Detection of leukocytes in contact with the vessel wall from in vivo microscope recordings using a neural network

Leukocytes play an important role in the host defense as they may travel from the blood stream into the tissue in reacting to inflammatory stimuli. The leukocyte-vessel wall interactions are studied in post capillary vessels by intravital video microscopy during in vivo animal experiments. Sequences of video images are obtained and digitized with a frame grabber. A method for automatic detection and characterization of leukocytes in the video images is developed. Individual leukocytes are detected using a neural network that is trained with synthetic leukocyte images generated using a novel stochastic model. This model makes it feasible to generate images of leukocytes with different shapes and sizes under various lighting conditions. Experiments indicate that neural networks trained with the synthetic leukocyte images perform better than networks trained with images of manually detected leukocytes. The best performing neural network trained with synthetic leukocyte images resulted in an 18% larger area under the ROC curve than the best performing neural network trained with manually detected leukocytes.     

Phosphorus speciation in manure and manure-amended soils using XANES spectroscopy

Previous studies suggested an increase in the proportion of calcium phosphates (CaP) of the total phosphorus (P) pool in soils with a long-term poultry manure application history versus those with no or limited application histories. To understand and predict long-term P accumulation and release dynamics in these highly amended soils, it is important to understand what specific P species are being formed. We assessed forms of CaP formed in poultry manure and originally acidic soil in response to different lengths of mostly poultry manure applications using P K-edge X-ray absorption near-edge structure (XANES) spectroscopy. Phosphorus K-edge XANES spectra of poultry manure showed no evidences of crystalline P minerals but dominance of soluble CaP species and free and weakly bound phosphates (aqueous phosphate and phosphate adsorbed on soil minerals). Phosphate in an unamended neighboring forest soil (pH 4.3) was mainly associated with iron (Fe) compounds such as strengite and Fe-oxides. Soils with a short-term manure history contained both Fe-associated phosphates and soluble CaP species such as dibasic calcium phosphate (DCP) and amorphous calcium phosphate (ACP). Long-term manure application resulted in a dominance of CaP forms confirming our earlier results obtained with sequential extractions, and a transformation from soluble to more stable CaP species such as beta-tricalcium calcium phosphate (TCP). Even after long-term manure application (> 25 yr and total P in soil up to 13,307 mg kg(-1)), however, none of the manure-amended soils showed the presence of crystalline CaP. With a reduction or elimination of poultry manure application to naturally acidic soils, the pH of the soil is likely to decrease, thereby increasing the solubility of Ca-bonded inorganic P minerals. Maintaining a high pH is therefore an important strategy to minimize P leaching in these soils.     

On-chip high voltage charge pump in standard low voltage CMOS process

An on-chip high voltage tolerant 4VDD charge pump with symmetrical architecture in a standard low voltage 1.8 V 0.18 /spl mu/m CMOS process is presented. For a 250 k/spl Omega/ load, circuit efficiency of the charge pump is approximately 71%. All the MOS transistors satisfy typical voltage stress related reliability requirements for standard low voltage CMOS devices.     

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Distribution of protein phosphatases type 1 and 2A in RINm5F cells

In the insulin producing cell line RINm5F distribution of serine/threonine specific protein phosphatases type 1 (PP1) and 2A (PP2A) was studied. Using different agents which inhibit or stimulate PP1 and PP2A we found that in membrane and nuclear fractions phosphatase activity was inhibited by okadaic acid (OA), protamine, heparin, and inhibitor-2 in a concentration-dependent manner. C2-ceramide had no effect. In the cytosolic fraction the inhibitory effect of okadaic acid was tenfold higher. Protamine stimulated phosphatase activity at low concentrations and became inhibitory at higher concentrations. Inhibitor-2 and heparin caused a decrease in phosphatase activity whereas C2-ceramide led to a slight activation. The data suggest that in membrane and nuclear fractions of RINmSF cells predominantly PP1 is present, whereas in the cytosol PP1 as well as PP2A can be detected.     

Terlipressin (glypressin) versus somatostatin in the treatment of bleeding esophageal varices--final report of a placebo-controlled, double-blind study

One hundred and six episodes of bleeding from esophageal or gastric varices in 72 patients with cirrhosis of the liver were randomized to treatment either with intravenous terlipressin 2 mg initially and 1 mg every four hours for 24 hours together with bolus injection and continuous infusion of placebo, or with somatostatin 250 micrograms as a bolus and continuous infusion of 250 micrograms/h somatostatin for 24 hours and placebo injections. Standard treatment with transfusions, fluid and electrolyte correction, and lactulose was administered in both groups. In the terlipressin group, 48 out of 53 bleeding episodes (91%) and in the somatostatin group 43 out of 53 bleeds (81%) were initially stopped by the vasoactive drugs. Four of the five bleeds not arrested by terlipressin, and nine of the ten bleeds not arrested by somatostatin, were stopped by balloon tamponade. In one patient in each group variceal bleeding could not be stopped initially, and both patients died. The failure rate of the vasoactive treatment alone, including rebleeds within the study period, was 17% in the terlipressin, and 28% in the somatostatin, group. The initial hemostasis, including balloon tamponade, were 98%, and the definitive bleeding control rates were 89% in both groups. The hospital mortality rate was 21% (11/53) in the terlipressin, and 21% (11/53) in the somatostatin, group. Blood transfusions and duration of bleeding did not differ significantly. The study indicates that a large proportion of bleeds from esophageal and fundic varices can be stopped initially (86%) and definitively controlled (77%) by vasoactive drugs alone.     

