TGF-beta(1) down-regulates inflammatory cytokine-induced VCAM-1 expression in cultured human glomerular endothelial cells.

BACKGROUND: Endothelial cells are active participants in the processes controlling coagulation, inflammation and the immune response. Variations are recognized between endothelia isolated from different vascular beds as well as from different species. Though transforming growth factor-beta(1) (TGF-beta(1)) has been known to have an anti-inflammatory action, little is known about its effect on expression of cellular adhesion molecules during the inflammatory process in human glomerular endothelial cells. The aim of this study was to determine the effect of TGF-beta(1) on the inflammatory cytokine-induced expression of vascular cell adhesion molecule-1 (VCAM-1) in cultured human glomerular endothelial cells. METHODS: The culture of human glomerular endothelial cells was established using the normal portion of nephrectomized renal tissues and identified by factor VIII staining and cellular uptake of fluorescent-labelled acetylated low-density lipoprotein (LDL). The endothelial cells were stimulated by interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) with or without TGF-beta(1). Cellular expression of VCAM-1 was measured by enzyme-linked immunosorbent assay (ELISA) and flow cytometry, and VCAM-1 mRNA was measured by Northern blot analysis. RESULTS:TGF-beta(1) (1, 10 and 25 ng/ml) blunted IL-1beta- (5 ng/ml) induced VCAM-1 expression significantly (OD=1.08+/-0.14, 1. 10+/-1.16 and 1.05+/-0.14 vs IL-1beta=1.97+/-0.29, n=6, P<0.05) in ELISA. The addition of TGF-beta(1) (1, 10 and 25 ng/ml) also suppressed TNF-alpha- (10 ng/ml) induced VCAM-1 expression (OD=1. 14+/-0.15, 1.17+/-0.17 and 1.18+/-0.16 vs TNF-alpha=1.96+/-0.26, n=6, P<0.05). The same results were obtained by flow cytometry. TGF-beta(1) (10 ng/ml) inhibited both IL-1beta- (5 ng/ml) and TNF-alpha-(10 ng/ml) induced expression of VCAM-1 (MFI: IL-1beta=90. 8+/- 17.6, IL-1beta+TGF-beta(1)=37.8+/-14.9, TNF-alpha=113.6+/- 12.4, TNF-alpha+TGF-beta(1)=64.3+/-13.8, mean+/-SD, n=3, P<0.05). By Northern blot analysis, TGF-beta(1) (10 ng/ml) significantly suppressed the stimulatory effect of IL-1beta and TNF-alpha. CONCLUSIONS: These results show that TGF-beta(1) down-regulates the inflammatory cytokine-induced expression of VCAM-1 in human glomerular endothelial cells, which could be a novel mechanism for the anti-inflammatory action of TGF-beta(1) during the inflammatory processes in human glomerular diseases.     

The effect of aprotinin on hypoxia-reoxygenation-induced changes in neutrophil and endothelial function

BACKGROUND AND OBJECTIVE: An acute inflammatory response associated with cerebral ischaemia-reperfusion contributes to the development of brain injury. Aprotinin has potential, though unexplained, neuroprotective effects in patients undergoing cardiac surgery. METHODS: Human neutrophil CD11 b/CD18, endothelial cell intercellular adhesion molecule-1 (ICAM-1) expression and endothelial interleukin (IL)-1beta supernatant concentrations in response to in vitro hypoxia-reoxygenation was studied in the presence or absence of aprotinin (1600 KIU mL(-1)). Adhesion molecule expression was quantified using flow cytometry and IL-1beta concentrations by enzyme-linked immunosorbent assay. Data were analysed using ANOVA and post hoc Student-Newman-Keuls test as appropriate. RESULTS: Exposure to 60-min hypoxia increased neutrophil CD11b expression compared to normoxia (170+/-46% vs. 91+/-27%, P = 0.001) (percent intensity of fluorescence compared to time 0) (n = 8). Hypoxia (60 min) produced greater upregulation of CD11b expression in controls compared to aprotinin-treated neutrophils [(170+/-46% vs. 129+/-40%) (P = 0.04)] (n = 8). Hypoxia-reoxygenation increased endothelial cell ICAM-1 expression (155+/-3.7 vs. 43+/-21 mean channel fluorescence, P = 0.0003) and IL-1beta supernatant concentrations compared to normoxia (3.4+/-0.4 vs. 2.6+/-0.2, P = 0.02) (n = 3). Hypoxia-reoxygenation produced greater upregulation of ICAM- 1 expression [(155+/-3.3 vs. 116+/-0.7) (P = 0.001)] and IL-1beta supernatant concentrations [(3.4+/-0.3 vs. 2.6+/-0.1) (P = 0.01)] in controls compared to aprotinin-treated endothelial cell preparation (n = 3). CONCLUSIONS: Hypoxia-reoxygenation-induced upregulation of neutrophil CD11b, endothelial cell ICAM-1 expression and IL-1beta concentrations is decreased by aprotinin at clinically relevant concentrations.     

