Long-term weekly treatment of colorectal metastatic cancer with fluorouracil and leucovorin: results of a multicentric prospective trial of fluorouracil dosage optimization by pharmacokinetic monitoring in 152 patients.

PURPOSE: A relationship between fluorouracil (5-FU) dose and response has been previously shown in advanced colorectal cancer. In a previous study with 5-FU stepwise dose escalation in a weekly regimen, and pharmacokinetic monitoring, we defined a therapeutic range for 5-FU plasma levels: 2,000 to 3,000 microg/L (area under the concentration-time curve at 0 to 8 hours [AUC0-8], 16 to 24 mg x h/L). The current study investigated 5-FU therapeutic intensification with individual dose adjustment in a multicentric phase II prospective trial. PATIENTS AND METHODS: Weekly high-dose 5-FU was administered by 8-hour infusion with 400 mg/m2 leucovorin. The initial dose of 5-FU (1,300 mg/m2) was adapted weekly according to 5-FU plasma levels, to reach the therapeutic range previously determined. RESULTS: A total of 152 patients entered the study from December 1991 to December 1994: 117 patients with measurable metastatic disease and 35 with assessable disease. Toxicity was mainly diarrhea (39%, with 5% grade 3) and hand-foot syndrome (30%, with 2% grade 3). Among 117 patients with measurable disease, 18 had a complete response (CR), 48 a partial response (PR), 35 a minor response (MR) and stable disease (SD), and 16 progressive disease (PD). Median overall survival time was 19 months. The 5-FU therapeutic plasma range was rapidly reached with a variable 5-FU dose in the patient population: mean, 1,803 +/- 386 mg/m2/wk (range, 950 to 3,396). Thirteen patients were immediately in the toxic zone, whereas 51 required a > or = 50% dose increase. CONCLUSION: Individual 5-FU dose adjustment with pharmacokinetic monitoring provided a high survival rate and percentage of responses, with good tolerance.

Partial characterization of eosinophilic granule cells (EGCs) and identification of mast cells of the intestinal lamina propria in rainbow trout (Oncorhynchus mykiss). Biochemical and cytochemical study.

The role of intestinal eosinophilic granule cells (EGCs) is still a subject of discussion. The aim of this study was to obtain additional functional data for a better characterization of these cells. Biochemical studies indicated the presence of small amounts of histamine, a characteristic and consistent marker of mast cells, in the posterior gut. On the other hand, histamine is always absent from homogenates of isolated EGCs. Using colorimetric assays, we were able to show aryl sulphatase B activity (18.5 +/- 3.7 nM nitrocatechol/10(6) cells) and detected peroxidase (1.86 +/- 0.03 ng/10(6) cells) in EGC homogenates. A cytochemical study enabled us to localize peroxidase in the granules of EGCs. These cells can also phagocytose latex beads. EGCs should thus be considered as homologous with mammalian eosinophils and not with mast cells. The screening for cells in the mucosae containing chondroitin sulphate revealed sparsely represented cells in the loose connective tissue in immediate proximity to blood capillaries. These cells could be mucosal mast cells.

Endocytosis and intracellular degradation of heterologous protein by eosinophilic granulocytes isolated from rainbow trout (Oncorhynchus mykiss) posterior intestine.

Adherence capacity to tissue substrate, endocytosis capacity for heterologous and homologous proteins, and proteolytic activity were determined in intestinal granulocytes (EGCs) isolated from healthy adult rainbow trout. The percentage of cells that could adhere to a smooth plastic surface increased with increasing incubation time. Endocytosis was effective for heterologous (human immunoglobulin G, IgGh; ovine somatotropin, oST) but not homologous proteins (recombinant trout somatotropin, rtST). The activity of cathepsin D increased significantly after the endocytosis of a heterologous protein. Finally, the analysis of immunoblots of homogenates of granulocytes incubated in the presence of the two different proteins was used to show the endocytosis and degradation of heterologous proteins. These results show that isolated EGCs can endocytose and degrade heterologous proteins.

Absorption of intact proteins by the intestinal epithelium of trout, Salmo gairdneri. A luminescence enzyme immunoassay and cytochemical study.

In the rainbow trout, a carnivorous species with a stomach, orally administered horseradish peroxidase (HRP) crossed the intestinal epithelium into the circulation. The protein first appeared in the blood 7 to 8 h after intubation, and could be assayed up to 24 h after administration. The method used, which combines ELISA (enzyme-linked immunoabsorbant assay) and chemiluminescence, enabled the transfer to be measured quantitatively. There was a direct correlation between the quantity ingested and the quantity transferred to the plasma within the experimental limits chosen. The clearance was monophasic and exponential (clearance rate: 3% per minute). Up to 6% of the ingested HRP was transferred to the blood. By cytochemistry it was possible to demonstrate that the protein crossed the intestinal cells at the level of the posterior segment, escaping the particularly intense intracellular lysosomal digestion. After entering the intercellular space, HRP was transferred to the interstitial space of the subepithelial lamina propria. During this transfer the HRP was in close contact with infiltrated macrophages and leukocytes resembling lymphoid cells. Thus, the passage of these protein particles could be the first indispensable step in the possible triggering of a local and/or a systemic immune response.

