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Severe alcoholic hepatitis (sAH) is associated with a poor prognosis. There is no proven effective treatment for sAH, which is why early transplantation has been increasingly discussed. Hepatoblastoma‐derived C3A cells express anti‐inflammatory proteins and growth factors and were tested in an extracorporeal cellular therapy (ELAD) study to establish their effect on survival for subjects with sAH. Adults with sAH, bilirubin ≥8 mg/dL, Maddrey's discriminant function ≥ 32, and Model for End‐Stage Liver Disease (MELD) score ≤ 35 were randomized to receive standard of care (SOC) only or 3‐5 days of continuous ELAD treatment plus SOC. After a minimum follow‐up of 91 days, overall survival (OS) was assessed by using a Kaplan‐Meier survival analysis. A total of 203 subjects were enrolled (96 ELAD and 107 SOC) at 40 sites worldwide. Comparison of baseline characteristics showed no significant differences between groups and within subgroups. There was no significant difference in serious adverse events between the 2 groups. In an analysis of the intent‐to‐treat population, there was no difference in OS (51.0% versus 49.5%). The study failed its primary and secondary end point in a population with sAH and with a MELD ranging from 18 to 35 and no upper age limit. In the prespecified analysis of subjects with MELD < 28 (n = 120), ELAD was associated with a trend toward higher OS at 91 days (68.6% versus 53.6%; P = .08). Regression analysis identified high creatinine and international normalized ratio, but not bilirubin, as the MELD components predicting negative outcomes with ELAD. A new trial investigating a potential benefit of ELAD in younger subjects with sufficient renal function and less severe coagulopathy has been initiated. Liver Transplantation 24 380–393 2018 AASLD.

Tissue-engineered aortic valves, known as recellularized heart valves, were developed by seeding human neonatal fibroblasts onto decellularized, porcine aortic valves. Recellularized heart valves were cultured up to 8 weeks in a novel bioreactor that imposed dynamic pulsatile fluid flow to expose the dermal fibroblasts to mechanical forces. Our data showed that, under static or dynamic flow conditions, dermal fibroblasts attached to and migrated into the decellularized, porcine valve scaffolding. The human cells remained viable as indicated by MTT viability staining. Gradual colonization of the decellularized porcine scaffolding by the human dermal fibroblasts was shown histologically by hematoxylin & eosin staining, immunocytochemically using a monoclonal antibody directed against prolyl-4-hydroxylase (an intracellular enzyme expressed by human fibroblasts synthesizing collagen), and quantitative digital image analyses. Thymidine and proline radiolabeled analog studies at 1, 2 and 4 weeks of individual leaflets cultured statically demonstrated that the human fibroblasts were mitotic and synthesized human extracellular matrix proteins, thereby supplementing the existing porcine matrix. The overall approach results in a heart valve populated with viable human cells. In the development of valves that perform in a similar manner as natural biological structures, this approach may present some unique benefits over current medical therapies.

Background-We have developed techniques to implant angiogenic patches onto the epicardium over regions of infarcted cardiac tissue to stimulate revascularization of the damaged tissue. These experiments used a scaffold-based 3D human dermal fibroblast culture (3DFC) as an epicardial patch. The 3DFC contains viable cells that secrete angiogenic growth factors and has previously been shown to stimulate angiogenic activity. The hypothesis tested was that a viable 3DFC cardiac patch would stimulate an angiogenic response within an area of infarcted cardiac tissue. Methods and Results-A coronary occlusion of a branch of the left anterior descending coronary artery was performed by thermal ligation in severe combined immunodeficient mice. 3DFCs with or without viable cells were sized to the damaged area, implanted in replicate mice onto the epicardium at the site of tissue injury, and compared with animals that received infarct surgery but no implant. Fourteen and 30 days after surgery, hearts were exposed and photographed, and tissue samples were prepared for histology and cytochemistry. Fourteen and 30 days after surgery, the damaged myocardium receiving viable 3DFC exhibited a significantly greater angiogenic response (including arterioles, venules, and capillaries) than nonviable and untreated control groups. Conclusions-In this animal model, viable 3DFC stimulates angiogenesis within a region of cardiac infarction and can augment a repair response in damaged tissue. Therefore, a potential use for 3DFC is the repair of myocardial tissue damaged by infarction.

Water disinfection systems utilizing electrolytically generated copper and silver ions (200 and 20, 400 and 40, or 800 and 80 ,ug/liter) and low levels of free chlorine (0.1 to 0.4 mg/liter) were evaluated at room (21 to 23°C) and elevated (39 to 40°C) temperatures in filtered well water (pH 7.3) for their efficacy in inactivating Legionella pneumophila (ATCC 33155). At room temperature, a contact time of at least 24 h was necessary for copper and silver (400 and 40 ,ug/liter) to achieve a 3-log1o reduction in bacterial numbers. As the copper and silver concentration increased to 800 and 80 pg/liter, the inactivation rate significantly (P s 0.05) increased from K = 2.87 x 10-3 to K = 7.50 x 10-3 (loglo reduction per minute). In water systems with and without copper and silver (400 and 40 ,g/liter), the inactivation rates significantly increased as the free chlorine concentration increased from 0.1 mg/liter (K = 0.397 log1o reduction per min) to 0.4 mg/liter (K = 1.047 log1o reduction per min). Compared to room temperature, no signfficant differences were observed when 0.2 mg of free chlorine per liter with and without 400 and 40 ,ug of copper and silver per liter was tested at 39 to 40°C. All disinfection systems, regardless of temperature or free chlorine concentration, showed increase inactivation rates when 400 and 40 ,ug of copper and silver per liter was added; however, this trend was significant only at 0.4 mg of free chlorine per liter.

