The anti-fibrotic effect of liver growth factor is associated with decreased intrahepatic levels of matrix metalloproteinases 2 and 9 and transforming growth factor beta 1 in bile duct-ligated rats

Liver growth factor (LGF), a mitogen for liver cells, behaves as an anti-fibrotic agent even in extrahepatic sites, but its mechanistic basis is unknown. We aimed to determine the intrahepatic expression pattern of key modulators of liver fibrosis in bile duct-ligated rats (BDL) after injection of LGF. BDL rats received either LGF (4.5 microg/ratXdose, two doses/week, at time 0 or 2 or 5w after operation, depending on the group (BDL+LGF groups, n=20) or saline (BDL+S groups, n=20). Groups were compared in terms of fibrosis (histomorphometry), liver function (aminopyrine breath test), matrix metalloproteinases MMP-2 and MMP-9, transforming growth factor beta 1 (TGF-beta1) and liver endoglin content (Western blotting), and serum tissue inhibitor of metalloproteinases 1 (TIMP-1) levels (ELISA). In BDL+LGF rats, the fibrotic index was significantly lower at 5w, p=0.006, and at 8w, p=0.04, than in BDL+S rats. Liver function values in BDL+LGF rats were higher than those obtained in BDL+S rats (80% at 5w and 79% at 8w, versus 38% and 29%, p<0.01, taking healthy controls as 100%). Notably, in BDL+LGF rats the intrahepatic expression levels of both MMPs were lower at 2w (MMP-2, p=0.03; MMP-9, p=0.05) and 5w (MMP-2, p=0.05, MMP-9, p=0.04). In addition, the hepatic TGF-beta1 level in BDL+LGF rats was lower at 2w (36%, p=0.008), 5w (50%) and 8wk (37%), whereas intrahepatic endoglin expression remained constant in all BDL rats studied. LGF ameliorates liver fibrosis and improves liver function in BDL rats. The LGF-induced anti-fibrotic effect is associated with a decreased hepatic level of MMP-2, MMP-9 and TGF-beta1 in fibrotic rats.     

Response of cytokinin pool and cytokinin oxidase/dehydrogenase activity to abscisic acid exhibits organ specificity in peas

Changes in cytokinin pool and cytokinin oxidase/dehydrogenase activity (CKX EC: 1.5.99.12) in response to increasing abscisic acid (ABA) concentrations (0.5–10 μM) were assessed in the last fully expanded leaves and secondary roots of two pea (Pisum sativum) varieties with different vegetation periods. Certain organ diversity in CKX response to exogenous ABA was observed. Treatment provoked altered cytokinin pool in the aboveground parts of both studied cultivars. Specific CKX activity was influenced significantly basically in roots of the treated plants. Results suggest that ABA-mediated cytokinin pool changes are leaf-specific and involve certain root signals in which CKX activity presents an important link. This enzymatic activity most probably regulates vascular transport of active cytokinins from roots to shoots.     

Pulmonary fibrosis in L-NAME-treated mice is dependent on an activated endothelin system

Activation of the endothelin (ET) system promotes vasoconstriction, inflammation, and fibrosis in various tissues, including the lung. Therefore, ET-1 transgenic mice overexpressing ET-1 develop pulmonary fibrosis in a slow, age-dependent manner. In vivo, NO is the most important counterregulatory mediator of the ET system and decreases ET-1 promoter activity. The aim of our study was to elucidate the impact on pulmonary inflammation and fibrosis of the interaction between NO and the ET system in young ET-1 transgenic mice before the onset of pulmonary fibrosis. Male ET-1 transgenic mice and wild-type littermates at the age of 8 weeks were randomly allocated to the following 6 groups: WT (n = 11), wild-type animals without treatment; WT + l-NAME (n = 14), wild-type animals receiving l-NAME, an inhibitor of NO synthase; WT + l-NAME + LU (n = 13), wild-type animals receiving l-NAME and LU 302872, a dual ETA/ETB-receptor antagonist; ET1tg (n = 10), ET-1 transgenic mice; ET1tg + l-NAME (n = 13); and ET1tg + l-NAME + LU (n = 13). After 6 weeks, animals were euthanized, and hearts and lungs were harvested for histology and immunohistochemistry. No differences in pulmonary inflammation, as indicated by macrophage infiltration, or in interstitial fibrosis were observed between WT and ET1tg mice at baseline; however, inflammation and interstitial fibrosis were significantly enhanced in ET1tg mice, but not in WT groups, after l-NAME treatment. The combined ETA/ETB-receptor antagonist LU 302872 abolished inflammation and interstitial fibrosis in l-NAME-treated ET1tg mice. Perivascular fibrosis and media/lumen ratio of pulmonary bronchi and arteries did not differ between all study groups. In our study l-NAME induced pulmonary fibrosis and inflammation only in young ET1tg mice. Additional treatment with LU 302872 abolished these effects. We thus conclude that an imbalance between an activated ET system and a suppressed NO system contributes to pulmonary inflammation and fibrosis.     

