Sensitive and rapid immunoassay for parathyroid hormone using magnetic particle labels and magnetic actuation.

A rapid method for the sensitive detection of proteins using actuated magnetic particle labels, which are measured with a giant magneto-resistive (GMR) biosensor, is described. The technique involves a 1-step sandwich immunoassay with no fluid replacement steps. The various assay binding reactions as well as the bound/free separation are entirely controlled by magnetic forces induced by electromagnets above and below the sensor chip. During the assay, particles conjugated with tracer antibodies are actuated through the sample for target capture, and rapidly brought to the sensor surface where they bind to immobilized capture antibodies. Weakly or unbound labels are removed with a magnetic force oriented away from the GMR sensor surface. For the measurement of parathyroid hormone (PTH), a detection limit in the 10 pM range is obtained with a total assay time of 15 min when 300 nm particles are used. The same sensitivity can be achieved in 5 min when 500 nm particles are used. If 500 nm particles are employed in a 15-minute assay, then 0.8 pM of PTH is detectable. The low sample volume, high analytical performance and high speed of the test coupled with the compact GMR biosensor make the system especially suitable for sensitive testing outside of laboratory environments.

Collagen synthesis in explants from rat intestine.

Collagen is the major structural component of the intestinal wall and its metabolism is of special interest for intestinal strength. We describe collagen synthesis in short-term (3 h) incubations of rat intestinal tissue, as measured in terms of incorporation of [3H]proline in collagenase-digestible protein and percentage relative collagen synthesis. In this time span, incorporation of [3H]proline in collagen increases linearly with time and tissue weight. Addition of unlabeled proline during incubation, in excess of the 0.1 microM [3H]proline always present, strongly increases both total protein and collagen synthesis, suggesting that proline transport is rate limiting. Further experiments have been performed in the presence of labeled proline alone and with the addition of 0.35 mM unlabeled proline. Collagen synthesis is significantly higher in colon than in ileum, comprising 0.37 and 0.21%, respectively, of total protein synthesis. Also, collagen synthesis decreases considerably with age, both in ileum and colon. The results presented here demonstrate that rat intestinal explants synthesize measurable amounts of collagen in vitro and that the system used is able to detect changes in in vivo synthetic capability such as those induced by ageing.

Collagen synthetic capacity throughout the uninjured and anastomosed intestinal wall.

The capacity to synthesize collagen was measured throughout the intestinal tract of the rat, using an in vitro technique. In addition, the effect of anastomotic construction in either the ileum or the colon on collagen synthetic capacity at specific distant locations both 1 and 4 days after operation was investigated. Collagen synthetic capacity is relatively uniform throughout the large bowel. However, in the small bowel in which synthesis is lower than in the large bowel, synthesis is significantly higher in the most proximal and distal segments than in the intermediate tissue. Anastomotic construction in either part of the bowel strongly increases collagen synthetic capacity at the immediate wound site. The synthetic response is not restricted to the anastomotic site but appears to be more generalized and is, in the small bowel, to some extent specific for collagen since the collagen synthetic capacity is increased far more than the capacity to produce noncollagenous protein. Anastomotic construction in the colon has only minor, although sometimes significant, effects on collagen synthetic capacity in the ileum, and vice versa.

Postoperative changes in collagen synthesis in intestinal anastomoses of the rat: differences between small and large bowel.

Collagen synthesis is an essential feature of anastomotic healing in the intestine. Postoperative collagen synthesis, measured in vitro in intestinal anastomoses was studied from three hours to 28 days after operation. For this purpose, an ileal and a colonic anastomosis were constructed within the same animal and the results in both intestinal segments were compared. In the ileum, collagen synthesis was significantly increased, with respect to unoperated controls, three hours after operation. It remained raised during the period of study, with a maximal 10-fold stimulation four days after operation, and had nearly returned to the preoperative level after four weeks. The general pattern was the same in the colon, although quantitatively different: the increase in synthetic activity was delayed in comparison with the ileum. Maximal stimulation was approximately six-fold. In addition, we calculated the ratio for each rat between anastomotic collagen synthesis and the average value found in non-operated control animals. Postoperative stimulation in the ileum was higher than in the colon in almost every animal examined. The results show that the ileum responds more quickly and strongly to wounding than the colon, at least as far as the production of new collagen is concerned. Possibly, this phenomenon contributes to the lower failure rate apparent for anastomoses in the small bowel.