19-nor vitamin-D analogs: a new class of potent inhibitors of proliferation and inducers of differentiation of human myeloid leukemia cell lines

We have studied the in vitro biological activities and mechanisms of action of 1,25-dihydroxyvitamin D3 (1,25D3) and nine potent 1,25D3 analogs on proliferation and differentiation of myeloid leukemia cell lines (HL-60, retinoic acid-resistant HL-60 [RA-res HL-60], NB4 and Kasumi-1). The common novel structural motiff for almost all the analogs included removal of C-19 (19-nor); each also had unsaturation of the side chain. All the compounds were potent; for example, the concentration of analogs producing a 50% clonal inhibition (ED50) ranged between 1 x 10(-9) to 4 x 10(-11) mol/L when using the HL-60 cell line. The most active compound [1, 25(OH)2-16,23E-diene-26-trifluoro-19-nor-cholecalciferol (Ro 25-9716)] had an ED50 of 4 x 10(-11) mol/L; in contrast, the 1,25D3 produced an ED50 of 10(-9) mol/L with the HL-60 target cells. Ro 25-9716 (10(-9) mol/L, 3 days) was a strong inducer of myeloid differentiation because it caused 92% of the HL-60 cells to express CD11b and 75% of these cells to reduce nitroblue tetrazolium (NBT). This compound (10(-8) mol/L, 4 days) also caused HL-60 cells to arrest in the G1 phase of the cell cycle (88% cells in G1 v 48% of the untreated control cells). The p27(kip-1), a cyclin-dependent kinase inhibitor which is important in blocking the cell cycle, was induced more quickly and potently by Ro 25-9716 (10(-7) mol/L, 0 to 5 days) than by 1,25D3, suggesting a possible mechanism by which these analogs inhibit proliferation of leukemic growth. The NB4 promyelocytic leukemia cells cultured with the Ro 25-9716 were also inhibited in their clonal proliferation (ED50, 5 x 10(-11) mol/L) and their expression of CD11b was enhanced (80% positive [10(-9) mol/L, 4 days] v 27% untreated NB4 cells). Moreover, the combination of Ro 25-9716 (10(-9) mol/L) and all-trans retinoic acid (ATRA, 10(-7) mol/L) induced 92% of the NB4 cells to reduce NBT, whereas only 26% of the cells became NBT positive after a similar exposure to the combination of 1,25D3 and ATRA. Surprisingly, Ro 25-9716 also inhibited the clonal growth of poorly differentiated leukemia cell lines (RA-res HL-60 [ED50, 4 x 10(-9) mol/L] and Kasumi-1 [ED50, 5 x 10(-10) mol/L]). For HL-60 cells, Ro 25-9716 markedly decreased the percent of the cells in S phase of the cell cycle and increased the expression of the cyclin-dependent kinase inhibitor, p27(kip-1). In summary, 19-nor vitamin D3 compounds strongly induced differentiation and inhibited clonal proliferation of various myeloid leukemia cell lines, suggesting a therapeutic niche for their use in myeloid leukemia.     

Phosphoinositide 3-kinase induces scattering and tubulogenesis in epithelial cells through a novel pathway

Hepatocyte growth factor/scatter factor (HGF/SF) treatment of the Madin-Darby canine kidney epithelial cell line causes scattering of cells grown in monolayer culture and the formation of branching tubules by cells grown in collagen gels. HGF/SF causes prolonged activation of both the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase 2 (ERK2) and the phosphoinositide 3-OH kinase (PI 3-kinase) target protein kinase B (PKB)/Akt; inhibition of either the MAP kinase pathway by the MAP kinase/ERK kinase inhibitor PD98059 or the PI 3-kinase pathway by LY294002 blocks HGF/SF induction of scattering, although in morphologically distinct ways. Expression of constitutively activated PI 3-kinase, Ras, or R-Ras will cause scattering, but activated Raf will not, indicating that activation of the MAP kinase pathway is not sufficient for this response. Downstream of PI 3-kinase, activated PKB/Akt and Rac are both unable to induce scattering, implicating a novel pathway. Scattering induced by Ras or PI 3-kinase is sensitive to PD98059, as well as to LY294002, suggesting that basal MAP kinase activity is required, but not sufficient, for the scattering response. Induction of MDCK cell tubulogenesis in collagen gels by HGF/SF is inhibited by PD98059; expression of activated Ras and Raf causes disorganized growth in this system, but activated PI 3-kinase or R-Ras causes branching tubule formation similar to that seen with HGF/SF treatment. These data indicate that multiple signaling pathways acting downstream of Met and Ras are needed for these morphological effects; scattering is induced primarily by the PI 3-kinase pathway, which acts through effectors other than PKB/Akt or Rac and requires at least basal MAP kinase function. Elevated PI 3-kinase activity induces tubulogenesis, but total inhibition and excess activation of the MAP kinase pathway both oppose this effect.