Michael M. Lipsky

The TLR4 Antagonist, Eritoran, Protects Mice from Lethal Influenza Infection

There is a pressing need to develop alternatives to annual influenza vaccines and antiviral agents licensed for mitigating influenza infection. Previous studies reported that acute lung injury caused by chemical or microbial insults is secondary to the generation of host-derived, oxidized phospholipid that potently stimulates Toll-like receptor 4 (TLR4)-dependent inflammation. Subsequently, we reported that Tlr4−/− mice are highly refractory to influenza-induced lethality, and proposed that therapeutic antagonism of TLR4 signalling would protect against influenza-induced acute lung injury. Here we report that therapeutic administration of Eritoran (also known as E5564)—a potent, well-tolerated, synthetic TLR4 antagonist—blocks influenza-induced lethality in mice, as well as lung pathology, clinical symptoms, cytokine and oxidized phospholipid expression, and decreases viral titres. 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 signalling represents a novel therapeutic approach for inflammation associated with influenza, and possibly other infections.

Neutral red (NR) assay for cell viability and xenobiotic-induced cytotoxicity in primary cultures of human and rat hepatocytes

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., Waymouths, 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 µM for AFB1 in rat hepatocytes with 24 hr of exposures and reduced to 12.5 mM for DMN and 0.053 µ uM for AFB1 with 48 fr exposures. Human hepatocytes were more resistant to the toxicity of both chemicals; NR50 values were 100 mM DMN and 1.8 µM 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.

CXCR3 expression is associated with poor survival in breast cancer and promotes metastasis in a murine model

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 node-negative 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 gland–implanted 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-γ, 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]

Oxidative stress induced by lead, cadmium and arsenic mixtures: 30-day, 90-day, and 180-day drinking water studies in rats: an overview.

Humans are frequently exposed to combinations of lead (Pb), cadmium (Cd) and Arsenic (As) but there is a paucity of actual data on the molecular effects of these agents at low dose levels. The present factorial design studies were undertaken in rats to examine the effects of these agents at LOEL dose levels on a number of molecular parameters of oxidative stress in hematopoietic and renal organ systems following oral exposure in drinking water at 30, 90 and 180 day time points. Results of these studies demonstrated dynamic, time-dependent alterations in both molecular targets and inducible oxidative stress protective systems in target cell populations. In general, cellular protective systems, which protected against oxidative damage at the 90 day time point, appeared to be finite such that molecular manifestations of oxidative stress became statistically significant at the 180 day time point for several of the combination exposure groups. These data demonstrate the importance of duration of exposure in assessing the toxic potential of Pb, Cd and As mixtures at low dose levels.

MOUSE LIVER CELL CULTURE II. Primary Culture

SummaryMouse hepatocytes in primary culture were characterized. Hepatocytes were isolated by the two-step hepatic portal vein perfusion method described previously. An optimal cell attachment of 43% was noted after 2 h incubation in 10% fetal bovine serum. Minimal attachment (lees than 7%) occurred in serumless medium. Serum concentrations above 10% and attachment durations greater than 2 h resulted in no increased attachment of viable cells. Nonviable cells, however progressively attached when both of these parameters were increased. Survival data of the cells in culture resembled those reported for rat hepatocytes in primary culture. A progressive decrease in survival was noted following initial attachment until only approximately 15% of initially plated cells remained viable and attached after 8 d culture. The decrease in survival was accompanied by morphologic changes including flattening and elongation of the cells, some multinucleation, and disruption of monolayer groups.

Alteration in protein synthesis in primary cultures of rat kidney proximal tubule epithelial cells by exposure to gallium, indium, and arsenite☆

Patterns of protein synthesis in primary cultures of rat kidney proximal epithelial tubule cells were examined following exposure to gallium (Ga) chloride, indium (In) chloride, and sodium arsenite. After incubation with these chemicals for 20 hr, newly synthesized proteins were labeled with [35S]methionine. 35S-labeled proteins in the cells were separated by SDS/polyacrylamide gel and two-dimensional gel electrophoresis and detected by fluorography. A protein with molecular weight (Mr) of 30,000 was markedly induced by exposure to 300 microM Ga or 10 microM arsenite, and synthesis of proteins with Mr of 85,000, 71,000, 65,000, 51,000, 38,000, and 28,000 was also increased by Ga or arsenite. Arsenite exposure increased synthesis of eight different proteins, which were not induced by Ga. No significant changes in protein synthesis were observed with 300 microM In exposure. Release of lactate dehydrogenase from the cells was not significantly increased by exposure to concentrations of 300 microM Ga and 3 microM arsenite or less. In the absence of overt cell injury, the induction of these proteins may be useful as an early indicator for assessing exposure to Ga.

