Combination of in ovo electroporation and time‐lapse imaging to study migrational events in chicken embryos

Background : During embryonic development cell migration plays a principal role in several processes. In past decades, many studies were performed to investigate migrational events, occurring during embryonic organogenesis, neurogenesis, gliogenesis or myogenesis, just to name a few. Although different common techniques are already used for this purpose, one of their major limitations is the static character. However, cell migration is a sophisticated and highly dynamic process, wherefore new appropriate technologies are required to investigate this event in all its complexity. Results and Conclusions: Here we report a novel approach for dynamic analysis of cell migration within embryonic tissue. We combine the modern transfection method of in ovo electroporation with the use of tissue slice culture and state‐of‐the‐art imaging techniques, such as confocal laser scanning microscopy or spinning disc confocal microscopy, and thus, develop a method to study live the migration of myogenic precursors in chicken embryos. The conditions and parameters used in this study allow long‐term imaging for up to 24 hr. Our protocol can be easily adapted for investigations of a variety of other migrational events and provides a novel conception for dynamic analysis of migration during embryonic development. Developmental Dynamics 243:690–698, 2014. © 2013 Wiley Periodicals, Inc.     

Pharmacosimulation of delays and interruptions during administration of tirofiban: a systematic comparison between EU and US dosage regimens

Journal of Thrombosis and Thrombolysis - Tirofiban is a glycoproteine (GP) IIb/IIIa receptor antagonist, which inhibits platelet-platelet aggregation and is a potential adjunctive antithrombotic...     

Perfused rat intrahepatic bile ducts secrete and absorb water, solute, and ions

BACKGROUND & AIMS: We report a novel approach to study biliary water, bile acid, and HCO(3)(-) transport: the microperfusion of intrahepatic bile duct units (IBDUs) isolated from normal rat liver. METHODS: To study water transport, IBDUs were perfused in vitro with a membrane-impermeant fluorescent volume marker, fluorescein sulfonate; net water movement (J(v)) and osmotic water permeability (P(f)) were then calculated. To study solute transport, IBDUs were perfused with taurocholic acid (TCA) and bile acid uptake was calculated from the concentrations of TCA in the perfused and collected solutions. To study ion transport, IBDUs were perfused with the cell-impermeant pH-sensitive dye BCECF dextran; luminal pH was determined from fluorescence excitation ratios. RESULTS: When inward (secretory) or outward (absorptive) osmotic gradients were established across IBDUs, water movement was observed from bath to lumen (i.e., secretion) and from lumen to bath (i.e., absorption). The perfused IBDUs absorbed TCA in a saturable, sodium-dependent manner; in addition, TCA absorption was blocked in a dose-dependent fashion by S0960, a specific inhibitor of the Na(+)/bile acid cotransporter. Addition of forskolin to HCO(3)(-)-containing (but not HCO(3)(-)-free) bath buffer resulted in lumen alkalinization reflecting HCO(3)(-) transport into the lumen of perfused IBDUs. CONCLUSIONS: The results provide direct functional evidence that intrahepatic bile ducts both secrete and absorb water in response to osmotic gradients, actively absorb bile acid, and transport HCO(3)(-).     

Hepatic artery and portal vein remodeling in rat liver: vascular response to selective cholangiocyte proliferation

Three-dimensional reconstruction of the biliary tree, hepatic artery, and portal vein in normal rats and rats fed alpha-naphthylisothiocyanate (ANIT), a compound that causes selective proliferation of epithelial cells (ie, cholangiocytes) that line the bile ducts, was performed. All hepatic structures in ANIT-fed rats branched 1.5 times more often than in normal rats, reflecting an increased number of segments, whereas the length of the biliary tree, hepatic artery, and portal vein remain unchanged. The length of the proximal vessel segments was uniform in both groups of rats whereas the length of distal segments decreased twofold in ANIT-fed rats, suggesting that small vessels preferentially undergo proliferation. In contrast, the length of all bile duct segments decreased twofold, suggesting that ANIT induced proliferation of all compartments of the biliary tree. The total volume of the biliary tree, hepatic artery, and portal vein was increased 18, 4, and 3 times, respectively, after ANIT feeding. The diameters of the bile ducts (range, 20 to 259 microm) and arterial (range, 21 to 276 microm) segments in ANIT-fed rats did not differ from normal rats (range, 21 to 245 microm and 20 to 265 microm, respectively). In contrast, the diameters of proximal venous segments in ANIT-fed rats were significantly less (316 +/- 68 micro m versus 488 +/- 89 micro m, P < 0.001). The data suggest that after experimentally induced cholangiocyte proliferation, the hepatic artery and portal vein also undergo marked proliferation, presumably to support the increased nutritional and functional demands of the proliferated bile ducts. The molecular mechanisms of these vascular changes remain to be determined.     

