Irena Kirman

Upregulation of Interleukin-12 and -17 in Active Inflammatory Bowel Disease

Background: Cytokines are essential mediators of the intestinal inflammation during active episodes of inflammatory bowel disease (IBD). Interleukin (IL)-12 and IL-17 are potent immunoregulatory cytokines whose roles in the pathogenesis of IBD are unknown. The aim of this study was to evaluate the colonic expression of IL-12 and IL-17 genes in IBD. Methods: Fifty-one patients (22 with ulcerative colitis (UC), 17 with Crohn disease (CD), and 12 controls) who underwent colonoscopy were included. IBD disease activity was determined using a clinical grading scale. The degree of inflammation, as well as the content of CD4+ T cells (synthesizing IL-17) and CD68+ macrophages (synthesizing IL-12) in colonic biopsies, was determined. The amounts of IL-12 and IL-17 mRNA were assessed by RT-PCR, using GAPDH as an internal standard. Results: In colonic specimens, IL-17 mRNA expression was increased in moderately and severely active UC (P = 0.03) and in all degrees of activity in CD (P < 0.04). Levels of IL-12 mRNA were upregulated in both active UC and active CD compared to controls (P < 0.02). In cases of remission, IL-12 mRNA expression was similar to that found in control samples. Compared to controls, histological examination showed significant differences in signs of chronic and acute inflammation in UC (P < 0.01) and CD (P < 0.02), revealing a high correlation between clinical disease activity and histological scoring (r 2  = 0.92, P < 0.005). Whereas CD4+ T cells were observed in lymphocyte aggregates located profound in the lamina propria, CD68+ macrophages were primarily found just underneath the surface epithelium. The density of CD4+ and CD68+ cells correlated significantly with the amounts of IL-17 and IL-12 mRNA, respectively (P < 0.05). Conclusion: The expression of both IL-12 and IL-17 mRNA is induced in active UC and CD and may thus be involved in sustaining the intestinal inflammation in IBD. Inhibition of IL-12 or IL-17 might be future therapeutic targets in IBD.

Major abdominal surgery increases plasma levels of vascular endothelial growth factor: open more so than minimally invasive methods.

Introduction:Vascular endothelial growth factor (VEGF) is a potent inducer of angiogenesis that is necessary for wound healing and also promotes tumor growth. It is anticipated that plasma levels would increase after major surgery and that such elevations may facilitate tumor growth. This studys purpose was to determine plasma VEGF levels before and early after major open and minimally invasive abdominal surgery. Methods:Colorectal resection for cancer (n = 139) or benign pathology (n = 48) and gastric bypass for morbid obesity (n = 40) were assessed. Similar numbers of open and laparoscopic patients were studied for each indication. Plasma samples were obtained preoperatively and on postoperative days (POD) 1 and 3. VEGF levels were determined via ELISA. The following statistical methods were used: Fisher exact test, unmatched Student t test, Wilcoxons matched pairs test, and the Mann Whitney U Test with P < 0.05 considered significant. Results:The mean preoperative VEGF level of the cancer patients was significantly higher than baseline level of benign colon patients. Regardless of indication or surgical method, on POD3, significantly elevated mean VEGF levels were noted for each subgroup. In addition, on POD1, open surgery patients for all 3 indications had significantly elevated VEGF levels; no POD1 differences were noted for the closed surgery patients. At each postoperative time point for each procedure and indication, the open groups VEGF levels were significantly higher than that of the matching laparoscopic group. VEGF elevations correlated with incision length for each indication. Conclusion:As a group colon cancer patients prior to surgery have significantly higher mean VEGF levels than patients without tumors. Also, both open and closed colorectal resection and gastric bypass are associated with significantly elevated plasma VEGF levels early after surgery. This elevation is significantly greater and occurs earlier in open surgery patients. The duration and clinical importance of this finding is uncertain but merits further study.

Interleukin-15 and its role in chronic inflammatory diseases

Abstract. This review focuses on the biological effects of the newly discovered cytokine, interleukin 15 (IL-15), in chronic inflammatory disorders. IL-15 shares biological activities with IL-2, and like IL-2 it is a member of the four-helix bundle cytokine family. IL-15 interacts with a heterotrimeric receptor that consists of the β and γ subunits of the IL-2 receptor (IL-2R) as well as a specific, high-affinity IL-15-binding subunit, IL-15Rα. IL-15 is produced by macrophages and various other cells in response to environmental stimuli and infectious agents, and it is important for the growth and differentiation of T and B lymphocytes, natural killer cells, macrophages, and monocytes as well as it activates a number of important intracellular signaling molecules, including the Janus kinases and members of the transcription factor family of signal transducers and activators of transcription. These facts suggest that IL-15 may play a pivotal role both in protective immune responses and in the pathogenesis of various chronic immuno-inflammatory disorders. The important new insight into the role of IL-15 in diseases such as rheumatoid arthritis, sarcoidosis, chronic hepatitis C, and ulcerative colitis are reviewed in this paper.