Syntheses of N3-substituted thymine acyclic nucleoside phosphonates and a comparison of their inhibitory effect towards thymidine phosphorylase

A series of N(3)-substituted thymine acyclic nucleoside phosphonates bearing a number of (phosphonomethoxy)alkyl groups were synthesized and investigated for their ability to inhibit the human thymidine phosphorylase expressed in V79 Chinese hamster cells, as well as thymidine phosphorylase from SD-lymphoma, Escherichia coli and human placenta. In comparison to N(1)- substituted analogues which possess a considerable inhibitory activity towards thymidine phosphorylase from SD-lymphoma, the results showed a marginal inhibitory effect of these compounds. None of the presented N(3)-substituted derivatives possess a significant cytostatic activity.     

Gamma-lactones alpha,beta- and beta,gamma-fused to carbocycles as novel antiproliferative drugs

A set of gamma-lactones alpha,beta-fused and beta,gamma-fused to carbocycles have been synthesized and evaluated for their in vitro antiproliferative activities using the human cancer cell lines SW1573 (lung), T-47D (breast) and WiDr (colon). The compounds are obtained by intramolecular ring closing metathesis of the corresponding dienes. Active compounds exhibited GI(50) values in the range 8-18 microM. A structure-activity relationship is also discussed.     

Cytological changes and alterations in polyamine contents induced by cadmium in tobacco BY-2 cells.

Changes in cell viability, proliferation, cell and nuclear morphology including nuclear and DNA fragmentation induced by 0.05 and 1 mM CdSO4 (Cd2+) in tobacco BY-2 cell line (Nicotiana tabacum L.) were studied in the course of 7 days. Simultaneously changes in endogenous contents of both free and conjugated forms of polyamines (PAs) were investigated for 3 days. The application of 0.05 mM Cd2+ evoked decline of cell viability to approximately 60% during the first 24 h of treatment. Later on degradation of cytoplasmic strands, formation of the stress granules and vesicles, modifications in size and shape of the nuclei, including their fragmentation, were observed in the surviving cells. Their proliferation was blocked and cells elongated. Beginning the first day of treatment TUNEL-positive nuclei were detected in cells cultivated in medium containing 0.05 mM Cd2+. Treatment with highly toxic 1 mM Cd2+ induced fast decrease of cell viability (no viable cells remained after 6-h treatment) and cell death occurred before DNA cleavage might be initiated. The exposure of tobacco BY-2 cells to 0.05 mM Cd2+ resulted in a marked accumulation of total PAs (represented by the sum of free PAs and their perchloric acid (PCA)-soluble and PCA-insoluble conjugates) during 3-day treatment. The increase in total PA contents was primarily caused by the increase in putrescine (Put) concentration. The accumulation of free spermidine (Spd) and spermine (Spm) at 12 and 24 h in 0.05 mM Cd2+ treated BY-2 cells and high contents of Spd and especially Spm determined in dead cells after I mM Cd2+ application was observed. The participation of PA conjugation with hydroxycinnamic acids and PA oxidative deamination in maintaining of free PA levels in BY-2 cells under Cd2+-induced oxidative stress is discussed.     

