Hogkin's disease suggests that some lymphoproliferative disorders discovered subsequent to therapy are not the result of therapy but recurrences of previously undetected and therapeutically suppressed disease.

An automatic leukocyte analyzer. Validity of its results.

A statistical study was undertaken to evaluate the Corning Larc Leukocyte Analyzer by comparing its results with those of 20 medical technologists with regard to reproducibility and degree of agreement. A special effort was made to use statistically quantitative comparisons, large numbers of samples, and a large group of technologists. The results of the Larc are frequently more reproducible than those of technologists, especially for bands and segmented neutrophils. Other comparative studies show that the Larc reports clinically equivalent proportions of the different cell types normal and abnormal. This is true for case by cases as well as cumulative comparisons. All known cases of leukemia was detected by the Larc during the study, although the number of samples from leukemia patients was small (11 of 700).

The white blood cell differential. Evaluation of rapid impression scanning versus the routine manual count.

A quick, one and a half-minute qualitative microscopic scan was investigated as an alternative approach to the more labor-intensive 100-cell differential white blood cell count. The scanning results of 400 randomly selected hospital cases were compared with the on-line results of the 100-cell counts. Additionally, 50 cases selected to have a high percentage of abnormal results were each scanned and manually counted by four different readers. The results indicate that the scanning differential is equivalent to the 100-cell manual count in the detection of the presence of abnormal cell types such as immature granulocytes and blasts. Its ability to properly estimate the relative proportions of normal cells, especially lymphocytes, however, does not appear as reliable as the manual count. Most importantly, the analysis demonstrates that the scanning differential count exhibits a set of advantages and disadvantages that is complementary to those of the "three-part differential" technic provided by the newer generation automated hematologic analyzers. The authors therefore propose that these two procedures used in combination offer a suitable alternative to the manual 100-cell differential count.

Immunophenotypic identification of Sezary cells in peripheral blood.

Despite anecdotal literature that Sezary cells express the CD4+ CD7- immunophenotype, no formal validation has been published establishing the use of this immunophenotype for clinical or experimental purposes. Consequently, the only method presently available for Sezary cell identification is nuclear contour analysis, a labor-intensive procedure not generally available at most major medical centers. In this study, the accuracy of CD4+ CD7- subset quantitation for the identification of Sezary cells was examined. The study found that the percentage of CD4+ CD7- cells is elevated in many Sezary syndrome/MF patients relative to normal, healthy individuals. In addition, CD4+ CD7- enumeration correlates with enumeration by nuclear contour analysis in most patients (11 of 15) with elevated CD4/CD8 ratios. The CD4+ CD7- subset also correlates with the expression of other aberrant immunophenotypes, such as CD3low or CD4low. Lastly, CD4+ CD7- subset quantitation correlates with the number of clonal T lymphocytes, as measured using V beta-specific T-cell receptor monoclonal antibodies. The study found this method to be exceptionally accurate, with two caveats: (1) the absence of an expanded CD4+ CD7- subset in patients with a normal CD4/CD8 ratio is uninformative; and (2) in approximately 25% of patients with an elevated CD4/CD8 ratio, the Sezary cells are CD7+.

The preoperative bleeding time test lacks clinical benefit: College of American Pathologists' and American Society of Clinical Pathologists' position article.

The major conclusions of this position article are as follows: (1) In the absence of a history of a bleeding disorder, the bleeding time is not a useful predictor of the risk of hemorrhage associated with surgical procedures. (2) A normal bleeding time does not exclude the possibility of excessive hemorrhage associated with invasive procedures. (3) The bleeding time cannot be used to reliably identify patients who may have recently ingested aspirin or nonsteroidal anti-inflammatory agents or those who have a platelet defect attributable to these drugs. The best preoperative screen to predict bleeding continues to be a carefully conducted clinical history that includes family and previous dental, obstetric, surgical, traumatic injury, transfusion, and drug histories. A history suggesting a possible bleeding disorder may require further evaluation; such an evaluation may include performance of the bleeding time test, as well as a determination of the platelet count, the prothrombin time, and the activated partial thromboplastin time. In the absence of a history of excessive bleeding, the bleeding time fails as a screening test and is, therefore, not indicated as a routine preoperative test.