INTERACTION OF NITRIC OXIDE AND TRANSFORMING GROWTH FACTOR-β1 INDUCED BY ANGIOTENSIN II AND MECHANICAL STRETCH IN RAT RENAL TUBULAR EPITHELIAL CELLS

PURPOSE Changes in intrarenal pressure accompanying unilateral ureteral obstruction can result in tubular mechanical stretch and mediator release from renal tubules. Therefore, we examined the synthesis of nitric oxide and transforming growth factor-beta (TGF-beta), and their interaction in rat renal epithelial cells (NRK-52E) exposed to either angiotensin II or mechanical stretch. MATERIALS AND METHODS NRK-52E were exposed to either angiotensin II or mechanical stretch. Nitrite and TGF-beta in the supernatant were assessed by the Greiss reaction and bioassay, respectively. The level of cell hypertrophy and intracellular TGF-beta protein was determined by flow cytometry. TGF-beta messenger RNA and inducible nitric oxide synthase protein were detected by reverse transcriptase polymerase chain reaction and Western blot, respectively. RESULTS Angiotensin II stimulated TGF-beta1 and nitric oxide. The nitric oxide synthase inhibitor, N-nitro-L-arginine (L-NAME) or angiotensin II type I receptor blocker, losartan, inhibited nitric oxide and TGF-beta1 induced by angiotensin II. Flow cytometry showed that either L-NAME or losartan inhibited angiotensin II induced cell hypertrophy. TGF-beta1 inhibited iNOS protein and nitric oxide, whereas an anti-TGF-beta antibody enhanced iNOS. Mechanical stretch induced TGF-beta, inducible NOS protein and nitric oxide. However, TGF-beta expression was not affected by L-arginine or L-NAME when cells were exposed to mechanical stretch. CONCLUSIONS These results demonstrate that nitric oxide is an intermediate in angiotensin II stimulated TGF-beta1 expression but not in stretch induced TGF-beta expression, and that TGF-beta1 is a negative regulator of nitric oxide in rat renal epithelial cells. The complex interaction of these cytokines may be a target for intervention in the fibrotic and apoptotic processes in the obstructed kidney.

Laparoscopic-assisted cecectomy is associated with decreased formation of postoperative pulmonary metastases compared with open cecectomy in a murine model

BACKGROUND It was shown in a murine model that sham laparotomy is associated with a higher incidence of postoperative lung metastases when compared with results seen after carbon dioxide pneumoperitoneum. Using the same tumor model, the present study was undertaken to determine if the addition of bowel resection to the operative procedure would impact the results. METHODS Sixty mice underwent anesthesia alone (anesthesia control [AC]), laparoscopic-assisted cecectomy (LC), or open cecectomy (OC). After surgery, all animals received tail vein injections of 105 TA3-Ha tumor cells. On postoperative day 14, the lungs and trachea were excised en bloc and processed, and surface lung metastases were counted and recorded by a blinded observer. RESULTS The mean number of surface lung metastases in the AC, LC, and OC groups was 30.9, 76.3, and 134.5, respectively. Significantly more metastases were documented after OC (P<.001) and LC (P<.05) than after anesthesia alone. Mice in the LC group had significantly fewer lung metastases (43% less) than mice in the OC group (P<.01). CONCLUSIONS OC was associated with significantly more lung metastases than either LC or AC. Surgery-related immune suppression or trophic tumor cell stimulation occurring after surgery may contribute to this phenomenon.

Open surgery induces a dramatic decrease in circulating intact IGFBP-3 in patients with colorectal cancer not seen with laparoscopic surgery

BackgroundAs shown earlier by the authors via Western blot analysis, open (OS) but not laparoscopic surgery (LS) induces a qualitative decrease in plasma insulin-like growth factor–binding protein 3 (IGFBP-3) levels on postoperative day 1 (POD 1). Intact IGFBP-3 has tumor suppressive effects, but its degradation products do not. Enzyme linked immunoassay (ELISA) inevitably measures both. In this study, using a novel combined Western blot and ELISA analysis method, precise plasma levels of intact IGFBP-3 on POD2 after open and closed colorectal cancer resection (stage I–III) were determined.MethodsThis study included 15 OS patients with a mean incision length of 26.7 ± 15.5 cm and 16 LS patients with a mean incision length of 5.3 ± 3.1 cm. Intact IGFBP-3 levels were determined via ELISA and Western blot analysis in plasma collected preoperatively and postoperatively.ResultsIn the OS patients, the mean preoperative concentration of intact 43–45 kDa IGFBP-3 protein was 1920 ± 1430 ng/ml. It decreased dramatically on POD2 to 355 ± 545 ng/ml (p < 0.005). In the LS group, no significant difference was noted between the preoperative level (1305 ± 807 ng/ml) and the POD2 level (922 + 714 ng/ml).ConclusionsOpen cancer resection, unlike its minimally invasive alternative, induces a dramatic decrease in concentration of intact IGFBP-3, which may have important implications with regard to colon cancer recurrence.