Blood loss after fluid resuscitation with isotonic or hypertonic saline for the initial treatment of uncontrolled hemorrhage induced by spleen rupture

BACKGROUND: It has been suggested that fluid resuscitation for the prehospital management of hypotensive trauma victims increases bleeding. In a model of uncontrolled hemorrhage induced by complete splenic laceration with a hilar vascular injury, we hypothesized that small-volume hypertonic saline or large-volume lactated Ringer's solution may provide sustained hemodynamic benefits despite promoting increases in intra-abdominal bleeding. METHODS: Forty anesthetized, spontaneously breathing dogs (18 +/- 1 kg) underwent laparotomy. A suture line was placed around the spleen to produce a splenic rupture with hilar vascular injury by pulling the exteriorized lines after incision closure. Intra-abdominal blood loss was measured directly, immediately after the animal was killed. Dogs were randomly assigned to four groups (n = 10 per group): Untreated controls were killed 20 (CT20) or 40 (CT40) minutes after splenic rupture to measure blood loss directly. Treatment groups received (20 minutes after spleen rupture) lactated Ringer's (LR), 33 mL/kg over 15 minutes, or 7.5% NaCl/6% dextran 70 (HSD), 4 mL/kg over 4 minutes. Blood loss was measured 40 minutes after spleen rupture. RESULTS: Mean arterial pressure dropped from an average value of 103 +/- 3 mm Hg to 67 +/- 5 mm Hg during the first 20 minutes and was partially restored afterward in all groups, with no significant differences between them. No resuscitation was associated with low cardiac output, whereas HSD restored and LR overshot baseline cardiac output. Intra-abdominal blood loss was 30 +/- 4, 38 +/- 4, 43 +/- 5, and 42 +/- 5 mL/kg for groups CT20, CT40, HSD, and LR, respectively, with no statistical significance between groups. CONCLUSION: No-fluid resuscitation in uncontrolled hemorrhage from splenic rupture resulted in a low-flow state, whereas resuscitation with small volumes of HSD or large volumes of LR produced hemodynamic benefits without significant increases in bleeding.     

Reduction of the inflammatory response following coronary bypass grafting with total minimal extracorporeal circulation.