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Landeen LK, Dederko DA, Kondo CS, Hu BS, Aroonsakool N, Haga JH, Giles WR. Mechanisms of the negative inotropic effects of sphingosine-1-phosphate on adult mouse ventricular myocytes. Am J Physiol Heart Circ Physiol 294: H736-H749, 2008. First published November 16, 2007 doi:10.1152/ajpheart.00316.2007.-Sphingosine-1-phosphate (S1P) induces a transient bradycardia in mammalian hearts through activation of an inwardly rectifying K ϩ current (IK ACh ) in the atrium that shortens action potential duration (APD) in the atrium. We have investigated probable mechanisms and receptor-subtype specificity for S1P-induced negative inotropy in isolated adult mouse ventricular myocytes. Activation of S1P receptors by S1P (100 nM) reduced cell shortening by ϳ25% (vs. untreated controls) in fieldstimulated myocytes. S1P 1 was shown to be involved by using the S1P 1-selective agonist SEW2871 on myocytes isolated from S1P3-null mice. However, in these myocytes, S1P 3 can modulate a somewhat similar negative inotropy, as judged by the effects of the S1P 1 antagonist VPC23019. Since S1P1 activates Gi exclusively, whereas S1P 3 activates both Gi and Gq, these results strongly implicate the involvement of mainly G i. Additional experiments using the IK ACh blocker tertiapin demonstrated that IK ACh can contribute to the negative inotropy following S1P activation of S1P 1 (perhaps through Gi␤␥ subunits). Mathematical modeling of the effects of S1P on APD in the mouse ventricle suggests that shortening of APD (e.g., as induced by I K ACh ) can reduce L-type calcium current and thus can decrease the intracellular Ca 2ϩ concentration ([Ca 2ϩ ]i) transient. Both effects can contribute to the observed negative inotropic effects of S1P. In summary, these findings suggest that the negative inotropy observed in S1P-treated adult mouse ventricular myocytes may consist of two distinctive components: 1) one pathway that acts via G i to reduce L-type calcium channel current, blunt calcium-induced calcium release, and decrease [Ca 2ϩ ]i; and 2) a second pathway that acts via Gi to activate IK ACh and reduce APD. This decrease in APD is expected to decrease Ca 2ϩ influx and reduce [Ca 2ϩ ]i and myocyte contractility.calcium; contraction; cell shortening; inhibitory G protein; acetylcholine-sensitive potassium; myocyte SPHINGOSINE-1-PHOSPHATE (S1P) is a biologically active, cell membrane-associated sphingolipid that binds with high affinity to five distinct G-coupled protein receptor isoforms (S1P 1-5 ). S1P 1 has been detected in abundance in neonatal rat cardiomyocytes (39). In these cells, exposure to S1P (500 nM) results in an initial negative inotropic effect (reduction of systolic calcium). However, this may be followed by calcium overload (increased diastolic calcium) and then a cessation of contractility. In isolated atrial myocytes, S1P has been shown to activate a weakly inwardly rectifying potassium (K ϩ ) current. This K ϩ conductance is very similar to the K ϩ current activated by ACh (I K ACh ). Activation of this current can shorte...

Cryptosporidium and Giardia species are enteric protozoa which cause waterborne disease. The detection of these organisms in water relies on the detection of the oocyst and cyst forms or stages. Monoclonal and polyclonal antibodies were compared for their abilities to react with Giardia cysts and Cryptosporidium oocysts after storage in water, 3.7% formaldehyde, and 2.5% potassium dichromate, upon exposure to bleach, and in environmental samples. Three monoclonal antibodies to Cryptosporidium parvum were evaluated. Each test resulted in an equivalent detection of the oocysts after storage, after exposure to bleach, and in environmental samples. Oocyst levels declined slightly after 20 to 22 weeks of storage in water, and oocyst fluorescence and morphology were dull and atypical. Oocyst counts decreased after exposure to 2,500 mg of sodium hypochlorite per liter, and fluorescence and phase-contrast counts were similar. Sediment due to algae and clays found in environmental samples interfered with the detection of oocysts on membrane filters. Two monoclonal antibodies and a polyclonal antibody directed against Giardia lamblia cysts were evaluated. From the same seeded preparations, significantly greater counts were obtained with the polyclonal antibody. Of the two monoclonal antibodies, one resulted in significantly lower cyst counts. In preliminary studies, the differences between antibodies were not apparent when used on the environmental wastewater samples. After 20 to 22 weeks in water, cyst levels declined significantly by 67%. Cysts were not detected with monoclonal antibodies after exposure to approximately 5,000 mg of sodium hypochlorite per liter. The enteric protozoa Giardia and Cryptosporidium species are well-documented causes of waterborne disease. From 1971 to 1985, Giardia species have been responsible for 92 outbreaks of disease associated with both animal and human fecal contamination of water supplies in the United States (2). Cryptosporidium species, newly recognized in 1980 as agents of diarrhea, have been documented in a number of outbreaks of waterborne disease in both the United States and the United Kingdom (4, 5, 8, 17). Zoonotic transmission has been demonstrated for Cryptosporidium parvum; therefore, many animals may also serve as sources of Cryptosporidium contamination of water supplies with the potential for human infection (3). Methods to study the occurrences of Cryptosporidium and Giardia species in water by detection of oocysts and cysts, respectively, have been developed. Large volumes of water are filtered, and the concentrate is examined by microscopic techniques. Developments in methods for Giardia and Cryptosporidium detection over the last few years have included the use of yam-wound filters for sample collection (9), various density gradients for sample clarification (15), and membrane filtration coupled with immunofluorescence techniques for cyst and oocyst detection and enumeration (10-16). Limitations of these methods include (i) poor recoveries influenced by water qualit...