There was only a 20% increase in the weight of the rem-A reproducible experimental animal model of fulminant liver lobes due to swelling. No hepatocytes stained nant hepatic failure (FHF) resembling the clinical condipositively for BrdU and PCNA, and none showed mitotic tion is needed. We have developed such a model in the figures. In contrast, all PH controls showed vigorous rat by combining resection of the two anterior liver lobes liver regeneration. function tests, prothrombin time) and to assessDespite improvement in our understanding of hepatic failliver regenerative response in the residual omental liver ure and determination of the inciting etiology in the majority lobes (weight, protein content, incorporation of bromoof cases, it is unclear which specific biochemical abnormalideoxyuridine [BrdU], expression of proliferation cell nuties are pathognomonic for FHF and which elements are esclear antigen [PCNA], mitotic activity), plasma levels of sential for patient survival. A reproducible experimental hepatocyte growth factor (HGF) and transforming model of FHF relevant to the clinical setting is therefore growth factor b (TGF-b 1 ), and tissue expression of the highly desirable because it would allow studies to improve HGF and it's receptor c-met. Rats undergoing partial our insight into the full gamut of metabolic and physiological hepatectomy of 68% (PH; n Å 42) and a sham operation derangements in FHF and would facilitate the design of new (SO; n Å 42) served as controls. All SO and PH controls therapeutic modalities. survived. PH rats showed only transient decreases inWe have developed a novel experimental model of FHF in body temperature, signs of modest early hepatic dysthe rat. In this simple and highly reproducible preparation, function (hyperlactemia, hyperammonemia, prolonged resection of the two anterior liver lobes (68% liver mass) is PT time), and normal restitution of liver mass. All FHF combined with ligation of the common right liver lobes pedicle rats became comatose by 24 hours postoperatively. Most (24% liver mass). As a result, the functional liver mass is animals (90%) died within 24-48 hours postoperatively greatly reduced, there is a significant amount of liver tissue (mean, 39 { 11 hours). Changes in blood chemistry reundergoing necrosis leading to toxemia, and animal survival flected rapid development of liver failure. Plasma HGF is dependent on the ability of the residual omental liver lobes levels were markedly elevated and at all time points (8% liver mass) to regenerate. Here, we present data on aniwere higher than in PH controls (P õ .05). At the same mal survival, sequential changes in blood chemistry, and retime, expression of HGF and c-met messenger RNA in generative response in the residual viable liver tissue. the remnant liver was delayed. Plasma TGF-b 1 levels increased early (18 hours) and remained twofold to three-MATERIALS AND METHODS fold higher than that of PH and SO controls (P õ .05).Animal studies were performed in compliance with institutional and National R...

Natural killer cells can be separated into three major subsets (free, binder, and killer) based on their ability to bind and kill sensitive target cells. The nonbinder, nonkiller free cells are the most immature and can be activated to become binders and killers. Natural killer (NK) cells synthesize and secrete several cytokines that are intimately involved in NK activation. This study investigated the secretion of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) by purified NK cells and NK subsets following activation by various stimuli. K562 target cells stimulated secretion of both TNF-alpha and IFN-gamma by both the binder and the killer subsets but not by the free subset. IFN-alpha activated the secretion of IFN-gamma only, whereas IL-2 activated the secretion of both TNF-alpha and IFN-gamma by the binder and killer subsets and secretion was augmented by the addition of K562 to the cultures. Phorbol myristate acetate (PMA) and ionophore stimulated TNF-alpha and IFN-gamma secretion in both the binder and the killer subsets, though IFN-gamma secretion was more pronounced in the binder subset. Activation of TNF-alpha and IFN-gamma secretion was dependent on de novo protein synthesis. Analysis at the single-cell level demonstrated that the binder subset had the highest frequency of cells secreting IFN-gamma. These results demonstrate that both the binder and the killer subsets can be activated to secrete TNF-alpha and IFN-gamma, whereas the free NK subset secretes little or no TNF-alpha and IFN-gamma following activation. These data suggest that the ability of NK cells to secrete TNF-alpha and IFN-gamma following activation correlates with the functional stage of maturation of NK cells.

