Endothelial cells (ECs) not only are important for oxygen delivery but also act as a paracrine source for signals that determine the balance between tissue regeneration and fibrosis. Here we show that genetic inactivation of flow-induced transcription factor Krüppel-like factor 2 (KLF2) in ECs results in reduced liver damage and augmentation of hepatocyte proliferation after chronic liver injury by treatment with carbon tetrachloride (CCl4). Serum levels of GLDH3 and ALT were significantly reduced in CCl4-treated EC-specific KLF2-deficient mice. In contrast, transgenic overexpression of KLF2 in liver sinusoidal ECs reduced hepatocyte proliferation. KLF2 induced activin A expression and secretion from endothelial cells in vitro and in vivo, which inhibited hepatocyte proliferation. However, loss or gain of KLF2 expression did not change capillary density and liver fibrosis, but significantly affected hepatocyte proliferation. Taken together, the data demonstrate that KLF2 induces an antiproliferative secretome, including activin A, which attenuates liver regeneration.

Therapy resistance and tumor recurrence are often linked to a small refractory and highly tumorigenic subpopulation of neoplastic cells, known as cancer stem cells (CSCs). A putative marker of CSCs is CD133 (prominin-1). We have previously described a CD133-targeted oncolytic measles virus (MV-CD133) as a promising approach to specifically eliminate CD133-positive tumor cells. Selectivity was introduced at the level of cell entry by an engineered MV hemagglutinin (H). The H protein was blinded for its native receptors and displayed a CD133-specific single-chain antibody fragment (scFv) as targeting domain. Interestingly, MV-CD133 was more active in killing CD133-positive tumors than the unmodified MV-NSe despite being highly selective for its target cells. To further enhance the antitumoral activity of MV-CD133, we here pursued arming technologies, receptor extension, and chimeras between MV-CD133 and vesicular stomatitis virus (VSV). All newly generated viruses including VSV-CD133 were highly selective in eliminating CD133-positive cells. MV-CD46/CD133 killed in addition CD133-negative cells being positive for the MV receptors. In an orthotopic glioma model, MV-CD46/CD133 and MVSCD-CD133, which encodes the super cytosine deaminase, were most effective. Notably, VSV-CD133 caused fatal neurotoxicity in this tumor model. Use of CD133 as receptor could be excluded as being causative. In a subcutaneous tumor model of hepatocellular cancer, VSV-CD133 revealed the most potent oncolytic activity and also significantly prolonged survival of the mice when injected intravenously. Compared to MV-CD133, VSV-CD133 infected a more than 104-fold larger area of the tumor within the same time period. Our data not only suggest new concepts and approaches toward enhancing the oncolytic activity of CD133-targeted oncolytic viruses but also raise awareness about careful toxicity testing of novel virus types.

Classical Hodgkin lymphoma (cHL) is a hematopoietic malignancy with a characteristic cellular composition. The tumor mass is made up of infiltrated lymphocytes and other cells of hematologic origin but only very few neoplastic cells that are mainly identified by the diagnostic marker CD30. While most patients with early stage cHL can be cured by standard therapy, treatment options for relapsed or refractory cHL are still not sufficient, although immunotherapy-based approaches for the treatment of cHL patients have gained ground in the last decade. Here, we suggest a novel therapeutic concept based on oncolytic viruses selectively destroying the CD30+-positive cHL tumor cells. Relying on a recently described CD30-specific scFv we have generated CD30-targeted measles virus (MV-CD30) and vesicular stomatitis virus (VSV-CD30). For VSV-CD30 the VSV glycoprotein G reading frame was replaced by those of the CD30-targeted MV glycoproteins. Both viruses were found to be highly selective for CD30-positive cells as demonstrated by infection of co-cultures of target and non-target cells as well as through blocking infection by soluble CD30. Notably, VSV-CD30 yielded much higher titers than MV-CD30 and resulted in a more rapid and efficient killing of cultivated cHL-derived cell lines. Mouse tumor models revealed that intratumorally, as well as systemically injected VSV-CD30, infected cHL xenografts and significantly slowed down tumor growth resulting in a substantially prolonged survival of tumor-bearing mice. Taken together, the data support further preclinical testing of VSV-CD30 as novel therapeutic agent for the treatment of cHL and other CD30+-positive malignancies.

Abstract. Spontaneous cholelithiasis was found in a male African green monkey (Chlorocebus aethiops) at necropsy. Choleliths varied in size, shape and colour. Gallstones were analysed using accepted analytical methods. Results showed that the gallstones were composed of cholesterol and protein in varying proportions. Histologically, the gallbladder showed diffuse mild to moderate lymphocytic infiltration. The etiology of the cholelithiasis in the examined individual remains unknown.

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