Immunological demonstration of intestinal absorption and digestion of protein macromolecules in the trout (Salmo gairdneri).

An immunofluorescence technique using antibodies against the Fc and Fab fragments of human IgG (IgGH) was used to study the absorption of proteins by the intestinal epithelial cells of rainbow trout after oral or anal administration. Cellular absorption of a high molecular weight protein, hepatitis-B surface antigen (HBsAg), was also studied by using two monoclonal antibodies, one specific for the confirmation of the antigen (implying disulfide bridges), and the other that reacts with the constituent polypeptides. Both absorbed IgGH and HBsAg were seen to be segregated in the apical vacuolar system, a characteristic feature of intestinal epithelial cells. The same antibodies were used with an everted sac technique in conjunction with immunofluorescence, to show the intravacuolar degradation of IgGH and HBsAg following absorption. By using an antibody against cathepsin D, it was possible to demonstrate, by immunofluorescence, the localization of this enzyme in the same vacuolar system. After coupling the antibody to peroxidase or to the protein A/colloidalgold complex, the ultrastructural antigenic sites of cathepsin D could be seen to be localized in the interior of the vacuoles. The vacuolar localization of a cathepsin B activity was determined by incubating sections of intestinal mucosa, or isolated epithelial cells, with a specific synthetic substrate (Z-Ala-Arg-Arg-methoxynaphthylamide). The supranuclear hyaloplasmic vacuoles of intestinal epithelial cells may be considered to be phagolysosomes that assure the degradation of absorbed proteins. This function may be of fundamental importance in the in the nutritional processes of this species.

Is the Golgi apparatus the obligatory final step for lipoprotein secretion by intestinal cells?

It is currently admitted that the synthesis and excretion of triglyceride-rich lipoproteins (chylomicrons and 'small chylomicrons') by intestinal epithelial cells involves the Golgi apparatus as an obligatory final step before exocytosis. The cells of the proximal intestine of the trout are an excellent model for investigating functional compartmentalization in the course of lipid absorption. Using this model, our data invalidate morphological data which were the basis for considering the Golgi apparatus as the mandatory final stage for their secretion. In particular, we show that triglyceride-rich particles can be transported directly from the endoplasmic reticulum to the intercellular space. Two pathways of intestinal lipoprotein excretion appear to coexist. One follows the classical export route, the second functions in a manner that bypasses the Golgi apparatus. The arguments used to affirm the requirement for the Golgi apparatus as a final step (glycosylation of apoprotein B, membrane vehicle for exocytosis) are discussed.

New views on intestinal absorption of lipids in teleostean fishes: an ultrastructural and biochemical study in the rainbow trout.

Lipid absorption in rainbow trout was studied after gastric administration of [1-14C]linoleic and [1-14C]-palmitic acids. The intestinal epithelial cells were isolated at various times of absorption and the major lipid classes were isolated. Radioactivity was found primarily in the triglycerides. Blood radioactivity was measured at different times after administration of the labeled acids. It was very low until after 6 hr. After 4 hr when it was detectable, it was located essentially in the triglyceride fraction. At various times after feeding (a meal with 60% of unsaturated long chain fatty acids) the absorptive epithelium of the anterior intestine and pyloric caeca were examined by electron microscopy. Surprisingly, the esterification of fatty acids corresponded to the formation of VLDL-like particles, seen in SER, RER, Golgi apparatus, lamellar structures, intercellular space, interstitial space of lamina propria and lumen of lymphatic vessels. The respective roles of endoplasmic reticulum and Golgi apparatus in the lipoprotein synthesis are discussed. The particles are delivered in the intercellular spaces by way of the lamellar structures, whose role until now was unknown. Though the absorption of dietary triglycerides is much slower than in mammals, the mechanism does not differ fundamentally. The long chain fatty acids are esterified by the intestinal cells and transferred as VLDL-like particles to lymph. X

Evolution of the glycogen content and of glucose-6-phosphatase activity in the liver of Salmo gairdneri during development.

The evolution of the liver's glycogenic content, cytochemical characterization of the glycogen and glucose-6-phosphatase activity enable us to define three successive phases up to stage 36, just before the first feed. The grade which was low up to stage 24 is then due to beta-particles of ovule origin. Then, up to stage 27, there is a storage phase: alpha-particles appear and accumulate while the enzymatic activity remains non-existent. From the stage 28 to 36 the grade is progressively increasing, the enzymatic activity appears and increases. When the phase ends the liver is able to ensure glycemic regulation and to deal with exogenous nutritional contributions.