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Matrix metalloproteinases (MMPs) are a family of endoproteases that break down extracellular matrix and whose upregulation contributes to several diseases. A liquid chromatography/tandem mass spectrometry (LC/ MS/MS) method was developed to quantify MMP-1 and MMP-9 substrates and their N-terminal peptide products in samples obtained from implanted microdialysis sampling probes. In vitro studies with purified human MMP-1 and MMP-9 were used to optimize the assay and determine the effectiveness of the local delivery of a broadspectrum MMP inhibitor, GM 6001. Localized delivery of GM 6001 at 10 [micro]M was sufficient to completely inhibit product formation in vitro. In vivo studies in male Sprague--Dawley rats were performed with microdialysis probes implanted into the subcutaneous tissue. Directly after microdialysis probe implantation, infusions of the MMP-1 and MMP-9 substrates (50 [micro]M each) resulted in recovered product concentrations of approximately 2 [micro]M. During a 50 [micro]M GM 6001 coinfusion with the substrates, a 30% and 25% reduction in product formation for the MMP-1 and MMP-9 substrates was obtained, respectively. Blank dialysates were negative for enzymatic activity that could cleave the MMP substrates. This method allowed for the activity of different MMPs surrounding the microdialysis probe to be observed during in vivo sampling. 10.1021/ac901703g

Byline: Daniel J. Loegering (1), Michelle R. Lennartz (2) Keywords: tumor necrosis factor; Kupffer cells; splenic macrophages; peritoneal macrophages. FcI3 receptor Abstract: Lipopolysaccharide (LPS) increases serum TNF-[alpha] levels due to TNF-[alpha] secretion by macrophages. The serum TNF-[alpha] response to LPS was augmented 10x when FcI3R ligation was induced by the intravenous injection of Gig-coated erythrocytes (IgG) prior to the administration of LPS. The macrophage population responsible for the augmented TNF-[alpha] secretion was determined by isolating Kupffer cells, splenic macrophages and peritoneal macrophages from mice that had been given ElgG prior to LPS and determining TNF-[alpha] secretion ex vivo. The intravenous injection of ElgG augmented LPS-stimulate TNF-[alpha] secretion by Kupffer cells and splenic macrophages. In contrast, LPS-stimulated TNF-[alpha] secretion by peritoneal macrophages was not altered by either the intravenous or intraperitoneal injection of ElgG. In vitro phagocytosis of ElgG by isolated peritoneal macrophages also did not augment LPS-stimulated TNF-[alpha] secretion. These results show that FcI3R ligation augments LPS-stimulated TNF-[alpha] secretion by Kupffer cells and splenic macrophages but not by peritoneal macrophages. Thus, the ability of FcI3R ligation to influence TNF-[alpha] secretion may be specific to the tissue source of the macrophages. Author Affiliation: (1) Center for Cardiovascular Sciences, Albany Medical College, Albany, NY, 12208 (2) Center for Cell Biology and Cancer Research, Albany Medical College, Albany, NY, 12208 Article History: Registration Date: 12/10/2004

Past investigations have indicated that the injection of IgC-coated erythrocytes (EIgC) causes a rise in the mortality rate because of bacterial lipopolysaccharide (LPS). The effect of EIgC on LPS-stimulated increase in secretion by macrophages was studied. Isolated macrophages that had ingested EIgC or were adherent to immobilized IgC indicated increased tumor necrosis factor-alpha levels that is partly caused by complement activation and signaling through FclambdaR.

The contribution of blood flow to the development of insulin resistance in denervated muscles was investigated in a number of muscles from the denervated limb of rats. Results indicate that blood flow to muscle is dependent on the type of muscle fiber and the duration of denervation. Changes in blood flow to denervated muscles is not a contributory factor in the development of insulin resistance.

Refici, Marion L., Dennis W. Metzger, Bernard P. Arulanandam, Michelle R. Lennartz, and Daniel J. Loegering. Fc[Gamma]-receptor signaling augments the LPS-stimulated increase in serum tumor necrosis factor-[Alpha] levels. Am J Physiol Regulatory Integrative Comp Physiol 280: R1037-R1044, 2001.--The phagocytosis of IgG-coated erythrocytes (EIgG) has been shown to augment the bacterial lipopolysaccharide (LPS)-stimulated increase in serum tumor necrosis factor-[Alpha] (TNF-[Alpha]) levels. The present study evaluated the role of Fc[Gamma]-receptor (Fc[Gamma]R) signaling and complement activation in the effect of EIgG on the TNF-[Alpha] response to LPS. The role of Fc[Gamma]R was determined using FcR [Gamma]-chain knockout mice that lack functional Fc[Gamma]RI and Fc[Gamma]RIII. In wild-type animals, EIgG caused a 16-fold augmentation of the serum TNF-[Alpha] response to LPS, whereas there was no augmentation in the Fc[Gamma]R-deficient animals. Heat-damaged erythrocytes also augmented the TNF-[Alpha] response to LPS. This effect was absent in Fc[Gamma]R-deficient animals. An IgG antibody against heated erythrocytes was detected in mouse serum. The complement activation caused by EIgG had little effect on the LPS-stimulated increase in serum TNF-[Alpha] levels as indicated by activation of complement with cobra venom factor or IgM-coated erythrocytes as well as studies with C5-deficient mice. These results indicate that Fc[Gamma]R signaling primarily mediates the augmented serum TNF-[Alpha] response to LPS caused by EIgG. Fc receptor [Gamma]-chain knockout mice; sepsis; heat-damaged erythrocytes; complement activation; C5 knockout mice; cobra venom factor