Two cathepsin L proteinases, cathepsin L1 and cathepsin L2, secreted by liver flukes may be involved in tissue penetration, nutrition, and protection from immune attack. To ascertain the immunoprophylactic potential of these proteinases, and of another molecule, liver fluke hemoglobin (Hb), we performed vaccine trials in cattle. In the first vaccine trial various doses of cathepsin L1 were tested. The mean protection level obtained was 53.7%. In a second vaccine trial cathepsin L1 and Hb elicited 42.5 and 43.8% protection levels, respectively, while a combination of the two molecules induced a significantly higher level of protection (51.9%). Cathepsin L2 was not examined alone; however, vaccination of cattle with a combination of cathepsin L2 and Hb elicited the highest level of protection (72.4%). The animals that received cathepsin L1-Hb or cathepsin L2-Hb showed reduced liver damage as assessed by serum glutamic dehydrogenase and gamma-glutamyl transferase levels. Furthermore, a reduced viability was observed for fluke eggs recovered from all vaccine groups. This anti-embryonation effect of vaccination was particularly evident in the group that received cathepsin L2-Hb where >98% of the eggs recovered did not embryonate to miracidia. Although all vaccine preparations induced high antibody titers which were boosted following the challenge infection, there was no correlation between antibody titers and protection. The results of these trials demonstrate that cathepsin Ls and Hb could form the basis of a molecular vaccine that would not only reduce parasite burden but would also prevent transmission of liver fluke disease.

Inhibitors of Rho kinase (ROCK) are a relatively new class of drugs with potential benefits in oncology, neurology, and fibrotic and cardiovascular diseases. ROCK-inhibitors modulate many cellular functions, some of which are similar to the pleiotropic effects of statins, suggesting additive or synergistic properties. Studies to date have used compounds which inhibit both isoforms of ROCK, ROCK1 and ROCK2. This study was designed to compare gene expression profiles of atorvastatin with the newly developed ROCK2-inhibitor SLx-2119 in primary cultures of normal human endothelial cells, smooth muscle cells, and fibroblasts. Cells were treated with each compound for 24 hours, after which total RNA was isolated and genome-wide gene expression profiles were obtained with Illumina arrays. Because of the known effect of statins on the actin cytoskeleton and on connective tissue growth factor (CTGF), a prominent growth factor involved in tissue fibrosis, the effects of SLx-2119 and atorvastatin on the actin cytoskeleton and CTGF mRNA were also examined in cultures of smooth muscle cells with a fibrotic phenotype, isolated from biopsies of human intestine with radiation-induced fibrosis. Although SLx-2119 and atorvastatin affected expression of genes belonging to the same biological processes, individual genes were mostly different, consistent with synergistic or additive properties. Both SLx-2119 and atorvastatin reduced CTGF mRNA and remodeled the actin cytoskeleton in fibrosis-derived smooth muscle cells, suggesting that both compounds have anti-fibrotic properties. These results form the basis for further studies to assess the possible therapeutic benefit of combined treatments.

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A 29.5-kDa cysteine proteinase was purified from medium in which mature Fasciola hepatica parasites were maintained. The N-terminal sequence (14 residues) of the purified protein is similar to known cathepsin L proteinases, including a 27-kDa cathepsin L proteinase, also secreted by this parasite, which had been isolated previously in our laboratory [Smith, A. M., Dowd, A. J., Mc Gonigle, S., Keegan, P. S., Brennan, G., Trudgett, A. & Dalton, J. P. (1993) Mol. Biochem. Parasitol. 62,. The N-terminal sequences of the 29.5-kDa and 27-kDa cathepsin L proteinases differ only in residue number seven (arginine and proline, respectively). Immunoblot studies, using antiserum that reacts with both cathepsin L proteinases, rule out the possibility of both enzymes arising from a higher molecular sized parent molecule. The reaction kinetics of the two F. hepatica cathepsin L proteinases on a variety of peptide substrates revealed that the two enzymes differ in their substrate specificity. Five peptide substrates that are cleaved with high affinity by the 29.5-kDa cathepsin L isolated in this study are not cleaved by the previously purified 27-kDa cathepsin L. The protein-modifying reagent, tetranitromethane, affected the 29.5-kDa cathepsin L proteinase only, causing inactivation of the enzyme and changing its migration in polyacrylamide gel electrophoresis. Our studies suggest that the two F. hepatica cysteine proteinases represent two distinct subclasses within the cathepsin L class.Cathepsin L proteases are lysosomal cysteine proteinases which, along with cathepsins B, H and S, belong to the papain superfamily [ l -31. Cathepsin L can hydrolyse a variety of substrates such as extracellular matrix proteins, collagen and elastin [l, 41 and type-IV collagen which is a major component of basement membranes [l]. Cathepsin B can indirectly cause the degradation of collagen since it is capable of converting inactive procollagenase IV to its enzymically active form [5]. It is suggested that where certain malignant tissues secrete cathepsin-like proteinases, these enzymes may be involved in extracellular matrix destruction, in the facilitation of cellular detachment from primary tumour masses, and in reinvasion of tissue [4].Cathepsins L and B are synthesised as inactive, alkalistable, precursors (proenzymes) of 39 kDa prior to their targeting to the lysosomes [6, 71. Cathepsin H is synthesised as a 41-kDa proenzyme [7]. The proenzymes are subseCorrespondence to J. P. Dalton, School of Biological Sciences,

Inhibition of Poly(ADP-ribose) Polymerase1 (PARP1) impairs DNA damage repair, and early generation PARP1/2 inhibitors (olaparib, niraparib, etc.) have demonstrated clinical proof of concept for cancer treatment. Here, we describe the development of the novel PARP inhibitor E7449, a potent PARP1/2 inhibitor that also inhibits PARP5a/5b, otherwise known as tankyrase1 and 2 (TNKS1 and 2), important regulators of canonical Wnt/β-catenin signaling. E7449 inhibits PARP enzymatic activity and additionally traps PARP1 onto damaged DNA; a mechanism previously shown to augment cytotoxicity. Cells deficient in DNA repair pathways beyond homologous recombination were sensitive to E7449 treatment. Chemotherapy was potentiated by E7449 and single agent had significant antitumor activity in BRCA-deficient xenografts. Additionally, E7449 inhibited Wnt/β-catenin signaling in colon cancer cell lines, likely through TNKS inhibition. Consistent with this possibility, E7449 stabilized axin and TNKS proteins resulting in β-catenin de-stabilization and significantly altered expression of Wnt target genes. Notably, hair growth mediated by Wnt signaling was inhibited by E7449. A pharmacodynamic effect of E7449 on Wnt target genes was observed in tumors, although E7449 lacked single agent antitumor activity in vivo, a finding typical for selective TNKS inhibitors. E7449 antitumor activity was increased through combination with MEK inhibition. Particularly noteworthy was the lack of toxicity, most significantly the lack of intestinal toxicity reported for other TNKS inhibitors. E7449 represents a novel dual PARP1/2 and TNKS1/2 inhibitor which has the advantage of targeting Wnt/β-catenin signaling addicted tumors. E7449 is currently in early clinical development.

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Telehealth incorporated the cost-saving ability for HPN patients to maintain proper medical care, support, and collaboration of specialists inaccessible to their local community. Thus, its strongly positive role in HPN care deserves further consideration for a national application.