Safety, Tumor Reduction, and Clinical Impact of Zika Virus Injection in Dogs with Advanced-Stage Brain Tumors.

Malignant brain tumors are among the most aggressive cancers with poor prognosis and no effective treatment. Recently, we reported the oncolytic potential of Zika virus infecting and destroying the human central nervous system (CNS) tumors in vitro and in immunodeficient mice model. However, translating this approach to humans requires pre-clinical trials in another immunocompetent animal model. Here, we analyzed the safety of Brazilian Zika virus (ZIKVBR) intrathecal injections in three dogs bearing spontaneous CNS tumors aiming an anti-tumoral therapy. We further assessed some aspects of the innate immune and inflammatory response that triggers the anti-tumoral response observed during the ZIKVBR administration in vivo and in vitro. For the first time, we showed that there were no negative clinical side effects following ZIKVBR CNS injections in dogs, confirming the safety of the procedure. Furthermore, the intrathecal ZIKVBR injections reduced tumor size in immunocompetent dogs bearing spontaneous intracranial tumors, improved their neurological clinical symptoms significantly, and extended their survival by inducing the destruction specifically of tumor cells, sparing normal neurons, and activating an immune response. These results open new perspectives for upcoming virotherapy using ZIKV to destroy and induce an anti-tumoral immune response in CNS tumors for which there are currently no effective treatments.

Delayed osteoblastic differentiation and bone development in Cx43 knockout mice.

GJA1 gene (Connexin43, also known as Cx43) is the most abundant gap junction protein isoform in animal cells and is associated with bone development in embryos. The objective of the present work was to evaluate in vivo osteal development in GJA1-deficient fetal mice through determination of the histological and molecular alterations induced by partial or total deletion of the GJA1 gene. Heterozygous C57BL/6 mice (HT) harboring a null mutation of the GJA1 gene were mated, and pregnant females were submitted to euthanasia and Caesarean section from 12.5 to 19.5 days post coitum (dpc). HT (GJA1(+/-)) and homozygous (GJA1(-/- )) knockout (KO) mutants and wild-type (WT) fetuses were identified by polymerase chain reaction (PCR), and development curves were constructed on the basis of fetus weight and crown-rump length. Histopathological, histochemical, and real-time PCR analyses were performed in order to assess the expression of markers associated with bone development, namely, osteocalcin, osteopontin, alkaline phosphatase, RUNX2, GJA1, GJC1 (Cx45), and GJA3 (Cx46). HT and KO fetuses exhibited delays in the differentiation of osteoblasts and, consequently, in bone development in comparison with the WT group. Additionally, less deposition of mineralized and osteoid matrix was observed in GJA1-deficient fetuses. Bone development in KO fetuses was delayed through the moment of birth, but in HT animals the delay only extended until 17.5 dpc, following which development was normalized. The expression of genes coding for osteocalcin, osteopontin, alkaline phosphatise, and RUNX2 were also delayed in GJA1-deficient fetuses. Animals that exhibited a lower expression of GJA1 presented delayed expression of the GJC1 and GJA3 genes and their corresponding protein products in the bone tissue. The results of the present study contribute to our understanding of the function of GJA1 during bone development and suggest that GJC1 could play a role in restoring intercellular communication in GJA1-deficient mice.

Pfaffosidic Fraction from Hebanthe paniculata Induces Cell Cycle Arrest and Caspase-3-Induced Apoptosis in HepG2 Cells.

Hebanthe paniculata roots (formerly Pfaffia paniculata and popularly known as Brazilian ginseng) show antineoplastic, chemopreventive, and antiproliferative properties. Functional properties of these roots and their extracts are usually attributed to the pfaffosidic fraction, which is composed mainly by pfaffosides A-F. However, the therapeutic potential of this fraction in cancer cells is not yet entirely understood. This study aimed to analyze the antitumoral effects of the purified pfaffosidic fraction or saponinic fraction on the human hepatocellular carcinoma HepG2 cell line. Cellular viability, proliferation, and apoptosis were evaluated, respectively, by MTT assay, BrdU incorporation, activated caspase-3 immunocytochemistry, and DNA fragmentation assay. Cell cycle was analyzed by flow cytometry and the cell cycle-related proteins were analyzed by quantitative PCR and Western blot. The cells exposed to pfaffosidic fraction had reduced viability and cellular growth, induced G2/M at 48 h or S at 72 h arrest, and increased sub-G1 cell population via cyclin E downregulation, p27(KIP1) overexpression, and caspase-3-induced apoptosis, without affecting the DNA integrity. Antitumoral effects of pfaffosidic fraction from H. paniculata in HepG2 cells originated by multimechanisms of action might be associated with cell cycle arrest in the S phase, by CDK2 and cyclin E downregulation and p27(KIP1) overexpression, besides induction of apoptosis through caspase-3 activation.

Morphological and molecular pathology of CCL4-induced hepatic fibrosis in connexin43-deficient mice.

Gap junction channels, formed by connexins (Cx), are involved in the maintenance of tissue homeostasis, cell growth, differentiation, and development. Several studies have shown that Cx43 is involved in the control of wound healing in dermal tissue. However, it remains unknown whether Cx43 plays a role in the control of liver fibrogenesis. Our study investigated the roles of Cx43 heterologous deletion on carbon tetrachloride (CCl(4))-induced hepatic fibrosis in mice. We administered CCl(4) to both Cx43-deficient (Cx43(+/-)) and wild-type mice and examined hepatocellular injury and collagen deposition by histological and ultrastructural analyses. Serum biochemical analysis was performed to quantify liver injury. Hepatocyte proliferation was analyzed immunohistochemically. Protein and messenger RNA (mRNA) expression of liver connexins were evaluated using immunohistochemistry as well as immunoblotting analysis and quantitative real-time PCR. We demonstrated that Cx43(+/-) mice developed excessive liver fibrosis compared with wild-type mice after CCl(4) -induced chronic hepatic injury, with thick and irregular collagen fibers. Histopathological evaluation showed that Cx43(+/-) mice present less necroinflammatory lesions in liver parenchyma and consequent reduction of serum aminotransferase activity. Hepatocyte cell proliferation was reduced in Cx43(+/-) mice. There was no difference in Cx32 and Cx26 protein or mRNA expression in fibrotic mice. Protein expression of Cx43 increased in CCl(4)-treated mice, although with aberrant protein location on cytoplasm of perisinusoidal cells. Our results demonstrate that Cx43 plays an important role in the control and regulation of hepatic fibrogenesis.