We investigated regulation of macrophage prostaglandin production during activation by interferon gamma (IFN-gamma) and lipopolysaccharide (LPS). An in vitro model was established using the mouse macrophage-like cell line RAW 264.7. Cells were cultivated in the presence of IFN-gamma and LPS for up to 48 h and changes in the secretion of nitric oxide (NO.) and tumor necrosis factor alpha (TNF-alpha) were observed as activation markers. Under these conditions a prompt and strong increase in PGE2 production was found in the first 8 h followed by nearly constant generation of PGE2 during the next 40 h. In contrast, the activity of prostaglandin endoperoxide synthase (PGHS), measured as PGE2 production of microsomal protein fractions, was also increased, but reached a clear maximum at 24 h. Recently a second form of PGHS was cloned (PGHS-2) and specific antibodies and mRNA probes for both isoforms are available. PGHS-2 enzyme was expressed maximally after 24 h of activation whereas PGHS-1 was not influenced. In the presence of IFN-gamma and LPS, PGHS-2 mRNA expression reached a maximum at 8 h but PGHS-1 mRNA was not induced during the whole time period. These data indicate that changes in PG synthesis following macrophage activation are due to regulation of PGHS-2 expression.

The monocyte/macrophage plays a key role in the network of immune reactions. Dependent on activation, it is able to produce various cytokines which act on other cells of the immune system in the sense of upregulation or downregulation. In addition, it presents antigens by the HLA-DR molecule as an initial trigger of an antigen-specific T-cell response. Monocyte function is affected by surgical disease and further affected by surgical trauma. We found the monocyte to be activated in a subgroup of patients before the operation, related to an increased rate of postoperative septic complications. After the operation, plasma concentrations of IL-6 and IL-10 were increased indicating the activation of an immune response. After surgery HLA-DR expression decreased as well as LPS-stimulated TNFα and IL-6 production, the latter indicating a hyporesponsiveness of peripheral blood cells (presumably monocytes) to further stimulation. On the other hand, continuously high plasma concentrations of activation markers like neopterin and IL-6 in the postoperative course were associated with complications and poor outcome. In postoperative septic shock monocytes may be almost areactive towards natural stimuli like bacteria and endotoxin, since IL-6 and TNFα production decreased to very low amounts. Adequate pre- and postoperative monocyte function is related to an uneventful postoperative course after major surgical operations. Surgical trauma affects monocyte function rendering it less reactive, which is a potential risk factor for postoperative septic complications.

Attachment of microcrystallites to cellular membranes may be an important component of the pathophysiology of many diseases including urolithiasis. This study attempts to characterize the interaction of calcium oxalate (CaOx) crystals and apatite (AP) crystals with renal papillary collecting tubule (RPCT) cells in primary culture. Primary cultures of RPCT cells showed the characteristic monolayer growth with sporadically interspersed clumped cells. Cultures were incubated with [14C]CaOx crystals, and the crystals that bound were quantified by microscopy and adherent radioactivity. Per unit of cross-sectional area, 32 times more CaOx crystals were bound to the clumps than to the monolayer. CaOx adherence demonstrated concentration-dependent saturation with a beta value (fraction of cell culture area binding CaOx crystals) of 0.179 and a 1/alpha ox value (maximum micrograms of crystallites adhering to 1 cm2 of binding area) of 287 micrograms/cm2. On coincubation with AP crystals, CaOx binding demonstrated concentration-dependent inhibition with a 1/alpha AP value of 93 micrograms/cm2. Microcrystallite adherence to RPCT cells demonstrates selectivity for cellular clumps, saturation, and inhibition. These features suggest specific binding.

New diagnostic and prognostic markers are needed in colorectal cancer. They can be found by differential analysis at DNA, RNA or protein level. The accuracy of phenotypic comparisons of tumor and normal tissues depends on the purity of the samples. We present an effective method to identify and isolate proteins that are differentially expressed under altered conditions, and a two-dimensional reference protein map of the normal human colonic epithelium. Normal colonic mucosa, primary tumors and liver metastases were prepared in the operating room. After washing in an ice-cold medium containing protease inhibitors, crypts were isolated by mechanical preparation without using metalloproteinases. Epithelial cells were then selected using Ber-EP4 Dynabeads. The samples were denaturated before processing for immobilized pH gradient two-dimensional polyacrylamide gel electrophoresis according to SWISS-2DPAGE standards. The samples contained more than 95% epithelial cells as confirmed by fluorescence-activated cell sorting using pan-anticytokeratin antibodies. Cell surfaces were not damaged, as assessed by scanning electronic microscope. A protein reference map of the normal colonic epithelium was defined. Using gel matching, N-terminal sequencing and/or immunoblotting techniques, 60 polypeptides - including proteins specifically expressed in colorectal epithelium - have now been identified. This reproducible method of sample preparation permits the comparison of protein patterns found in various pathological states with the present reference map (http://www.expasy.ch). Some of these patterns might provide diagnostic or prognostic markers, or even molecular targets for therapy in the future.

Currently used appropriateness criteria for diagnostic colonoscopy increase the yield of relevant findings but lead to a miss rate for relevant findings in the range of 10 to 15 percent. Simple selection criteria based on age and symptoms could be more suitable and should be tested in a larger group of patients.