The biological effects of monocyte chemoattractant protein (MCP) 1 are mediated by binding to C-C chemokine receptor (CCR) 2. In the present studies, we used CCR2 knockout (CCR2؊/؊) mice to examine the role of MCP-1 in acetaminophen-induced macrophage accumulation in the liver, expression of inflammatory cytokines, and hepatotoxicity. We found that hepatic expression of CCR2 and MCP-1 was increased 10-fold and 20-fold, respectively, 12 to 72 hours after administration of acetaminophen to wild-type mice. Expression of these proteins was localized in centrilobular regions of the liver. Whereas MCP-1 was expressed by both hepatocytes and macrophages, CCR2 was identified in inflammatory macrophages. F4/80 is a marker of mature macrophages expressed in large quantities by Kupffer cells. In wild-type mice, a 75% decrease in F4/80-positive macrophages was observed 24 to 48 hours after administration of acetaminophen. In contrast, expression of macrosialin (CD68), a marker of activated macrophages, increased 2-fold 24 to 72 hours after administration of acetaminophen and was associated with inflammatory cells. Although there was a decrease in the overall severity of inflammation and in the number of macrosialin-positive macrophages 72 hours after administration of acetaminophen in CCR2؊/؊ mice, the number of F4/80-positive cells did not change. Loss of CCR2 was also found to alter acetaminophen-induced expression of tumor necrosis factor ␣, monocyte chemoattractant protein 3, and KC/gro. However, the overall outcome of acetaminopheninduced hepatic injury was not affected. In conclusion, these data indicate that MCP-1 and CCR2 contribute to the recruitment of a subset of activated macrophages into the liver during acetaminophen-induced hepatotoxicity that may be important in resolution of tissue injury.

Development of pink salmon (Oncorhynchus gorbuscha) incubating in gravel contaminated with weathered Prudhoe Bay crude oil was retarded at concentrations as low as 55.2 μg oil/g gravel. Larvae exposed to various levels of oil contamination were sampled 4 weeks before emergence, at emergence, and 13 days after emergence for histopathology (quantitative and semiquantitative) and cytochrome P4501A (CYP1A) induction (using immunohistochemical staining). A subset of postemergent fish was not fed. Hydrocarbon analysis by gas chromatography and mass spectroscopy revealed that tissue uptake of polynuclear aromatic hydrocarbons (PAH) was mediated by oil's dissolution in water, with significant biological effects when the peak total PAH concentration in water was as low as 4.4 μg/L. Oil-related effects included induction of CYP1A, development of ascites, and increased mortality. Several oil-related changes were indicative of premature emergence. Compared with control fish, for example, exposed fish of the same age and emerging on the same day had greater amounts of yolk and hepatocellular glycogen, increased apoptosis of gonadal cells and midventral skin cells, and less food in the gastrointestinal tract. Histological features were similar within groups of larvae sampled 4 weeks before and 13 days after emergence, and oil-induced changes were not affected by feeding during the first 13 days after emergence. Increased gonadal cell apoptosis may be related to later reproductive impairment documented in field studies of pink salmon up to 4 years after the Exxon Valdez oil spill.

