T. Hendriks

Soluble interleukin 6 receptor in biological fluids from human origin

OBJECTIVE measurement of baseline soluble interleukin 6 receptor (sIL-6R) and interleukin 6 (IL-6) levels in biological fluids in non-pathological conditions. SUBJECTS AND MATERIALS Blood and urine were obtained from healthy volunteers. Cerebrospinal fluid (CSF) and synovial fluid (SF) were obtained from patients during spinal puncture and athroscopy, respectively. Only CSF and SF of patients with proven non-pathological conditions were used in this study. Both sIL-6R and IL-6 were measured using ELISAs. It was shown that neither did sIL-6R interfere with the IL-6 ELISA nor did IL-6 interfere in the sIL-6R ELISA. Moreover, addition of recombinant sIL-6R to the IL-6 bio-assay (B9) did not influence IL-6 recovery. RESULTS using our sIL-6R ELISA we found baseline levels for sIL-6R in serum of 76.6 +/- 19.3 ng/ml in serum and 3.7 +/- 1.3 ng/ml in urine. In non-pathological conditions sIL-6R concentrations in CSF are 1.6 +/- 0.4 ng/ml, and in SF 11.6 +/- 3.3 ng/ml, while IL-6 concentrations are below detectable ranges in these fluids. CONCLUSIONS sIL-6R levels are detectable in serum, urine, CSF and SF during non-pathological conditions. sIL-6R levels in serum outrange levels in CSF, urine and SF and large interindividual differences in baseline concentrations for sIL-6R exist.

Post-operative levamisole may compromise early healing of experimental intestinal anastomoses.

There exists growing interest in immediate post-operative local adjuvant therapy after resection of intestinal malignancies. It is therefore necessary to assess it potential effect on the healing of intestinal anastomoses. Five groups (n = 20) of rats underwent resection and anastomosis of both ileum and colon: a control group and four experimental groups receiving intraperitoneal 5-fluorouracil (5-FU), 5-FU plus leucovorin, 5-FU plus levamisole or levamisole alone, on the day of surgery and the next 2 days. Animals were killed 3 or 7 days after operation. Another three groups (n = 6) of animals were used to compare anastomotic collagen synthetic capacity in control rats or rats receiving 5-FU or 5-FU plus levamisole. On the third post-operative day, the average anastomotic bursting pressure in the 5-FU/levamisole group was reduced by 36% as compared with the control group, both in ileum (P = 0.02) and in colon (P = 0.01). Values in the other groups were similar to those in the control group. Anastomotic breaking strength was significantly (P < 0.025) lowered in the ileum from the levamisole group at both days 3 and 7. Anastomotic collagen synthetic capacity was strongly reduced in the 5-FU and 5-FU/levamisole groups. However, there was no significant difference between the control group and the four experimental groups with regard to anastomotic hydroxyproline concentration and content, either 3 or 7 days after operation. Thus, limited use of levamisole, alone or in combination with intraperitoneal 5-FU, may compromise intestinal healing.

Hyperthermia and intraperitoneal chemotherapy for the treatment of peritoneal carcinomatosis: an experimental study

Objective:Hyperthermic intraperitoneal chemotherapy (HIPEC) with mitomycin C can improve survival if used as an adjunct to cytoreductive surgery (CS) for treatment of peritoneal carcinomatosis (PC). It remains unclear if both hyperthermia and chemotherapy are essential for the reported survival benefit. Methods:Eighty WAG/Rij rats were inoculated intraperitoneally with the rat colon carcinoma cell line CC-531. Animals were randomly assigned to 1 of the 4 treatment groups (n = 20): CS only, CS followed by HIPEC (mitomycin 35 mg/m2 at 41°C), CS followed by intraperitoneal mitomycin perfusion at 37°C, CS followed by intraperitoneal saline perfusion at 41°C. Survival was the primary outcome with a maximum follow up of 126 days. Results:Median survival was 62 days in rats treated with CS only and 57 days in rats treated with CS followed by hyperthermic saline perfusion. Rats receiving HIPEC had a median survival of 121 days (P = 0.022 when compared with CS only). In the group treated with chemotherapy at 37°C, 13 of 20 animals were still alive at the end of the experiment so median survival was not reached. (CS vs. IPEC: P = 0.002, hazard ratio 0.36, 95% CI 0.19–0.69) Rats treated with hyperthermic saline perfusion did not have an increased survival as compared with CS only. Conclusions:The effectiveness of intraoperative intraperitoneal perfusion after CS is highly dependent on the presence of chemotherapeutic agents in the perfusate but not on hyperthermia. The need to include hyperthermia in the adjuvant intraoperative treatment after CS for PC should be further investigated.

