Testing risk and protective factor assumptions in the Icelandic model of adolescent substance use prevention.

Iceland has witnessed a dramatic decline in adolescent substance use that may be partly the result of efforts related to the Icelandic prevention model (IPM). We sought to test risk and protective factor assumptions of the IPM using a prospective cohort study with 12 months separating baseline from follow-up. Participants were students in grades 8 and 9 in the national Icelandic school system enrolled in the spring of 2018 and 2019 (N=2165). Participants self-reported their experiences of cigarette smoking, alcohol consumption, and cannabis use and seven risk and protective factors. Analyses were conducted with generalized linear modeling with extension to general estimating equations with correlated outcomes data. Both individual main-effects models and collective models including all main-effects were tested. Out of 28 individual main-effects models, 23 produced findings consistent with study premises (P<0.05). Multiple main-effects models largely sustained the findings of the individual main-effects models. Findings support the assumption that the risk and protective factors commonly emphasized in the IPM are associated with the four different substance use outcomes in the hypothesized direction. Communities that plan to implement the IPM among adolescents might consider these factors in their work.

Prevention Is Possible: A Brief History of the Origin and Dissemination of the Icelandic Prevention Model.

In two decades, the Icelandic prevention model (IPM) has been employed to dramatically reduce rates of adolescent substance use in Iceland. Briefly, the IPM is a multisectoral, community-based, collaborative system where researchers, policy makers, administrative leaders, and practitioners join forces to reduce the odds of adolescent substance use over time. Comparatively, Iceland now ranks among the lowest in adolescent substance use in all of Europe. Since 2005, the IPM has garnered considerable international attention, and several countries or municipalities within them have adapted, or are presently adapting, the model to their needs. In this commentary, we first briefly review the history and formation of the IPM in Iceland from a school-based survey to a fully integrated prevention system. In the second part, we present a short overview of the national consensus building and institutional collaboration that led to the implementation of the model in Chile in Latin America, as a demonstrative example. In this volume of Health Promotion Practice, we also present a series of two practice-based articles that introduce the IPM. The first article, titled "Development and Guiding Principles of the Icelandic Model for Preventing Adolescent Substance Use," introduces the theoretical origins of the model, five guiding principles, and evidence of effectiveness to date. In the second article, titled "Implementing the Icelandic Model for Preventing Adolescent Substance Use," we outline 10 practice-based steps to guide model implementation in other countries. Both articles are available via open access, and both are also available online in Spanish.

Death of a living liver donor from illicit drugs.

In children with acute hepatic failure, it has been suggested to offer living donor transplantation to all parents when a deceased donor organ can not be provided. Ethically, living related donation is coercive by its very nature, especially in emergencies. We report a 36-year-old woman who died from a drug overdose 57 days after living donor liver resection. The recipient was her 3-year-old son, who experienced acute hepatic failure as a result of acetaminophen intoxication. A deceased donor organ had not become available within 2 days after listing. Was the death of this living donor preventable or unpreventable? Certainly if the mother had decided not to take drugs, she would not have died from an overdose. One could argue that this was her personal choice, and beyond our influence. On the other hand, if we had not performed the surgery, the recipient might have died without receiving a liver transplant in time.

Hypothermia inhibits Fas-mediated apoptosis of primary mouse hepatocytes in culture.

