Summary There is pressing need to develop alternatives to annual influenza vaccines and antiviral agents licensed for mitigating influenza infection. Previous studies reported that acute lung injury (ALI) caused by chemical or microbial insults is secondary to generation of host-derived, oxidized phospholipid that potently stimulates Toll-like Receptor 4 (TLR4)-dependent inflammation1. Subsequently, we reported that TLR4−/− mice are highly refractory to influenza-induced lethality2, and hypothesized that therapeutic antagonism of TLR4 signaling would protect against influenza-induced ALI. Herein, we report that therapeutic administration of Eritoran (E5564), a potent, well-tolerated, synthetic TLR4 antagonist3,4, blocks influenza-induced lethality in mice, as well as lung pathology, clinical symptoms, cytokine and oxidized phospholipid expression, and decreases viral titers. CD14 and TLR2 are also required for Eritoran-mediated protection, and CD14 directly binds Eritoran and inhibits ligand binding to MD2. Thus, Eritoran blockade of TLR signaling represents a novel therapeutic approach for inflammation associated with influenza, and possibly other, infections.

Normal human liver tissue and cultured human hepatocytes are valuable models to study xenobiotic metabolism and toxicity, but they only have a ilimited in vitro life-span and are not readily available. This report describes the establishment of replicative cultures of human adult liver epithelial cells in serum-free medium. The longevity of three of these cultures, derived from different donors, was cytes were transformed with adenovirus or adenovirus DNA, whereas transformation of rat fetal hepatocytes with 3'-methyl-4-dimethylaminoazobenzene led to cell lines that lack expression of albumin and P2-macroglobulin (7, 8).Metabolic activation of environmental carcinogens from several chemical classes have been studied in human liver tissue explants or microsomes and isolated human hepatocytes (4, 9-19). Furthermore, observed animal speciesspecific differences in aflatoxin B1 (AFB1) and 2-acetylaminofluorene metabolism indicate the need for studying human liver or hepatocytes (17, 18, 20). However, because tissue availability is limited, individuals vary in their propensity for xenobiotic metabolism, and standardized in vitro conditions are difficult to establish, a reproducible system with human liver cells for pharmacotoxicological studies has not been established.In this report, the immortalization of adult human liver epithelial cells from two different nondiseased donors with the SV40 T antigen is described. These cell lines are shown to be nontumorigenic, express hepatocyte differentiation markers, and possess enzymatic pathways responsible for xenobiotic metabolism. For example, AFB1, a known risk factor for human hepatocellular carcinoma (21), requires metabolic activation to exert its carcinogenic effects and is associated with a mutational hotspot in the p53 tumor-suppressor gene at the codon for 23). Thus, THLE-2 and -3 liver cell lines will be beneficial for multiple applications: standardized toxicological in vitro tests; investigations of species-specific nmechanisms of carcinogens and anticarcinogens; assessment ofthe mode ofaction ofbiologically active compounds; in vitro studies to assay factors involved in liver cancer, such as hepatitis infection; and protooncogene activation or tumorsuppressor gene inactivation. -7,t-8-dihydroxy-c-9,10epoxy-7,8,9,10tetrahydrobenzo[a]py- MATERIALS AND METHODS

Breast tumor cells express the chemokine receptor CXCR3, which binds the ligands CXCL9, CXCL10, and CXCL11. CXCR3 and other chemokine receptors may mediate tumor metastasis by supporting migration of tumor cells to sites of ligand expression including the lymph nodes, lungs, and bone marrow. We examined the relationship of CXCR3 expression to clinical outcome in 75 women diagnosed with early-stage breast cancer. We detected CXCR3 in malignant epithelium from all tumors. Twelve percent were weakly positive and 64% had moderate levels of CXCR3. Strong CXCR3-positive staining was observed in 24% of tumors. Kaplan-Meier survival curves showed that high CXCR3 expression was associated with poorer overall survival; the unadjusted hazard ratio was 1.56 and it was marginally significant (P = 0.07). When interactions between lymph node status and CXCR3 were considered, the adjusted hazard ratio for CXCR3 was 2.62 (P = 0.02) for women with nodenegative disease at diagnosis, whereas the hazard ratio for CXCR3 was not significant for those with node-positive disease. CXCR3 gene silencing inhibited lung colonization and spontaneous lung metastasis from mammary glandimplanted tumors in a murine model. The size or growth rate of the locally growing tumors was not affected. The antimetastatic effect of CXCR3 gene silencing was compromised in mice depleted of Natural Killer cells or with mutations in IFN-;, suggesting that the role of CXCR3 is not simply to mediate tumor cell trafficking. These studies support the continued examination of CXCR3 as a potential therapeutic target in patients with breast cancer. [Mol Cancer Ther 2009;8(3):490 -8]

We previously reported that TLR4-/- mice are refractory to mouse-adapted A/PR/8/34 (PR8) influenza-induced lethality and that therapeutic administration of the TLR4 antagonist, Eritoran, blocked PR8-induced lethality and acute lung injury (ALI) when given starting 2 days post-infection. Herein, we extend these findings: anti-TLR4- or TLR2-specific IgG therapy also conferred significant protection of wild-type (WT) mice from lethal PR8 infection. If treatment is initiated 3 h prior to PR8 infection and continued daily for 4 days, Eritoran failed to protect WT and TLR4-/- mice, implying that Eritoran must block a virus-induced, non-TLR4 signal that is required for protection. Mechanistically, we determined that (i) Eritoran blocks HMGB1-mediated, TLR4-dependent signaling in vitro and circulating HMGB1 in vivo, and an HMGB1 inhibitor protects against PR8; (ii) Eritoran inhibits pulmonary lung edema associated with ALI, (iii) IL-1β contributes significantly to PR8-induced lethality, as evidenced by partial protection by IL-1 receptor antagonist (IL-1Ra) therapy. Synergistic protection against PR8-induced lethality was achieved when Eritoran and the anti-viral drug, oseltamivir, were administered starting 4 days post-infection. Eritoran treatment does not prevent development of an adaptive immune response to subsequent PR8 challenge. Overall, our data support the potential of a host-targeted therapeutic approach to influenza infection.

Neutral red (NR) in medium was absorbed and concentrated in lysosomes of cultured rat and human hepatocytes. NR uptake increased with the time of incubation and reached a plateau in 2 hr. Uptake was proportional to the concentration of the NR solution and the numbers of viable liver cells. Prolonged culture of hepatocytes increased the numbers of lysosomes, and thus, the dye accumulation. The NR can be extracted from lysosomes for quantitative measurement of hepatocyte viability and cytotoxicity of xenobiotics. With this assay, several serum-free media (e.g., Waymouth's, MEM, LHC-8, etc.) were compared for the maintenance of viable hepatocytes in vitro. Interestingly, LHC-8 medium, which is used to grow human bronchial epithelial cells, best preserved viable rat hepatocytes. The cytotoxic effects of dimethylnitrosamine (DMN) and aflatoxin B1 (AFB1) were examined by NR assay on rat and human hepatocyte cultures and were found to be dependent on dose and time of the exposures. NR50 was 20 mM for DMN and 0.072 microM for AFB1 in rat hepatocytes with 24 hr of exposures and reduced to 12.5 mM for DMN and 0.053 mu microM for AFB1 with 48 hr exposures. Human hepatocytes were more resistant to the toxicity of both chemicals; NR50 values were 100 mM DMN and 1.8 microM AFB1 respectively, for 24 hr treatments. Compared with lactate dehydrogenase (LDH) leakage test, the NR assay was simpler and more sensitive in determining the viability and cytotoxicity of xenobiotics in primary cultures of hepatocytes.