Inhibition of fibroblast collagen synthesis and proliferation by levamisole and 5-fluorouracil

Experimental studies indicate that anastomotic healing in the intestine is compromised by the immediate postoperative administration of 5-fluorouracil and levamisole. Since fibroblast functions are crucial to healing, we investigated the effects of (combinations of) both drugs on proliferation and collagen synthesis of rat skin fibroblasts in vitro. Proliferation was measured in actively dividing cells by cellular [3H]thymidine uptake and collagen synthesis in non-dividing cells by [3H]proline incorporation into collagenase-digestible protein. 5-Fluorouracil strongly and significantly (P < 0.05) reduced DNA synthesis and collagen synthesis at concentrations of 1 microM or more. The latter effect was not specific for collagen since total protein production was affected similarly. Both effects depended on the duration of exposure to the drugs. Levamisole also inhibited fibroblast proliferation dose-dependently, but less effectively than 5-fluorouracil: 50% inhibition was observed at approximately 0.1 mM. Collagen synthesis was unaffected by levamisole. If levamisole was added together with a low (0.1 microM) concentration of 5-fluorouracil, which in itself did not decrease thymidine incorporation, levamisoles antiproliferative effects became apparent at concentrations as low as 1 microM. A similar effect, but at a much higher concentration (1 mM) was noted on fibroblast collagen synthesis. These results indicate that levamisole potentiates 5-fluorouracil effects in fibroblast cultures and that direct effects of these drugs, alone or in combination, on fibroblast proliferation and collagen synthesis may be responsible for their negative influence on wound repair.

Is early post-operative treatment with 5-fluorouracil possible without affecting anastomotic strength in the intestine?

SummaryEarly post-operative local or systemic administration of 5-fluorouracil (5-FU) is under investigation as a means to improve outcome after resection of intestinal malignancies. It is therefore quite important to delineate accurately its potentially negative effects on anastomotic repair. Five groups (n = 24) of rats underwent resection and anastomosis of both ileum and colon: a control group and four experimental groups receiving daily 5-FU, starting immediately after operation or after 1, 2 or 3 days. Within each group, the drug (or saline) was delivered either intraperitoneally (n = 12) or intravenously (n = 12). Animals were killed 7 days after operation and healing was assessed by measurement of anastomotic bursting pressure, breaking strength and hydroxyproline content. In all cases, 5-FU treatment from the day of operation or from day 1 significantly (P < 0.025) and severely suppressed wound strength; concomitantly, the anastomotic hydroxyproline content was reduced. Depending on the location of the anastomosis and the route of 5-FU administration, even a period of 3 days between operation and first dosage seemed insufficient to prevent weakening of the anastomosis. The effects of intravenous administration, though qualitatively similar, were quantitatively less dramatic than those observed after intraperitoneal delivery. Post-operative treatment with 5-FU, if started within the first 3 days after operation, is detrimental to anastomotic strength and may compromise anastomotic integrity.