OBJECTIVE: Cardiopulmonary bypass (CPB) is known to cause part of the systemic inflammatory reaction after cardiac surgery that can be responsible for organ failure. A novel technique based on a minimal extracorporeal circulation (MECC(R)) system has been evaluated with regard to the inflammatory response in a prospective study involving patients undergoing coronary artery bypass grafting. METHODS: Sixty consecutive patients were randomly assigned to either standard normothermic CPB (n=30) or the MECC system, with a reduced priming volume, no aortic venting and no venous reservoir, excluding the blood-air interface (n=30). Specific evaluation of cytokine release (IL-1beta, IL-6, TNF-alpha), as well as neutrophil elastase secretion and beta-thromboglobulin release from platelets and S100 protein assay were performed. Serial blood samples were taken prior to the onset, after initiation, at the end and after weaning of the CPB; further samples were collected 6 and 24h after the end of the CPB. RESULTS: All patients were similar with regards to pre- and intra-operative characteristics and clinical outcomes were comparable for both groups. MECC system allowed a reduced hemodilution with a mean drop of the hematocrit of 8.5 vs. 15.3% (P<0.05). Mononuclear phagocytes dropped in a more important manner under standard CPB conditions (247+/-151 vs. 419+/-168, P=0.002), but both groups demonstrated a rise in monocyte count at the end of the CBP. No significant release of IL-1beta was observed in either group. By the end of CPB, IL-6 levels were significantly lower in the MECC group (38.8+/-19.6 vs. 87.9+/-78.9, P=0.04), despite a higher monocyte count. Plasma levels of TNF-alpha rised significantly more during standard CPB than with the MECC system (17.8+/-15.4 vs. 10.1+/-5.6, P=0.002). With MECC, the neutrophil elastase release was reduced (72.7+/-47.9 vs. 219.6+/-103.4, P=0.001). Platelet count remained at higher values with the minimal compared to standard CPB. It is noteworthy to consider that beta-thromboglobulin levels showed slightly lower platelet activation in the MECC group at all times of CPB (110.5+/-55.6 vs. 134.7+/-46.8, P=0.10). The pattern of release of S100 protein showed higher values in patients undergoing standard CPB than after MECC. CONCLUSIONS: The MECC system is suitable to maintain total extracorporeal circulation and demonstrates a lower inflammatory reaction when compared to standard CPB.     

Interleukin-1 beta and tumor necrosis factor-alpha release in normal and diseased human infrarenal aortas.

The presence of chronic inflammatory cells in the adventitia and media of abdominal aortic aneurysms and aortic occlusive disease suggest an immunologic response. The purpose of this study is to determine whether normal or diseased infrarenal aortas liberate the inflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta). Twenty-six infrarenal aortic biopsies (5 aortic occlusive disease, 15 abdominal aortic aneurysms, and 6 cadaveric donors) were weighed, minced into small pieces, and incubated in media for 48 hours. Conditioned media was harvested at 48 hours and assayed for IL-1 beta or TNF-alpha with use of an ELISA assay. Comparison of groups was performed with a one-way analysis of variance. The constitutive IL-1 beta produced by abdominal aortic aneurysms was significantly different than that in cadaveric donors (908 +/- 194 pg/ml [SE] vs 100 +2- 30 pg/ml). There was no statistically significant difference between abdominal aortic aneurysms and aortic occlusive disease (908 +/- 194 pg/ml vs 604 +/- 256 pg/ml) or aortic occlusive disease and cadaveric donor (604 +/- 256 vs 100 +/- 30). In time-course studies for the release of IL-1 beta, abdominal aortic aneurysms demonstrated maximal release at 48 hours. IL-1 beta release was augmented by lipopolysaccharide in all categories. A dose response curve demonstrated maximal IL-1 beta release on stimulation with 5 micrograms/ml LPS. Constitutive TNF-alpha production was low, ranging from 13 +/- 1.5 pg/ml in cadaveric donor, to 20 pg/ml in aortic occlusive disease, and 24 +/- 11 pg/ml in abdominal aortic aneurysms. There was no augmentation in TNF-alpha with lipopolysaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)     

TNF Alpha−308 Genotype and Renin–Angiotensin System in Hemodialysis Patients: An Effect on Inflammatory Cytokine Levels?

BACKGROUND: Renin-angiotensin system (RAS) was suggested to modulate inflammatory cytokine production. Angiotensin II was consistently shown to increase production of tumor necrosis factor alpha (TNF-alpha). However, inflammatory cytokines and RAS were modulated by genetic polymorphisms such as TNF-alpha-308 G > A and angiotensin-converting enzyme (ACE) I/D gene polymorphisms. The aim of this study was to investigate the effects of ACE and TNF-alpha genotypes on inflammatory cytokines in hemodialysis (HD) patients. METHODS: ACE I/D and TNF-alpha-308 G > A genotypes, pre- and postdialysis plasma renin activity (PRA), serum ACE, interleukin-1 beta (IL-1beta), and TNF-alpha levels were determined in 22 HD patients. RESULTS: Predialysis serum ACE activity is correlated with TNF-alpha (r = 0.63; P = 0.01), and PRA was correlated with IL-1beta levels (r = 0.49; P = 0.02). Pre/postdialysis IL-1beta and TNF-alpha were similar in DD and II/ID ACE genotypes. Predialysis TNF-alpha and IL-1beta (32.4 +/- 5; 35.1 +/- 4.2 vs. 28.1 +/- 3.7; 26.5 +/- 6.2 pg/mL; P < 0.05) and postdialysis TNF-alpha levels (30.4 +/- 1.4 vs. 28.4 +/- 0.82 pg/mL; P < 0.05) were significantly higher in TNF1/2 than TNF1/1 patients. CONCLUSION: ACE and TNF-alpha-308 G > A (1/2) gene polymorphisms may contribute to modulation of proinflammatory cytokine production and hence chronic inflammation in HD patients.     