Effect of anticoagulation protocol on outcome in patients undergoing CABG with heparin-bonded cardiopulmonary bypass circuits.

BACKGROUND: We have demonstrated that the use of heparin-bonded cardiopulmonary bypass circuits (HBCs) combined with a lower anticoagulation protocol as an adjunct to an integrated blood conservation strategy decreases the incidence and magnitude of homologous transfusion and improves clinical outcome in patients undergoing primary coronary artery bypass grafting. It is not known whether it is the lower anticoagulation protocol that influences outcome in patients treated with HBCs. Furthermore, the thrombogenic risk of using lower anticoagulation with HBCs still is debated. METHODS: To answer these questions, a prospective randomized study was conducted in which 244 patients undergoing primary coronary artery bypass grafting were treated with HBCs and randomized to undergo either a full (activated clotting time, > 450 seconds) or a lower (activated clotting time, > 250 seconds) anticoagulation protocol. In addition to clinical outcome, levels of thrombin generation markers during and after cardiopulmonary bypass were assessed in a consecutive subset of 58 patients (full anticoagulation profile = 28, lower anticoagulation profile = 30) by measuring thrombin-antithrombin complexes and prothrombin fragment 1.2. Levels of these markers also were correlated with the activated clotting time during cardiopulmonary bypass. RESULTS: Preoperative and intraoperative risk profiles and other characteristics were similar in both groups, with more than 60% of patients undergoing nonelective operation. Compared with the full anticoagulation protocol group, patients in the lower anticoagulation protocol group were less likely to require blood products (24.2% versus 35.8%, respectively; p = 0.047) and received substantially fewer homologous donor units (0.50 +/- 0.92 versus 1.08 +/- 2.10 U, respectively; p = 0.005). Clinical outcomes were uniformly outstanding (but similar) in both treatment groups, with a modest reduction in the length of the hospital stay in the lower anticoagulation protocol group (5.26 +/- 1.23 versus 5.63 +/- 1.73 days, respectively; p = 0.05). The use of HBCs with a lower anticoagulation protocol was not associated with any adverse clinical events. Thrombin generation increased during cardiopulmonary bypass in both treatment groups, but was unrelated to the anticoagulation protocol or the activated clotting time (r2 = 0.03). No differences between the full and lower anticoagulation protocol groups were noted in the number of microemboli detected by transcranial Doppler analyses during cardiopulmonary bypass (n = 40) or in the postoperative neurologic and neuropsychologic outcomes (n = 30). CONCLUSIONS: This study definitively demonstrates that, when used appropriately, patients who are treated with HBCs and a lower anticoagulation protocol have a lower incidence and magnitude of homologous transfusion and are not at any added risk for clinical, hematologic (thrombin-antithrombin complex and fragment 1.2 measurements), or microscopic (transcranial Doppler analyses) thromboembolic complications or for neurologic or neuropsychologic deficits.

Response of protein C and protein C inhibitor to warfarin therapy in patient with combined deficiency of Factors V and VIII.

The role of Protein C in combined factor V/VIII deficiency was examined by reducing the Protein C concentration using warfarin therapy in a patient with the combined deficiency. The factor VIII deficiency was like Hemophilia-A, with deficiency of VIII:C and VIII:C(Ag), but normal VIIIR:Ag and VIIIR:cof. The factor V deficiency was due to loss of the V antigen. During warfarin therapy the Protein C level was reduced, but concentrations of factors V and VIII did not change. Protein C Inhibitor was normal throughout. Thus combined factor V/VIII deficiency is not related to Protein C levels.