Modulation of Adhesion Molecule Expression on Endothelial Cells after Induction by Lipopolysaccharide‐Stimulated Whole Blood

The relative contribution of the pro‐inflammatory cytokines tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β and the lipopolysaccharide (LPS)‐induced pathways that result in endothelial activation during sepsis are not fully understood. We have examined the effects of plasma obtained from LPS‐treated human whole blood on the expression of E‐selectin and intercellular adhesion molecule‐1 (ICAM‐1) on human endothelial cells. Stimulation of blood with 10 pg/ml of LPS is sufficient to produce plasma that induces E‐selectin and ICAM‐1 expression, while direct induction by LPS alone requires a 100‐fold higher concentration. Characteristics for the plasma‐induced adhesion molecule expression were similar to the LPS‐induced production of TNF‐α and IL‐1β in blood. A complete inhibition of E‐selectin and ICAM‐1 expression was observed when antibodies against TNF‐α and IL‐1β were added to plasma prior to the incubation to endothelial cultures. Significant inhibition was even observed if antibodies were added to the cultures up until 3 h after LPS‐conditioned plasma. The plasma‐induced adhesion molecule response could also be prevented with inhibitors of nuclear factor (NF)‐κB, such as pyrollidine dithiocarbamate. These findings emphasize the central role of TNF‐α and IL‐1β in LPS‐induced endothelial activation and suggest that simultaneous neutralization of these cytokines or their common pathways may, even after the initial stimulus, prevent endothelial response during sepsis.

Plasma obtained during human endotoxemia increases endothelial albumin permeability in vitro.

ABSTRACT To gain insight in the pathogenesis of increased vascular permeability during sepsis, we studied the effect of plasma obtained during human experimental endotoxemia on the permeability of cultured endothelial monolayers. Eight healthy subjects received an i.v. dose of 2 ng/kg Escherichia coli O:113 lipopolysaccharide (LPS). The concentration of various plasma mediators that supposedly induce vascular permeability was measured over time. Plasmas that were obtained before, and 2 and 4 h after the administration of LPS were added to human umbilical venular endothelial cells that were cultured on semipermeable membranes.The permeability of the endothelial monolayers to fluorescein isothiocyanate-labeled bovine serum albumin was determined and expressed as the relative concentration of fluorescein isothiocyanate-bovine serum albumin when compared with that measured across empty Transwell-COL (Corning Life Sciences B.V., Schiphol-Rijk, The Netherlands) membranes (i.e., without endothelial monolayers). The permeability levels were correlated with the concentrations of various mediators.Experimental endotoxemia resulted in elevated levels of tumor necrosis factor &agr;, interleukin (IL) 1&bgr;, IL-6, IL-8, IL-10, and vascular endothelial growth factor and a moderate increase of IL-12 and IFN-&ggr; (all P values < 0.01). Incubation of human umbilical venular endothelial cells with plasma obtained 2 and 4 h after the administration of LPS increased the relative permeability from a baseline level (median) of 17% (range, 14% - 31%) to 23% (range, 12% - 39%; P = not significant) and 28% (range, 11% - 40%; P < 0.05), respectively. Plasma levels of vascular endothelial growth factor and IL-10, but not TNF-&agr; or any other mediators, significantly correlated with the increase in endothelial permeability (r = 0.47, P = 0.038; r = 0.43, P = 0.038, respectively).The data presented here demonstrate that plasmas obtained from experimental human endotoxemia increase endothelial albumin permeability in vitro. Thus, cultured human endothelial monolayers provide a model to study sepsis-associated vascular changes.

Circulating Concentrations of Soluble Interleukin 6 Receptors gp80 an gp130 in Chronic Renal Failure and Effects of Renal Replacement Therapy