Interleukin-1beta induces human proximal tubule cell injury,alpha-smooth muscle actin expression and fibronectin production

BACKGROUND: Tubulointerstitial lesions, characterized by tubular injury, interstitial fibrosis and the appearance of myofibroblasts, are the strongest predictors of the degree and progression of chronic renal failure. These lesions are typically preceded by macrophage infiltration of the tubulointerstitium, raising the possibility that these inflammatory cells promote progressive renal disease through fibrogenic actions on resident tubulointerstitial cells. The aim of the present study, therefore, was to investigate the potentially fibrogenic mechanisms of interleukin-1beta (IL-1beta), a macrophage-derived pro-inflammatory cytokine, on human proximal tubule cells (PTC). METHODS: Confluent, quiescent, passage 2 PTC were established in primary culture from histologically normal segments of human renal cortex (N = 11) and then incubated in serum- and hormone-free media supplemented with either IL-1beta (0 to 4 ng/mL) or vehicle (control). RESULTS: IL-1beta significantly enhanced fibronectin secretion by up to fourfold in a time- and concentration-dependent fashion. This was accompanied by significant (2.5- to 6-fold) increases in alpha-smooth muscle actin (alpha-SMA) expression, transforming growth factor beta (TGF-beta1) secretion, nitric oxide (NO) production, NO synthase 2 (NOS2) mRNA and lactate dehydrogenase (LDH) release. Cell proliferation was dose-dependently suppressed by IL-1beta. NG-methyl-l-arginine (L-NMMA; 1 mmol/L), a specific inhibitor of NOS, blocked NO production but did not alter basal or IL-1beta-stimulated fibronectin secretion. In contrast, a pan-specific TGF-beta neutralizing antibody significantly blocked the effects of IL-1beta on PTC fibronectin secretion (IL-1beta, 268.1 +/- 30.6 vs. IL-1beta+alphaTGF-beta 157.9 +/- 14.4%, of control values, P < 0.001) and DNA synthesis (IL-1beta 81.0 +/- 6.7% vs. IL-1beta+alphaTGF-beta 93.4 +/- 2.1%, of control values, P < 0.01). CONCLUSION: IL-1beta acts on human PTC to suppress cell proliferation, enhance fibronectin production and promote alpha-smooth muscle actin expression. These actions appear to be mediated by a TGF-beta1 dependent mechanism and are independent of nitric oxide release.     

The effect of lidocaine on neutrophil CD11b/CD18 and endothelial ICAM-1 expression and IL-1beta concentrations induced by hypoxia-reoxygenation