Apoptosis occurs during the isolation and even short-term storage and culture of hepatocytes, and in the pathogenesis of liver diseases, such as hepatic failure and hepatitis. Therapeutic hypothermia has beneficial effects in experimental models of fulminant hepatic failure. The mechanisms underlying the potential benefits of mild hypothermia on the liver have not been well investigated. We examined the effects of temperature on soluble Fas ligand-induced apoptosis in freshly isolated mouse hepatocytes. Decreasing the culture temperature from 37 degrees C to 32 degrees C produced significant suppression of Fas-mediated apoptosis in cultured hepatocytes over a 12-h period. This observation was supported by cell morphology, flow cytometry analysis of cellular DNA content, and Annexin V-FITC staining of membrane phosphatidylserine translocation. In hypothermic conditions, Fas-mediated cytochrome c release from mitochondria of hepatocytes and the proximate downstream activation of caspase-9 were suppressed under mild hypothermic conditions. Effector caspase-7 activity was also inhibited at 32 degrees C. In contrast, the activation of initiator caspase-8 and cleavage of Bid were not affected after Fas-ligand stimulation. These findings suggest that mild hypothermia suppresses Fas-mediated apoptosis of liver cells by the partial inhibition of signaling events including mitochondrial damage, cytochrome c release, and subsequent apoptosome formation and effector caspase activation.

Hepatocyte transplantation in a 4-year-old girl with peroxisomal biogenesis disease: technique, safety, and metabolic follow-up.

Hepatocyte transplantation is an investigational alternative to orthotopic liver transplantation to treat liver based inborn errors of metabolism. We report successful hepatocyte transplantation in a 4-year-old girl with infantile Refsum disease. Hepatocytes were isolated from the left liver segment of two male donors using a classic two-step perfusion method. Fresh cells were transplanted first and then cryopreserved cells, for a total of 2 billion cells. Total bile acids and abnormal dihydroxycoprostanoïc acid markedly decreased in the patient's serum, indicating resolution of cholestasis and re-population of liver cells. Pipecholic acid decreased by 40% and c26:c22 fatty acid ratio by 36% after 18 months. Donor chromosomes sequences were detected on biopsy posttransplant, indicating engraftment. Hepatocyte transplantation is a safe and promising technique in the treatment of rare inborn errors of metabolism. Future improvements of cell viability and prevention of apoptosis may increase engraftment and subsequent re-population.

Engraftment assessment in human and mouse liver tissue after sex-mismatched liver cell transplantation by real-time quantitative PCR for Y chromosome sequences.

Transplanted hepatocytes can engraft, proliferate, and function permanently in host animals. After one cell infusion, however, engrafted hepatocytes constitute only between 1 in 200 to 1 in 3,000 host liver cells. Although transplanted cells can be identified using biochemical and molecular techniques, more accurate methods are needed to evaluate interventions that could improve cell engraftment rates. Real-time polymerase chain reaction (PCR) was done using primers and probes complementary to human testis determining gene (SRY) and mouse testis-specific Y-encoded protein (TSPY) pseudogene. Tissue samples from human or mouse recipients of liver cell transplantation were used to determine the test ability to detect transplanted cell DNA. Real-time PCR for the human SRY and mouse TSPY were species- and sex-specific. These two tests were sensitive in the detection of male DNA. Test sensitivity was consistently found at minimum 1:10,000 of male and female DNA mixing curve in both human SRY and mouse TSPY assays. The optimal amount of sample DNA per reaction to produce the highest sensitivity was 300 ng to 1 microg. Real-time PCR gave similar results whether standard male-female mixtures were prepared from liver cells or mononuclear cells. Engraftment of male liver cells in female liver tissues in mice and humans ranging from 0.125% to 0.257% was successfully measured using this method. Real-time PCR for SRY and TSPY affords a specific, sensitive, and reproducible tool for chimerism analysis in transplanted human and mouse liver tissues. This method could be used to optimize current models of cell transplantation.

Loss of cell anchorage triggers apoptosis (anoikis) in primary mouse hepatocytes.