Background: Circulating receptors modulate the biological effects of cytokines. Renal insufficiency is known to influence the concentrations of the soluble tumor necrosis factor (TNF) receptors p55 and p75. No data are available on the concentrations of the circulating interleukin 6 (IL-6) receptors gp80 and gp130 during chronic renal insufficiency. Methods: We compared the serum concentrations of the IL-6 receptors gp80 and gp130 to those of the TNF receptors p55 and p75 in end-stage chronic renal failure, continuous ambulatory peritoneal dialysis, and hemodialysis (HD). Results: In healthy controls the concentrations of gp80, gp130, p55, and p75 in serum were 82.1 ± 24.3, 87.9 ± 20.2, 1.1 ± 0.2, and 1.7 ± 0.3 ng/ml, respectively. These concentrations were increased to, respectively, 112.2 ± 18.0, 186.0 ± 37.7, 10.5 ± 4.3, and 15.0 ± 7.5 ng/ml in chronic renal failure, to 138.8 ± 18.0, 181.3 ± 46.1, 25.5 ± 5.2, and 19.1 ± 3.4 ng/ml in continuous ambulatory peritoneal dialysis, and to 107.9 ± 29.4, 146.6 ± 30.5, 22.9 ± 6.3, and 16.8 ± 6.0 ng/ ml in HD (before dialysis session). The concentrations after HD were higher for p75 only. Conclusions: The data show that the concentrations of the IL-6 receptors (gp80 and gp130) are elevated in chronic renal insufficiency. The increase is relatively low as compared with the elevation of the TNF receptors in this situation. HD does not result in a consistent change in serum concentrations of the various receptors.

Plasma gelatinase activity does not reflect disease activity after operation for colorectal cancer.

Objective: To investigate if plasma matrix metalloproteinase (MMP)-2 or -9 are better markers for disease activity than carcinoembryonic antigen (CEA) in the postoperative follow-up of colorectal cancer patients. Methods: A prospective study was performed including 61 patients operated for primary colorectal cancer. The follow-up was for at least 2 years and postoperative blood samples were obtained periodically with 3-month intervals. Plasma gelatinase activity was measured with quantitative gelatin zymography and serum CEA with a specific immunoassay. Results: Zymographic analysis of plasma samples revealed the presence of the proforms, but not the active forms, of both MMP-2 and -9. Prior to the detection of recurrent disease or metastasis in potentially curatively operated colorectal cancer patients, the changes in proMMP-2, -9 and CEA blood levels were determined. ProMMP-2 and -9 plasma levels changed little in this period and changes between patients with and without disease relapse were not statistically significant. In contrast, patients with disease relapse showed a significant increase (p = 0.002) in CEA in the two consecutive serum samples prior to the detection of recurrent disease or metastasis. Similarly, prior to death due to colorectal cancer, proMMP-2 and -9 plasma levels showed no significant change, whereas CEA levels increased considerably and significantly (p < 0.001) when compared to changes found in survivors. Conclusion: Plasma proMMP-2 and -9 activities show no potential value as prognostic markers in the follow-up of colorectal cancer.

Moderate Doses of Intraoperative Radiation Severely Suppress Early Strength of Anastomoses in the Rat Colon

Intraoperative irradiation appears to be a valuable addition to the modalities available to treat patients with large bowel cancer. However, its potential effect on healing of anastomoses has not been investigated extensively. For this purpose, male Wistar rats underwent colonic resection. Subsequently, 1 cm of each bowel end was irradiated with doses of 10, 15, 20 or 25 Gy and intestinal continuity was restored. After 3 or 7 days, animals were killed and the anastomoses were analyzed for bursting pressure (intraluminal force), breaking strength (longitudinal force) and hydroxyproline content. Intraoperative irradiation led to a massive (40-70%) and significant (P < 0.025) reduction in bursting pressure 3 days after operation compared to the control group for every dose used. After 7 days, the bursting site was outside the area of the anastomosis in all groups. The breaking strength at day 3 was also reduced, even after 10 Gy. At day 7, when tearing still occurred in the wound area, the breaking strength was still significantly lower in the 15- and 25-Gy groups than in the control group. The hydroxyproline content of the anastomoses was significantly reduced only after irradiation with the higher doses. Thus intraoperative irradiation constitutes a threat to early strength of anastomoses in the rat colon, and even at moderate doses it may threaten the integrity of the anastomosis.