Benzyladenine-induced inhibition of flowering in Chenopodium rubrum in vitro is not related to the levels of isoprenoid cytokinins

The effect of 6-benzylaminopurine (BAP) on floweringand on endogenous levels of isoprenoid cytokinins wasinvestigated in explanted terminal shoots of Chenopodium rubrum cultivated in vitro. Themother plants were grown under continuous light andexplants were cut off when the 6th leaf primordiumoriginated at the shoot apex. The explants wereexposed to one dark period of 13 hours inductive forflowering or to continuous light on medium with orwithout BAP (0.05;0.2;0.4 mg.l^-1). Undernon-inductive conditions no flowering was observedeither in the control or after BAP treatment. Afterreceiving one inductive dark period, the controlexplants flowered. However, BAP application either atthe beginning of the inductive dark period and/orduring the following light cultivation inhibitedflowering and stimulated initiation and growth of leafprimordia. In the case of the most efficient BAPconcentration (0.05 mg.l^-1) flowering wasinhibited by 80% and the number of leaf primordia wasincreased by 3. Explantation caused a significantincrease in the total amount of endogenous cytokininsin the explants within first 13 h, provided they werekept in light. When explants were kept in darkness,only a slight increase in cytokinin levels wasobserved. BAP treatment had no influence on the levelsof endogenous cytokinins either in light or indarkness. We may thus conclude, that BAP applicationinhibited flowering of photoperiodically inducedterminal shoot explants and stimulated leaf primordiaformation with no significant effect on changes inlevels of endogenous isoprenoid cytokinins. This maysuggest the direct ability of BAP to regulate morphogenesis.     

Identification of Glycoprotein gpTRL10 as a Structural Component of Human Cytomegalovirus

Human cytomegalovirus (HCMV) has a coding capacity for glycoproteins which far exceeds that of other herpesviruses. Few of these proteins have been characterized. We have investigated the gene product(s) of reading frame 10, which is present in both the internal and terminal repeat regions of HCMV strain AD169 and only once in clinical isolates. The putative protein product is a 171-amino-acid glycoprotein with a theoretical mass of 20.5 kDa. We characterized the protein encoded by this reading frame in the laboratory strain AD169 and a recent isolate, TB40E. The results from both strains were comparable. Northern blot analyses showed that the gene was transcribed with early/late kinetics. Two proteins of 22 and 23.5-kDa were detected in virus-infected cells and in cells transiently expressing recombinant TRL10. Both forms contained only high-mannose-linked carbohydrate modifications. In addition, virus-infected cells expressed small amounts of the protein modified with complex N-linked sugars. Image analysis localized transiently expressed TRL10 to the endoplasmic reticulum. Immunoblot analyses as well as immunoelectron microscopy of purified virions demonstrated that TRL10 represents a structural component of the virus particle. Immunoblot analysis in the absence of reducing agents indicated that TRL10, like the other HCMV envelope glycoproteins, is present in a disulfide-linked complex. Sequence analysis of the TRL10 coding region in nine low-passage clinical isolates revealed strain-specific variation. In summary, the protein product of the TRL10 open reading frame represents a novel structural glycoprotein of HCMV and was termed gpTRL10.     

Standardized generation of fully mature p70 IL-12 secreting monocyte-derived dendritic cells for clinical use

Dendritic cells (DC) have been shown to be efficient antigen-presenting cells (APC) and, as such, could be considered ideal candidates for cancer immunotherapy. Immature DC (iDC) efficiently capture surrounding antigens; however, only mature DC (mDC) prime naive T lymphocytes. Clinical trials using DC-based tumor vaccines have achieved encouraging, but limited, success, possibly due to the use of immature or incompletely mature DC. Thus, it was apparent that a method capable of generating large numbers of fully functional iDC, their pulsing with desired form of tumor antigens and the subsequent complete and reproducible maturation of iDC is needed. Therefore, we compared two different methods of producing large numbers of iDC. Both protocols yielded comparable numbers of cells with an iDC phenotype with phagocytic function. We next determined which of the clinically applicable activators could induce the complete and reproducible maturation of DC, in order to define the most suitable combination for future clinical trials. Only a combination of TNFalpha + Poly (I:C), or a previously described cytokine cocktail of TNFalpha + IL-1beta + IL-6 + prostaglandin E2, induced the complete activation of the whole DC population, as assessed by the cell surface expression of CD83 and costimulatory molecules. The matured DC were functionally superior to iDC in their ability to stimulate the proliferation of allogeneic lymphocytes and autologous keyhole limpet hemocyanin (KLH)-specific T lymphocytes. Furthermore, only the combination of TNFalpha + Poly (L:C) activated DC to produce large amounts of biologically active p70 IL-12. Thus DC maturation by TNFalpha + Poly (I:C) could efficiently bias T cell response towards Th1 response. Implementation of our results into clinical protocols used for DC generation could be beneficial for future immunotherapy trials.