Monocyte isolation

Although nitric oxide production has been extensively examined in rodents, the generation of nitric oxide by human tissue has been difficult. Nussler et al. (1992) have shown that human hepatocytes produce nitric oxide in response to IL-1, TNF, IFN, and LPS. Nitric oxide, produced by human monocytes treated with LPS, may play an important role in liver disease associated with alcoholism. Monocytes isolated from alcoholics with liver disease produce approximately the same level of nitric oxide as monocytes from control subjects simulated with LPS... 

While ASMase-deficient mice survive lethal doses of LPS (see above), the serum levels of TNF-a-induced by sublethal doses of LPS are similar to those in WT mice (Haimovitz-Friedman et ak, 1997) isolated ASMase-deficient macrophages respond to LPS with a normal pattern of cytokine secretion (Manthey and Schuchman, 1998). Thus, there is no evidence that ASMase is important for LPS-induced inflammatory responses. A recent report showed that imipramine, an inhibitor of cellular ASMase activity, blocked LPS-induced TNF-a secretion from differentiated human monocyte-like THP-1 cells (Cuschieri et ak, 2007). However, in our hands imipramine has no effect on cytokine secretion from these cells or from monocytes isolated from human blood (A.Billich, unpublished observations). 

In hydrophobic teflon bags, the test compound and blood monocytes isolated from pooled buffycoats at a density of 2 x 10 -cells/mL McCoy s medium supplemented with 20% fetal calf serum are cultured for 2 days. The monocytes are washed with... 

Weber, C., Erl, W., Weber, K. S., and Weber, P. C. (1997). HMG-CoA reductase inhibitors decrease CDllb expression and CDl lb-dependent adhesion of monocytes to endothelium and reduce increased adhesiveness of monocytes isolated from patients with hypercholesterolemia. J. Am. Coll. Cardiol. 30, 1212-1217. 

Newer tests that have not yet been introduced as diagnostic tests for MH are based on the enhanced release of calcium on exposure to halothane, e.g., in isolated monocytes from MH-susceptible patients, higher ratio of inorganic phosphate to... 

Tracy P. B Rorhbach M. S Mann K. G. Functional prothrombinase complex assembly on isolated monocytes and lymphocytes. J Biol Chem 1983 258,7264-7. 

Figure 9.5 Healthy volunteer monocytes after erythrophagocytosis. Rabbit erythrocytes were incubated with heat-inactivated mouse antirabbit erythrocyte serum. Human peripheral blood monocytes (MN) were obtained after Ficoll-Paque isolation and monocyte clumping with subsequent separation from lymphocytes, yielding a 95 % pure MN population. Non-ingested erythrocytes were removed by hypotonic lysis.

Determinations of NO in a variety of biological systems have been made. For example, measurement of NO has been made in eyes [79-81], gastrointestinal tract [82, 83], brain tissue [47, 50, 84-87], kidney and kidney tubule fluid [88-93], rat and guinea pig isolated and intact hearts [94, 95], rat spinal cord [96], human monocyte cells [97], human endothelial cells [98], mitochondria [99, 100], rat penis corpus cavemo-sum [101], granulocytes [102], invertebrate ganglia and immunocytes [103], choroidal endothelial cells [104], cancer cells [105, 106], peripheral blood [107], human blood [108], human leukocytes [109], platelets [110-112], ears [113, 114], plants [115-118], and pteropod mollusk [119]. 

Important studies were performed by Trush and coworkers [42], who showed the advantages of applying lucigenin-amplified CL for the measurement of superoxide production by mitochondria in unstimulated monocytes and macrophages as well as by isolated mitochondria [43,44]. Later on, these authors have shown that mitochondrial superoxide production measured by lucigenin-amplified CL increased in the liver of rats treated with the promoter of hepatocarcinogenesis ethinyl estradiol [45], in liver from obese mice [46], and in children with Down syndrome [47]. 

---