A. A. te Velde

IL-10 gene therapy prevents TNBS-induced colitis.

The transfer of genes encoding immunomodulatory proteins to the intestinal mucosa is a promising new approach to the treatment of Crohns disease (CD). This study investigates the therapeutic efficacy of an adenoviral vector encoding IL-10 (AdvmuIL-10) in experimental colitis. BALB/c mice were treated with a single intravenous injection of AdvmuIL-10, empty cassette virus (Adv0) or PBS prior to the induction of trinitrobenzene sulphonic acid (TNBS) colitis. AdvmuIL-10 treatment prevented the severe loss of body weight associated with TNBS administration. In addition, AdvmuIL-10 therapy led to a significant reduction in both stool markers of inflammation (IL-1β and TNFR-II) and acute phase response (serum amyloid protein). Finally, the histological scores of mice with TNBS colitis treated with AdvmuIL-10 were significantly lower than Adv0- or PBS-treated controls. The therapeutic efficacy of AdvmuIL-10 was associated with a decrease in the IFN-γ and IL-6 levels detected in colonic homogenates from mice with TNBS colitis, whereas no effect was observed on cytokine release from stimulated systemic lymphocytes. Thus, AdvmuIL-10 is an effective therapy in the TNBS model of colitis. Gene therapy strategies using adenoviral vectors encoding IL-10 may prove to be a potent therapy for chronic inflammatory conditions such as CD.

Upregulation of Monocyte Urokinase Plasminogen Activator Receptor during Human Endotoxemia

ABSTRACT The receptor for urokinase-type plasminogen activator (uPAR) (CD87) plays an important role in leukocyte adhesion and migration. To assess the effect of endotoxin on cellular uPAR, uPAR expression was determined on leukocytes by fluorescence-activated cell sorter analysis in seven healthy subjects following intravenous injection of endotoxin (lot G; 4 ng/kg). Endotoxin induced a transient increase in uPAR expression on monocytes, reaching a 92% ± 46% increase over baseline expression after 6 h (P < 0.05). Endotoxin did not influence uPAR expression on granulocytes, while uPAR remained undetectable on lymphocytes. Endotoxin also increased soluble uPAR levels in plasma (P < 0.05). Stimulation of human whole blood with endotoxin or gram-positive stimuli in vitro also resulted in an upregulation of monocyte uPAR expression. Although tumor necrosis factor alpha (TNF) upregulated monocyte uPAR expression, anti-TNF did not influence the endotoxin-induced increase in monocyte uPAR expression. These data suggest that infectious stimuli may influence monocyte function in vivo by enhancing the expression of uPAR.

Immunological consequences of the use of xenogeneic hepatocytes in a bioartificial liver for acute liver failure

The use of cells from xenogeneic origin in a bioartificial liver can have a number of immunological consequences, not only for the cells in the bioartificial liver but also for the patient receiving the bioartificial liver treatment. The impact of these consequences will depend on the immune status of the patient receiving bioartificial liver treatment, the duration and frequency of the treatment and on the extent of interaction between the patients blood (or plasma) and the xenogeneic liver cells. In an experimental model we infused rats with a culture supernatant of pig hepatocytes and demonstrated using Western blots and immunohistological techniques that antibodies are raised against the very small amounts of the pig hepatocyte-derived proteins present in the culture medium. Potential problems of bioartificial liver destruction and the possibility of hypersensitivity reactions due to the secretion of xenogeneic proteins into the circulation of the patient are discussed. Because the liver has an important role in the clearance of immune complexes it is concluded that precautions should be taken when (repeated) application of a xenogeneic bioartificial liver in patients with liver failure is considered.

Activation of mononuclear cells by interleukin-12: an in vivo study in chimpanzees

Interleukin (IL)-12 is considered a central regulator of host resistance against a variety of pathogens. Therefore, IL-12 has been advocated as a potential therapeutic agent in infections. To determine the in vivo effects of IL-12 on mononuclear cells involved in the host immune response, four chimpanzees received an intravenous injection of recombinant IL-12 (1 μg/kg). IL-12 induced a sustained decrease in lymphocyte counts, with decreases in CD3+/CD4+ and CD3+/CD8+ cells, while monocyte counts showed a transient increase. IL-12 injection resulted in a shift toward a Th1-mediated immune response as indicated by increased interferon-γ production during whole-blood stimulation, while not influencing IL-4 production. IL-12-induced activation of NK cells and phagocytes, as indicated by increased NK cell cytotoxicity and increased plasma levels of granzymes A and B and of chitotriosidase activity. These data support the hypothesis that IL-12 may serve as a useful therapeutic agent in infections where a cell-mediated response is protective.

Possible immunological problems of bioartificial liver support

In the development of a bioartificial liver for clinical application the use of hepatocytes from xenogeneic origin is the most promising alternative for human hepatocytes due to the shortage of human donor material and the lack of a well functioning non-tumorigenous human hepatocyte cell line. The best candidate for the supply of hepatocytes is the pig since pig livers are easily obtained and large numbers of cells can be isolated. However, biosafety and immunological consequences should be considered when humans are to be treated with bioartificial livers loaded with pig hepatocytes. Biosafety problems, such as transmission of porcine pathogens will not be addressed in this paper. Immunological problems may arise if the patient develops an immune response to the xenogeneic cells or to the xenogeneic proteins derived from these cells. An immunological reaction could result in either destruction of the bioartificial liver or in hypersensitivity reactions in the patient receiving bioartificial liver treatment.

Does the extend of the culture time of primary hepatocytes in a bioreactor affect the treatment efficacy of a bioartificial liver

The purpose of this study was to investigate whether the efficacy of our novel extracorporeal bioartificial liver (BAL) to support rats with complete liver ischemia (LIS) could be improved by extending the culture time of freshly isolated porcine hepatocytes from 14 hours to 38 hours. The results showed that survival as well as porcine hepatocyte integrity improved, the onset of coma delayed, and the ammonia levels decreased in LIS rats of the 38 hour group compared to the 14 hour group, but no statistically significant differences were observed. In the 38 hour group, but not the 14 hour group, the onset of hepatic encephalopathy was significantly delayed and ammonia metabolism significantly improved compared to the LIS rats in control groups that only received a glucose infusion or were connected to a BAL without cells. In conclusion, prolonged hepatocyte recovery favoured all investigated parameters, although not all observed effects were statistically significant. More research is required to find out how long primary hepatocytes should be cultured in a bioreactor for optimal BAL support.