Collagen synthesis in fibroblasts from human colon: regulatory aspects and differences with skin fibroblasts.

The purpose of this study was to examine regulation of collagen synthesis in human colon fibroblasts and compare the results from colon fibroblasts with those obtained in fibroblasts from human skin. The effects of interleukin-1 beta, tumour necrosis factor alpha, interferon gamma, transforming growth factor-beta, dexamethasone, and the calcium ionophore A23187 were investigated. All compounds were tested both in the absence and in the presence of fetal calf serum in the culture medium. The process of collagen synthesis in fibroblasts from colon and skin appears to be affected differently by these regulatory compounds. The most pronounced differences were that the relative collagen synthesis increased in dermal fibroblasts and decreased in colon fibroblasts upon addition of serum. In the presence of serum, interleukin-1 beta inhibited collagen synthesis in skin fibroblasts but not in colon fibroblasts. Dexamethasone suppressed the relative collagen synthesis in skin fibroblasts but not in colon fibroblasts. Transforming growth factor-beta stimulated the collagen synthesis in dermal fibroblasts in the presence of serum, but inhibited the process in colon fibroblasts. Because fibroblasts are the primary sources of collagen needed during wound repair, these results may offer (part of) the explanation why wounds in skin and intestine appear to behave differently under certain conditions.

Intraperitoneal cytostatics impair early post-operative collagen synthesis in experimental intestinal anastomosesP6.

Collagen synthesis in intestinal anastomoses has been measured in rats after in vivo administration of cytostatics. The cytostatics were administered during 5 consecutive days either intravenously or intraperitoneally. On day 3 of the course the rats received both an ileal and a colonic anastomosis. The animals were sacrificed 3 and 7 days after operation. The cytostatics regimen used was a combination of 5-fluorouracil, bleomycin and cisplatinum in a dose of 10, 2 and 0.35 mg kg-1 day-1, respectively. In an additional group, a twice higher dose was given intraperitoneally. Three days after operation a severe inhibition of the collagen synthesis was observed in all the cytostatics treated groups, both in ileum and in colon. The effects of intraperitoneal administration were much more pronounced than those observed after an equal dose given intravenously. Seven days after operation the collagen synthesis in the intravenously treated groups was restored to the level of the control group. However, in the intraperitoneal groups the collagen synthesis in ileal anastomoses was still inhibited. Thus, cytostatics suppress collagen synthesis in intestinal anastomoses. The effect is more severe after intraperitoneal than after intravenous administration, confirming our earlier hypothesis that the former mode of administration comprises a higher risk for anastomotic integrity.

Inhibition of basal and TGF beta-induced fibroblast collagen synthesis by antineoplastic agents. Implications for wound healing.

Antineoplastic drugs, given in the perioperative period, are thought to be a hazard to wound repair. Since fibroblast collagen synthesis is crucial to healing, we examined the effects of bleomycin, cisplatin and 5-fluorouracil on collagen synthesis in confluent cultures of fibroblasts from human colon and skin. The drugs were added in final concentrations between 0.1 and 50 microM. Bleomycin did not affect collagen synthesis in colon fibroblasts but inhibited synthesis in skin fibroblasts. Collagen synthesis in colon fibroblasts was strongly, and specifically, inhibited by cisplatin while synthesis in skin fibroblasts was affected only slightly. 5-Fluorouracil had no effect whatsoever on the collagen synthetic capacity in either colon or skin fibroblasts. If skin fibroblasts were cultured in the presence of transforming growth factor beta (TGF beta), the antineoplastic agents inhibited the TGF beta-stimulated collagen synthesis at far lower concentrations than those needed to suppress non-stimulated synthesis. This effect was not observed in fibroblasts from colon. The possible implications of these observations, as pertain to the use of perioperative chemotherapy, are discussed. Since 5-fluorouracil did not directly affect collagen synthesis in colon fibroblasts under any of the conditions tested it is suggested that the data support the contention that this drug is relatively harmless for intestinal healing.