Altered plasma matrix metalloproteinase-9/tissue metalloproteinase-1 concentration during the early postoperative period in patients with colorectal cancer

BackgroundThe authors have previously demonstrated that insulin-like growth factor binding protein-3 (IGFBP-3) is depleted in plasma for 1 to 3 days after major open surgery (OS), but not after laparoscopic surgery (LS). After surgery, IGFP-3 cleavage occurs rapidly and is likely attributable to altered plasma proteolytic activity. This study aimed to assess plasma proteolysis after both open and closed colorectal resection and, if possible, to identify a protease/protease inhibitor system affected by surgery.MethodsPlasma from 88 patients with colorectal cancer (stages I–III) who underwent resection was obtained preoperatively (pre-OP) and on postoperative days (POD) 1 to 3. Plasma proteolytic activity was assessed via zymography. On the basis of the results, specific protease and protease inhibitor concentrations were next measured via enzyme-linked immunoassay (ELISA). Statistical analysis was performed using Wilcoxon’s test.ResultsEarly after surgery, zymography showed a predominant band representing a 92-kDa gelatinase corresponding to a proform of matrix metalloproteinase-9 (MMP-9), a protease known to cleave IGFBP-3. In OS patients, the mean concentration of plasma MMP-9 was significantly higher on POD 1 than at pre-OP (p < 0.003). On POD 2 and 3, no differences were noted. In the LS group, the mean levels of MMP-9 before and after surgery were comparable. The levels of a natural MMP-9 inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), also were measured. In the OS group, the level of TIMP-1 was significantly higher on POD 1 (p < 0.0003) and POD 2 (p < 0.01) and 3 (p < 0.01) than at pre-OP. In the LS group, a smaller but significant increase in TIMP-1 levels was found between the pre-OP sample and the POD 1 (p < 0.01) and POD 2 (p < 0.01) samples. No difference was noted on POD 3 (p = 0.1).ConclusionsOpen surgery, but not laparoscopic surgery, is accompanied by a short-lived significant increase in MMP-9 levels, which likely accounts for the decrease in IGFBP-3 levels observed after OS. The transitory nature of MMP-9 imbalance may be attributable to the increase in TIMP-1 levels postoperatively.

Depletion of Circulating Insulin-Like Growth Factor Binding Protein 3 After Open Surgery is Associated With High Interleukin-6 Levels

PURPOSE:We have previously shown that plasma from open, but not laparoscopic-assisted, surgery patients has increased mitogenic activity for colon cancer cells. Decreased insulin-like growth factor binding protein 3 levels, most likely the result of an open surgery-induced proteolytic activity, may account for this finding. Plasma proteases are activated by interleukin-6. This study was designed to investigate plasma insulin-like growth factor binding protein 3 and interleukin-6 levels after major open or laparoscopic-assisted surgery.METHODS:EDTA plasma was obtained from 24 patients undergoing resection for colonic adenocarcinoma. Insulin-like growth factor binding protein 3 was detected by Western blot analysis and enzyme-linked immunosorbent assay. Interleukin-6 levels were determined by enzyme-linked immunosorbent assay. The effect of insulin-like growth factor binding protein 3 on tumor growth was tested using HCT116 cells.RESULTS:In patients undergoing open surgery, enzyme-linked immunosorbent assay revealed a significant decrease in total insulin-like growth factor binding protein 3 levels on postoperative Day 1 (915.6 ± 378.5 ng/ml) compared with preoperative levels (1,267.5 ± 407.9 ng/ml; P < 0.001). Western blots revealed a decrease in the levels of intact insulin-like growth factor binding protein 3. In patients undergoing laparoscopic-assisted surgery, levels of total and intact insulin-like growth factor binding protein 3 before surgery (1,088.9 ± 232.5 ng/ml) and on postoperative Day 1 (1,202.3 ± 285.6 ng/ml) were comparable with no significant changes in Western blot analysis. Interleukin-6 levels were undetectable preoperatively. On postoperative Day 1, interleukin-6 concentration was higher in open surgery group (434.8 ± 506.6 pg/ml) compared with laparoscopic-assisted surgery group (100.9 ± 60.2 pg/ml; P < 0.0001), and correlated significantly with a decrease in plasma insulin-like growth factor binding protein 3 after open surgery (r = 0.81; P < 0.0001).CONCLUSIONS:A significant decrease in both total and free insulin-like growth factor binding protein 3 occurs after open but not laparoscopic colectomy. There is an associated increase in the levels of interleukin-6. It remains to be proven that the interleukin-6 elevations are responsible for the low insulin-like growth factor binding protein 3 level seen after open surgery.