Exposure to Pb, Cd, and As mixtures potentiates the production of oxidative stress precursors: 30-day, 90-day, and 180-day drinking water studies in rats ☆

Exposure to chemical mixtures is a common and important determinant of toxicity and is of particular concern due to their appearance in sources of drinking water. Despite this, few in vivo mixture studies have been conducted to date to understand the health impact of chemical mixtures compared to single chemicals. Interactive effects of lead (Pb), cadmium (Cd) and arsenic (As) were evaluated in 30-, 90-, and 180-day factorial design drinking water studies in rats designed to test the hypothesis that ingestion of such mixtures at individual component Lowest-Observed-Effect-Levels (LOELs) results in increased levels of the pro-oxidant delta aminolevulinic acid (ALA), iron, and copper. LOEL levels of Pb, Cd, and As mixtures resulted in the increased presence of mediators of oxidative stress such as ALA, copper, and iron. ALA increases were followed by statistically significant increases in kidney copper in the 90- and 180-day studies. Statistical evidence of interaction was identified for six biologically relevant variables: blood delta aminolevulinic acid dehydratase (ALAD), kidney ALAD, urinary ALA, urinary iron, kidney iron, and kidney copper. The current investigations underscore the importance of considering interactive effects that common toxic agents such as Pb, Cd, and As may have upon one another at low-dose levels. The interactions between known toxic trace elements at biologically relevant concentrations shown here demonstrate a clear need to rigorously review methods by which national/international agencies assess health risks of chemicals, since exposures may commonly occur as complex mixtures.

Comparison of trout hepatocyte culture on different substrates

SummaryComparisons were made of attachment and viability of rainbow trout (Salmo gairdneri) hepatocytes in short-term (2 days), primary culture on plastic, collagen-coated or extracellular matrix (ECM) coated dishes. Hepatocyte isolation routinely yielded cells with good viability (96%). Cells plated on ECM attached with high efficiency (93%) in contrast to cells cultured on plastic or collagen (∼20%). The cells plated on ECM flattened out and formed monolayers, while the cells on plastic and collagen rounded up and formed multi-cell aggregates in suspension. Viability of cells in all substrates remained high over the 2 day culture period. ECM is the first substrate to support trout-hepatocyte attachment in primary culture. Differentiated liver function was maintained in cells cultured on ECM as evidence by the induction of tyrosine aminotransferase by hydrocortisone (200%).

Isolation and culture of hepatocytes from human liver of immediate autopsy.

SummaryHuman livers were removed at immediate autopsy (IA) from brain death patients within 1 h after cessation of cardiac function. Viable hepatocytes were isolated successfully from these IA livers by perfusion of an intack lobe with collagenase or by digestion of a small tissue wedge with collagenase-dispase. The yields of hepatocytes ranged from 1 to 3 × 106 cells/g liver in the five cases studied. Approximately 70 to 90% of the cells excluded trypan blue dye. In the isolated hepatocytes, 632 pmol/mg protein of cytochromep450 and 536. pmol/mg protein cytochromeb5 were measured. The cells attached to the dishes in 4 h and produced monolayer cultures with a high success rate. The cells maintained in primary cultures for several days and developed ultrastructural features characteristic of human hepatocytes in vivo. The cultured hepatocytes can hydroxylate benzo[a]pyrene, conjugate the metabolites, and have a benzo[a]pyrene hydroxylase activity of 48.7 pmol/mg DNA per h, which is comparable to that of rat hepatocytes. The liver cells repaired DNA damage caused by exposures to aminofluorene and acetylaminofluorene in culture.

STAT6 Expression in Multiple Cell Types Mediates the Cooperative Development of Allergic Airway Disease

Th2 cells induce asthma through the secretion of cytokines. Two such cytokines, IL-4 and IL-13, are critical mediators of many features of this disease. They both share a common receptor subunit, IL-4Rα, and signal through the STAT6 pathway. STAT6−/− mice have impaired Th2 differentiation and reduced airway response to allergen. Transferred Th2 cells were not able to elicit eosinophilia in response to OVA in STAT6−/− mice. To clarify the role of STAT6 in allergic airway inflammation, we generated mouse bone marrow (BM) chimeras. We observed little to no eosinophilia in OVA-treated STAT6−/− mice even when STAT6+/+ BM or Th2 cells were provided. However, when Th2 cells were transferred to STAT6×Rag2−/− mice, we observed an eosinophilic response to OVA. Nevertheless, the expression of STAT6 on either BM-derived cells or lung resident cells enhanced the severity of OVA-induced eosinophilia. Moreover, when both the BM donor and recipient lacked lymphocytes, transferred Th2 cells were sufficient to induce the level of eosinophilia comparable with that of wild-type (WT) mice. The expression of STAT6 in BM-derived cells was more critical for the enhanced eosinophilic response. Furthermore, we found a significantly higher number of CD4+CD25+Foxp3+ T cells (regulatory T cells [Tregs]) in PBS- and OVA-treated STAT6−/− mouse lungs compared with that in WT animals suggesting that STAT6 limits both naturally occurring and Ag-induced Tregs. Tregs obtained from either WT or STAT6−/− mice were equally efficient in suppressing CD4+ T cell proliferation in vitro. Taken together, our studies demonstrate multiple STAT6-dependent and -independent features of allergic inflammation, which may impact treatments targeting STAT6.