Objective. The transcription of several cytokines, cell adhesion molecules, and enzymes involved in the inflammatory and destructive mechanisms of rheumatoid arthritis is dependent on nuclear factor B (NF-B). Because IB kinase (IKK) is critical in transducing the signal-inducible activation of NF-B, we examined whether the highly selective and orally bioavailable IKK inhibitor BMS-345541 is efficacious against collagen-induced arthritis (CIA) in mice.Methods. Arthritis in DBA/1LacJ male mice was induced by subcutaneous immunization with bovine type II collagen on day 0 and day 21. BMS-345541 was administered perorally daily, either prophylactically (before disease onset) or therapeutically (after disease onset). Clinical assessment of the incidence and severity of disease was conducted throughout the study, and histologic evaluation was performed at the time of study termination (day 42).Results. When administered prophylactically, BMS-345541 (in a dose range of 10-100 mg/kg) was effective, in a dose-dependent manner, in reducing the incidence of disease and inhibiting clinical signs of disease. Histologic evaluation of the joints showed that both inflammation and joint destruction were blocked by the IKK inhibitor. Message levels of interleukin-1␤ in the joints were also dose-dependently inhibited in the mice that received BMS-345541. Dose-dependent efficacy in terms of both disease severity and histologic end points was observed with the therapeutic dosing regimen of BMS-345541, with use of the 100-mg/kg dose resulting in resolution of disease.Conclusion. IKK plays a key role in CIA in mice, and inhibitors of this enzyme represent a promising target for the development of novel agents to treat rheumatoid arthritis and other inflammatory diseases. BMS-345541 represents the first example of an inhibitor of IKK that has antiinflammatory activity in vivo.

Purpose: Trastuzumab-emtansine (T-DM1) is an antibodydrug conjugate (ADC) comprising the cytotoxic agent DM1 conjugated to trastuzumab with a stable linker. Thrombocytopenia was the dose-limiting toxicity in the phase I study, and grade !3 thrombocytopenia occurred in up to 13% of patients receiving T-DM1 in phase III studies. We investigated the mechanism of T-DM1-induced thrombocytopenia.Experimental Design: The effect of T-DM1 on platelet function was measured by aggregometry, and by flow cytometry to detect the markers of activation. The effect of T-DM1 on differentiation and maturation of megakaryocytes (MK) from human hematopoietic stem cells was assessed by flow cytometry and microscopy. Binding, uptake, and catabolism of T-DM1 in MKs, were assessed by various techniques including fluorescence microscopy, scintigraphy to detect T- [H 3 ]-DM1 and 125 I-T-DM1, and mass spectrometry. The role of FcgRIIa was assessed using blocking antibodies and mutant constructs of trastuzumab that do not bind FcgR.Results: T-DM1 had no direct effect on platelet activation and aggregation, but it did markedly inhibit MK differentiation via a cytotoxic effect. Inhibition occurred with DM1-containing ADCs but not with trastuzumab demonstrating a role for DM1. MKs internalized these ADCs in a HER2-independent, FcgRIIa-dependent manner, resulting in intracellular release of DM1. Binding and internalization of T-DM1 diminished as MKs matured; however, prolonged exposure of mature MKs to T-DM1 resulted in a disrupted cytoskeletal structure.Conclusions: These data support the hypothesis that T-DM1-induced thrombocytopenia is mediated in large part by DM1-induced impairment of MK differentiation, with a less pronounced effect on mature MKs.

The polypeptide (p)50 molecule, a subunit of nuclear factor (NF)-κB, is produced after proteolytic processing of the p105 precursor (NF-κB1). Although the p105 precursor has been postulated to play a role in the regulation of the Rel/NF-κB activity, its physiological relevance remains unclear. To investigate that, we generated mutant mice lacking the COOH terminal half of the p105 precursor, but expressing the p50 product (p105−/−). These mutant mice displayed an inflammatory phenotype composed of lymphocytic infiltration in lungs and liver, and an increased susceptibility to opportunistic infections. Enlargement of multiple lymph nodes, splenomegaly due to erythrocytic extramedullary hematopoiesis, and lymphoid hyperplasia were also observed in p105−/− mice. Cytokine production in p105−/− macrophages was severely impaired, whereas proliferative responses of p105−/− B cells were increased. T cell functions were only moderately impaired in mutant mice. Loss of p105 also led to enhanced constitutive p50 homodimer and inducible NF-κB activities in unstimulated and stimulated cells, respectively. As several genes regulated by Rel/NF-κB were upregulated in p105−/− thymus but downregulated in p105−/− macrophages, the enhanced p50 homodimers appear to function as transcriptional activators or repressors, depending on the cell type. Thus, the p105 precursor is indispensable in the control of p50 activity, and lack of the precursor has distinct effects on different cells.