The circulating common gamma chain (CD132) in inflammatory bowel disease.

Objective:Inflammatory bowel disease (IBD) is characterized by T cell activation. Activated T cells shed interleukin-2 receptors (IL-2R) in a soluble form. A positive correlation between sIL-2Rα (CD25) and disease activity is well documented in IBD, whereas IL-2Rγ (CD132) has not been investigated in this respect. Sera from 42 patients with ulcerative colitis (UC), 34 with Crohns disease (CD), 31 healthy volunteers, and 12 patients with infectious enterocolitis were obtained.Methods:Disease activity was scored according to a semiquantitative score for UC and CD. sIL-2R α chain and γ chain were assessed by sandwich ELISA techniques using monoclonal antibodies specific for CD25 and CD132, respectively.Results:The concentration of IL-2Rα chain (CD25) was found to be median 3.8 ng/ml in healthy volunteers versus 7.0 ng/ml in UC patients (p < 0.001), and 9.6 ng/ml in CD patients (p < 0.001). With respect to IL-2Rγ (CD132), significantly higher amounts were found in CD patients: 6.6 ng/ml as compared with healthy controls <1.0 ng/ml (p < 0.004). A Kruskal-Wallis test revealed a significant correlation between α chain and disease activity in CD (p < 0.001), and further significantly higher γ chain levels were found in active CD (p = 0.03). For UC patients, a statistically significant increase of the α chain with increasing disease activity (p < 0.01) was observed, whereas no significant changes of the γ chain levels were found (p > 0.05). A difference of γ chain levels were found between CD and UC in moderate and severe disease activity (p < 0.05). Further analyses revealed that mesalazine did not influence the IL-2Rα or -γ concentration either in UC or in CD patients.Conclusion:An increased circulating level of the soluble common γ chain (CD132) seems to be found in CD, and an overlap exists between CD and UC.

Perioperative GMCSF limits the proangiogenic plasma protein changes associated with colorectal cancer resection

AIMS Colorectal resection (CR) increases plasma VEGF levels which may promote residual tumor growth. This study assessed the effect of perioperative GMCSF on plasma levels of sVEGFR1, Ang-1 and Ang-2 and also the impact of post-GMCSF plasma on in vitro endothelial cell (EC) growth and invasion. Ang-2 increases while sVEGFR1 and Ang-1 impede angiogenesis. METHODS Fifty-nine CR cancer patients were randomized to 7 perioperative doses of GMCSF or saline for 3days prior and 4days after CR. Blood samples were taken pre-drug (PreRx) and on several postoperative days (POD). Protein levels were assessed and PreRx and POD 5 plasma added to EC cultures after which branch point formation (ECBPF) and invasion (ECI) were measured. RESULTS sVEGFR1 levels were significantly higher on POD 1 and POD 5 in both groups but the GMCSF POD 5 level was twice the control value (p=0.002). Ang-2 levels were higher on PODs 1 and 5 in both groups (p<0.05) but the control POD 5 value (vs. GMCSF) was greater (p=0.03). Ang-1 decreases were noted in all (p=not significant, ns). The control group POD 5 ECBPF was 35.8% greater than Pre Rx (p=0.001) while the GMCSF result was 18.0% lower (p=ns); the control POD 5 median percent change from baseline was greater than the GMCSF result(p=0.008). The POD 5 ECI was +12.2% for the control group vs. baseline (p=ns) and -17.2% for the GMCSF group (p=ns): the control median percent change was greater than in the GMCSF group(p=0.045). CONCLUSION CR-related plasma changes are proangiogenic (>Ang-2) and anti-angiogenic (>sVEGFR1); the net effect is promotion of in vitro ECBPF. GMCSF limits the proangiogenic changes (higher POD 5 sVEGFR1 levels and lower Ang-2 elevations, lower POD 5 ECBPF and ECI). The clinical import of these effects is unclear; perioperative GMCSF has anti-angiogenic plasma effects that may limit tumor growth. Further investigation is warranted.