Liver cell isolation and transplantation have been successfully performed in animal models and in humans. However, lack of initial engraftment due to cell death is a major roadblock to achieving clinical significance. Apoptosis was recently identified as an important cause of freshly isolated and banked hepatocyte cell death [Cell Transplant. 10 (2001) 59]. Pathways involving detachment-induced apoptosis (anoikis) are well characterized in other cell types. Loss of cell anchorage occurs during the hepatocyte isolation procedure prior to cell transplantation, but little is known about the role of this pathway in the survival of isolated hepatocytes. We report early occurrence of anoikis in primary mouse hepatocytes cultured under detached conditions on glass plates as compared to under attached conditions on plastic plates. Apoptosis in detached cells was determined using complementary techniques (DNA laddering, cell death ELISA assay, TUNEL assay and morphological analysis) and was detected as early as 15 min after culture under detached conditions. Further analysis of the mechanisms inducing apoptosis during liver cell isolation and transplantation and of ways to prevent them could lead to improved clinical protocols of liver cell therapies.

Liver repopulation after cell transplantation in mice treated with retrorsine and carbon tetrachloride.

BACKGROUND: Efficiency of engraftment after liver cell transplantation is less than 1% under conventional conditions. Our aim was to develop a high-efficiency, nonsurgical, no-genetic-advantage mouse model of liver repopulation with transplanted cells. METHODS: Mice were conditioned with nonlethal doses of a cell cycle inhibitor, retrorsine, 70 mg/kg, to irreversibly block proliferation of native hepatocytes. After the drug was eliminated, 2 million freshly isolated beta-galactosidase-labeled liver cells were transplanted into the spleens of C57BL/6J recipient mice. To stimulate donor cell proliferation, three doses of carbon tetrachloride (CCl4), 0.5 ml/kg, were given. Several control groups were studied to evaluate the contribution of each treatment to liver repopulation. RESULTS: Repopulation, as measured by cell isolation from recipient livers 1-7 months after transplantation, was on average 20%. Repopulation was 10% if CCl4 was given only once, between 0.5% and 1% if only retrorsine or CCl4 were used, and 0.05% if no conditioning was used. Phenotypically, whole livers turned blue on exposure to X-gal staining, whereas negative (control) livers remained pale brown. More than 55% of liver repopulation resulted from clusters containing 21 or more cells, some of which contained more than 200 cells, suggesting seven or more rounds of cell division in a subset of transplanted cells. CONCLUSION: This murine study demonstrates high levels of repopulation after liver cell transplantation into nongenetically modified livers, using a cell cycle inhibitor and chemical liver injury to provide transplanted cells a proliferative advantage. Liver repopulation was effected mostly by a small fraction of transplanted cells. Analogous nonsurgical liver cell transplantation strategies, but with clinically applicable drugs, could be devised for the treatment of liver-based metabolic diseases.

Apoptosis Occurs in Isolated and Banked Primary Mouse Hepatocytes.

Isolation and cryopreservation of freshly isolated hepatocytes is considered a standard procedure for the long-term storage of liver cells. However, most existing methods for banking hepatocytes do not allow sufficient recovery of viable cells to meet the needs of basic research or clinical trials of hepatocyte transplantation. The mechanisms underlying this poor rate of hepatocyte recovery are unknown. Although much of the cellular damage in freezing is caused by formation of ice crystals within the cells, this is largely prevented by the use of dimethyl sulfoxide (DMSO) and controlled rate freezing. As we demonstrated recently, necrosis does occur in primary hepatocytes following isolation and cryopreservation. In the present study, we explored the contribution of apoptosis, another form of cell death, in primary hepatocytes banked for transplantation. We evaluated apoptosis of C57BL/6J mouse primary hepatocytes using several different methods. Annexin binding and the TUNEL assay, in conjunction with flow cytometry and confocal laser scanning microscopy, revealed that the percentage of apoptotic cells was dramatically elevated in cryopreserved cells compared with that in the control group of unfrozen cells. DNA laddering detected by DNA electrophoresis in agarose gel also supported the presence of apoptosis in isolated and banked liver cells. Moreover, we found that the addition of glucose (from 10 to 20 mM) into the freezing solution (University of Wisconsin Solution) decreased the rate of apoptosis by 84% and improved the cell attachment at least fourfold in cryopreserved cells. These results suggest that apoptosis might contribute to cell death in isolated and banked primary hepatocytes.