Background, Low-molecular-weight heparin (LMWH) has been shown to prolong survival of rat cardiac allografts independently from immunosuppressive treatment. Furthermore, long-term treatment reduces the development of chronic graft vascular disease after experimental heart transplantation. The aim of the present study was to determine whether treatment with the LMWH reviparin has a beneficial effect on chronic rejection in a rat renal allograft; model.Methods. Kidneys of Fisher (F344) rats were transplanted into unilaterally nephrectomized Lewis (LEW) recipients. LEW --> LEW isografts served as controls. Animals were treated with cyclosporine (5 mg/kg/d) for the first 10 days. Nephrectomy of the remaining kidney was performed after 10 days. Allografted animals were treated either with reviparin (2 mg/kg/d subcutaneously) for 24 weeks (Allo-24), from week 12 to 24 (Allo-12), or with vehicle for 24 weeks. Proteinuria was determined at regular intervals. Kidneys were harvested after 24 weeks for histomorphological and immunohistochemical evaluation.Results. No major bleeding complications were observed in reviparin-treated animals. Proteinuria was significantly reduced in allografted animals both by early as well as by late-onset treatment with reviparin. Transplant glomerulopathy was diminished in Allo-24 and in Allo-12 groups compared to vehicle-treated animals, whereas tubulointerstitial inflammation was influenced only in animals immediately treated with reviparin. Immunohistochemical. studies demonstrated a marked reduction of renal monocyte and T-cell infiltration as well as expression of MHC II by treatment with reviparin.Conclusions. Treatment with the LMWH reviparin significantly improved chronic renal allograft rejection in the F344-to-LEW rat model, both after early and late start of therapy. Although the exact mechanisms of this beneficial effect remain unclear, our data offer a potential new therapeutical approach for prevention of chronic allograft nephropathy.

Background. Previously. it has been demonstrated that heparin inhibits major histocompatibility complex (MHC) class II and intercellular adhesion molecule-1. (ICAM-1) expression on interferon-gamma (IFN-gamma)-stimulated human umbilical vein endothelial cells (HUVECs). Inasmuch as proximal tubular epithelial cells (PTECs) are prime targets in acute renal allograft rejection, we investigated whether there is a difference in the ability of heparin to influence MHC and ICAM-1 expression on PTECs as compared to HUVECs, We also studied whether the degree of sulfation of heparin is of relevance for the binding to IFN-gamma and inhibition of MHC and ICAM-1 expression after IFN-gamma stimulation.Methods. Cultured HUVECs and PTECs were stimulated with IFN-gamma for 72 hr in the presence or absence of various heparinoids, MHC and ICAM-1 expression were thereafter determined by fluorescence-activated cell sorting.Results. Heparin was able to inhibit the up-regulation of MHC and ICAM-1 in a dose-dependent fashion on both IFN-gamma-stimulated HUVECs and PTECs. In PTEC cultures, higher concentrations of heparin were required for the inhibition of MHC class I. Heparin and supersulfated glycosaminoglycans (GAGs) were able to bind to IFN-gamma, whereas N-desulfated N-acetylated GAGs with a low amount of sulfate were not. Inhibition of cell-bound heparan sulfate proteoglycan sulfation with NaCIO3 resulted in an impaired MHC and ICAM-1 expression after IFN-gamma stimulation.Conclusion. We postulate that IFN-gamma binds to cell-bound heparan sulfate proteoglycan in a sulfation-dependent fashion. This binding mag facilitate the interaction of IFN-gamma with its receptor. Supersulfated GAGs with low anti-coagulant, activity could be used therapeutically to decrease MBC and ICAM-1 expression on organ grafts.

Trichoderma viride ITCC-1433 secretes a cellulase complex that is rich in β-glucosidase and therefore well suited for the saccharification of cellulosic materials. The cellulase was investigated with respect to optimum conditions of reaction and enzyme stability. Avicelase, CMCase, and β-glucosidase differed considerably in their physicochemical properties. At temperatures above 50°C, β-glucosidase is not very stable. Therefore, as a compromise the conditions of hydrolysis were chosen to be 50°C and pH 4.5. With the crude culture filtrate of T. viride ITCC-1433 a nearly pure glucose solution of 4% is reached from a 10% cellulose suspension. Wood pulp and newsprint are hydrolyzed to a much smaller extent. With an enzyme concentrate up to 8% glucose accumulated in the reaction fluid within 48 hr. At this time the glucose-cellobiose ratio was 75:1. Glucose was demonstrated to be the most potent inhibitor of total hydrolysis. The addition of glucose to the enzyme-substrate solution at zero time completely stopped its own formation and cellobiose and reducing groups (oligosaccharides) accumulated. By removing glucose through an ultrafilter device about 90% saccharification of cellulose to glucose was achieved in 48 hr without any accumulation of cellobiose.

Trichoderma viride ITCC‐1433 produces high yields of cellulase and especially β‐glucosidase when grown in submerged culture on different carbon sources. Cellulase synthesis was strongly repressed in the presence of glucose and only a low constitutive activity of β‐glucosidase and carboxymethylcellulase, but no Avicelase, could be demonstrated when culturing T. viride on glucose. With carboxymethylcellulose (CMC) as a substrate the secretion of enzyme as well as growth depended on the degree of substitution, but in general CMC cannot be regarded either as a powerful inducer or as a carbon source. With insoluble cellulose, maximum enzyme production and activities were obtained using an alkali‐treated cellulose powder. On this substrate the excretion of soluble protein into the culture broth increased and the protein concentration corresponded to cellulolytic activities.

In a special standard reaction system with active dried yeast as an enzyme source about 65% of added 5′-UMP was transformed into UDPG. In this system 2-deoxyglucose was not only demonstrated to be a substrate for yeast hexokinase, but was also shown to enter the “LELOIR-pathway”. Depending upon the ratio of glucose to glucose-analogue, 2-deoxyglucose was incorporated into uridine-sugar-nucleotides, deoxy- and dideoxytrahalose. The trahalose splitting enzyme, trehalase (EC 3.2.1.28) showed a different substrate specifity towards trehalose and its unnatural analogues. Although 2-deoxy-2-chloro-D-glucose and 2-deoxy-2-fluoro-D-glucose were good substrates for yeast hexokinase, these two deoxyhalogenglucopyranoses were not substrates for sugar-nucleotide and trehalose synthesis. 3-O-methylglucose was not phosphorylated and showed no influence on the course of reaction in the in vitro system.