Numerous studies have reported successful allotransplantation of hepatocytes. However, none have shown long-term correction of a liver-related metabolic defect. In this study, we used a method of regional hepatocyte transplantation and subsequent induction of transplanted cell proliferation by regeneration response in the transplant-bearing liver lobes. New Zealand White rabbits were used as cell donors and Watanabe heritable hyperlipidemic (WHHL) rabbits were used as cell recipients (2 x 10(8) cells/rabbit). All recipient rabbits were maintained on daily cyclosporine. Two weeks after baseline serum cholesterol determination, group I WHHL rabbits (n = 7) received an infusion of cells into the right lateral liver lobe, and a loose ligature was placed around the portal venous branch supplying the anterior lobe. After 1 week, to allow engraftment, the portal venous branch was ligated, which resulted in the atrophy of the affected liver parenchyma and induction of hyperplasia in the transplant-bearing liver tissue. Group II rabbits (n = 6) were transplanted with New Zealand White hepatocytes without portal branch ligation (PBL) and group III rabbits (n = 4) were subjected to sham transplantation (saline) and PBL. The experimental period extended to 150 days after transplantation. All WHHL rabbits transplanted with normal hepatocytes showed reduction in serum cholesterol and low-density lipoprotein (LDL) levels. Group I (PBL-stimulated) recipients demonstrated a more pronounced and sustained effect than group II animals (P < 0.05). Group III controls showed only a slight, typical for aging decrease in serum cholesterol. Group I recipient livers perfused with LDL labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) showed much higher numbers of DiI-LDL-positive hepatocytes than those of group II recipients. In conclusion, a liver regeneration stimulus enhanced the population of transplanted hepatocytes and their functional effect in a large animal model of inborn error of liver metabolism.

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Clinical islet transplantation (Tx) in type I diabetic patients has been successful so far only in a minority of cases, probably because of multiple factors, partly immunologic and partly nonimmunologic in nature. Preclinical studies of islet Tx in large animals are still needed to clarify the reasons and find possible solutions.In this study, we tested the feasibility of noninvasive, repeated intrahepatic allo-Tx of porcine pancreatic islets obtained from multiple donors, in pigs rendered diabetic by total pancreatectomy (Pct). In group I Yucatan miniature swine (n = 6), after induction of diabetes by Pct, repeated islet allo-Tx of ≥80% pure islets was performed. Islets obtained from two pigs of the Hanford breed were injected twice a week, half freshly isolated and half 48-h cultured, over a period of 11 days, for a total of 23,647 ± 1617 islet equivalents (IE)/kg recipient body weight (BW). In group II Yucatan miniature swine (n = 3), after Pct, a single allo-Tx of ≥80% pure islets, previously obtained from two donors of the Hanford breed, was performed, using a total of 22,416 ± 1124 IE/kg BW. In group III Yucatan miniature swine (n = 3), auto-Tx of 60-75% pure islets, averaging 2980 ± 424 IE/kg BW, was performed a few hours after Pct. Group IV Yucatan mini pigs (n = 3) underwent Pct and were used as diabetic controls. Group V animals (n = 3) were normal control Yucatan mini pigs. Porcine islets were isolated by a modification of the standard collagenase digestion and Ficoll gradient purification method. Donors and recipients were chosen on the basis of moderate to high mutual alloreactivity in mixed lymphocyte culture (MLC). In groups I and II, cyclosporine A (CsA) was started 4 days before allo-Tx, at the dose of 15 mg/kg IM, and then gradually reduced to 4 mg/kg IM. In all group I animals, normal fasting blood glucose (FBG) was restored within 2-3 weeks. Two normoglycemic pigs died of acute pneumonia at 33 and 112 days, respectively, and one animal became progressively hyperglycemic at 100 days. After 3 months, discontinuation of CsA treatment resulted in FBG increase in two group I animals. In one pig, CsA was stopped after 151 days, and normoglycemia persisted until euthanasia, after 8 months. In group II pigs, normoglycemia lasted 4-20 days, with a progressive increase of insulin requirement thereafter. In group III animals, after islet auto-Tx, normoglycemia lasted 7-10 days, while insulin daily requirement progressively increased thereafter, stabilizing at 0.4 IU/kg/day, corresponding to about one third of the amount required in diabetic controls. The single most important result in this series of experiments is that intraportal allo-Tx of a sufficient islet mass, divided in multiple subtherapeutic doses, produced a better metabolic long-term control in comparison to a single injection of the same amount of islets. The technique of multiple-donor repeated islet Tx may prove useful to overcome the problem of primary nonfunction or early graft failure, currently limiting the success of clinical islet Tx...