Development and characterization of a cholangiocyte cell line from the PCK rat, an animal model of Autosomal Recessive Polycystic Kidney Disease

In the PCK rat, a rodent model of Autosomal Recessive Polycystic Kidney Disease (ARPKD), a spontaneous splicing mutation of Pkhd1 initiates hepatic cyst development. Cystic cholangiocytes possess short and malformed cilia that do not express fibrocystin, the Pkhd1 protein. During the disease course, cysts continue to grow; however, the mechanisms underlying cyst progression are unclear due in part to the lack of suitable cell lines to study cystogenesis. Here, we describe the development of a PCK-derived cholangiocyte cell line (PCK-CCL). Normal rat cholangiocytes (NRCs) were used as a control. The PCK-CCL maintained a cholangiocyte phenotype as assessed by the expression of the CK-19, CK-7 and GGT. PCK-CCL grown on collagen formed a polarized monolayer with well-developed junctional complexes, and distinct apical and basolateral membranes. Compared to NRCs, cilia in the PCK-CCL were short and malformed and did not express fibrocystin. The PCK-CCL exhibited a higher rate of proliferation (P<0.05) with a doubling time approximately half that of NRCs. By RT-PCR analysis of exons 33-37, an approximately 800 bp product of Pkhd1 was amplified in NRCs. In contrast and as expected, in the PCK-CCL, the Pkhd1 amplicon was smaller ( approximately 630 bp) reflecting the IVS35-2A --> T mutation. PCK-CCL and NRCs seeded in 3-D cultures formed cystic structures; however, the PCK cysts expanded progressively up to day 21 while cysts formed by NRCs remained the same size after day 9. In summary, we have developed a cholangiocyte cell line from the PCK rat that retains properties of the cholangiocytes lining hepatic cysts in vivo. The cells have been grown continuously for approximately 18 month and 45 passages without crisis or senescence. The morphology and growth characteristics of the PCK-CCL are consistent with those seen in vivo in the PCK rat, suggesting that this cell line will be useful in dissecting the mechanisms of hepatic cyst formation.     

Effect of oral immunomodulator Dzherelo in TB/HIV co-infected patients receiving anti-tuberculosis therapy under DOTS

Open-label, phase II clinical trial was conducted in 40 HIV/TB dually infected patients to evaluate the effect of oral immunomodulator Dzherelo on immune and viral parameters. The anti-retroviral therapy naïve patients were randomized into two equal groups to be given anti-tuberculosis therapy (ATT) under DOTS. The arm A, which served as a control, received Isoniazid (H); Rimfapicin (R); Pyrazinamide (Z); Streptomycin (S); and Ethambutol (E), and arm B received 50 drops of Dzherelo twice per day in addition to the daily dose of HRZSE. After 2months the total CD3+ lymphocytes increased from 728 to 921cells/μl (P = 0.025) in Dzherelo recipients, whereas in the control group they decreased from 651 to 585 cells (P = 0.25). The population of CD4 T-cells expanded in Dzherelo arm (174 to 283; P = 0.00003) but declined in ATT group (182 to 174; P = 0.34). The CD8 cells fluctuated slightly upward in both groups: 159 > 180 (P = 0.17) and 159 > 183 (P = 0.13). The ratio between CD4/CD8 cells deteriorated in arm A (1.213 > 0.943; P = 0.002) but improved in arm B (1.244 > 1.536; P = 0.007). The percent of CD3+HLA-DR+ activated lymphocytes had fallen in ATT group (22.6 > 20.5; P = 0.004), but rose in Dzherelo recipients (21.5 > 30.5; P = 0.0001). The changes in CD20+ B lymphocytes were insignificant in both arms (28.4% > 28.6%; P = 0.4) and (27.2% > 26.7%; P = 0.38). No difference was seen in the amount of CD3-CD16+CD56+ natural killer (NK) cells in arm A (21.3% > 22.6%; P = 0.1), while in Dzherelo recipients they declined significantly (19.9% > 14.5%; P = 0.0026). The viral load, measured by plasma RNA-PCR, decreased in Dzherelo group (2174 > 1558; P = 0.002), but increased in ATT group (1907 > 2076 copies/ml; P = 0.03). Dzherelo has a favorable effect on the immune status and viral burden in HIV/TB patients when given as the immunomodulating adjunct to ATT.     

AQP4 transfected into mouse cholangiocytes promotes water transport in biliary epithelia

Rodent cholangiocytes express 6 of the 11 known channel proteins called aquaporins (AQPs) that are involved in transcellular water transport in mammals. However, clarifying the role of AQPs in mediating water transport in biliary epithelia has been limited in part because of the absence of physiologically relevant experimental models. In this study, we established a novel AQP4-transfected polarized mouse cholangiocyte cell line suitable for functional studies of transepithelial water transport, and, using this model, we define the importance of this AQP in water transport across biliary epithelia. Polarized normal mouse cholangiocytes (NMCs) lacking endogenous AQP4 were transfected stably with functional AQP4 or cotransfected with functional AQP4 and a transport-deficient AQP4 dominant negative mutant using a retroviral delivery system. In transfected NMCs, AQP4 is expressed on both the mRNA and protein levels and is localized at both the apical and basolateral membranes. In nontransfected NMCs, the transcellular water flow, P(f), value was relatively high (i.e., 16.4 +/- 3.2 microm/sec) and likely was a reflection of endogenous expression of AQP1 and AQP8. In NMCs transfected with AQP4, P(f) increased to 75.7 +/- 1.4 microm/sec, that is, by 4.6-fold, indicating the contribution of AQP4 in channel-mediated water transport across MNCs monolayer. In cotransfected NMCs, AQP4 dominant negative reduced P(f) twofold; no changes in P(f) were observed in NMCs transfected with the empty vector. In conclusion, we developed a novel polarized mouse cholangiocyte monolayer model, allowing direct study of AQP4-mediated water transport by biliary epithelia and generated data providing additional support for the importance of AQP4 in cholangiocyte water transport.