BACKGROUND: Lidocaine has actions potentially of benefit during ischaemia-reperfusion. Neutrophils and endothelial cells have an important role in ischaemia-reperfusion injury. METHODS: Isolated human neutrophil CD11b and CD18, and human umbilical vein endothelial cell (HUVEC) ICAM-1 expression and supernatant IL-1beta concentrations in response to hypoxia-reoxygenation were studied in the presence or absence of different concentrations of lidocaine (0.005, 0.05 and 0.5 mg mL(-1)). Adhesion molecule expression was quantified by flow cytometry and IL- 1beta concentrations by ELISA. Differences were assessed with analysis of variance and Student-Newman-Keuls as appropriate. Data are presented as mean+/-SD. RESULTS: Exposure to hypoxia-reoxygenation increased neutrophil CD11b (94.33+/-40.65 vs. 34.32+/-6.83 mean channel fluorescence (MCF), P = 0.02), CD18 (109.84+/-35.44 vs. 59.05+/-6.71 MCF, P = 0.03) and endothelial ICAM-1 (146.62+/-16.78 vs. 47.29+/-9.85 MCF, P < 0.001) expression compared to normoxia. Neutrophil CD18 expression on exposure to hypoxia-reoxygenation was less in lidocaine (0.005 mg mL(-1)) treated cells compared to control (71.07+/-10.14 vs. 109.84+/-35.44 MCF, P = 0.03). Endothelial ICAM-1 expression on exposure to hypoxia-reoxygenation was less in lidocaine (0.005 mg mL(-1)) treated cells compared to control (133.25+/-16.05 vs. 146.62+/-16.78 MCF, P = 0.03). Hypoxia-reoxygenation increased HUVEC supernatant IL-1beta concentrations compared to normoxia (3.41+/-0.36 vs. 2.65+/-0.21 pg mL(-1), P = 0.02). Endothelial supernatant IL-1beta concentrations in lidocaine-treated HUVECs were similar to controls. CONCLUSIONS: Lidocaine at clinically relevant concentrations decreased neutrophil CD18 and endothelial ICAM-1 expression but not endothelial IL-1beta concentrations.     

Less systemic cytokine response in patients following microendoscopic versus open lumbar discectomy.

The magnitude of the tissue damage from surgery impacts the trauma response. This response is proportional to the severity of surgical stress. Systemic cytokines are recognized as markers of postoperative tissue trauma. Microendoscopic discectomy (MED) recently has become popular for treating lumbar disc herniations, and is associated with favorable clinical outcomes compared with open discectomy (OD). This study postulates that MED is a less traumatic procedure, and therefore has a lower surgical stress response compared to OD. In this study, a quantitative comparison of the overall effects of surgical trauma resulting from MED and OD was performed through analyzing patient systemic cytokines response. From April, 2002 to June, 2003, 22 consecutive patients who had symptomatic lumbar disc herniations were prospectively randomized to undergo either intracanalicular MED (N=10) or OD (N=12). In this study, the Vertebroscope System (Zeppelin, Pullach, Germany) was used to perform the endoscopic discectomy procedure in all MED patients. Serum levels of tumor necrosis factor-alpha (TNF-alpha), Interleukin-1beta (IL-1beta), Interleukin-6 (IL-6), and Interleukin-8 (IL-8) were measured before surgery and at 1, 2, 4, 8 and 24h after surgery using an enzyme-linked immunosorbent assay. Serum C-reactive protein (CRP) was measured at the same time interval. The results showed the MED patients had shorter postoperative hospital stay (mean, 3.57+/-0.98 vs. 5.92+/-2.39 days, p=0.025) and less intraoperative blood loss (mean, 87.5+/-69.4 vs. 190+/-115 ml, p=0.042). The operating length, including the set-up time, was longer in the MED group (mean, 109+/-35.9 vs. 72.1+/-17.8 min, p=0.01). The mean size of skin incision made for the MED patients was 1.86+/-0.13 cm (range 1.7-2.0 cm); and 6.3+/-0.98 cm for the OD patients (range 5.5-8 cm), p=0.001. The patients' pain severity of the involved limbs on 10-point Visual Analog Scale before operation in MED group was 7.5+/-0.3 (range 6-9) and 8+/-0.2 (range 7-9) in OD group, p=0.17; and after surgery, 1.5+/-0.2 (range 1-2) in MED group and 1.4+/-0.1 (range 1-3) in OD group, p=0.91. CRP levels peaked at 24h in both groups, and OD patients displayed a significantly greater postoperative rise in serum CRP (mean, 27.78+/-15.02 vs. 13.84+/-6.25mg/l, p=0.026). Concentrations of TNF-alpha, IL-1beta, and IL-8 were detected only sporadically. Serum IL-6 increased less significantly following MED than after OD. In the MED group, IL-6 level peaked 8h after surgery, with the response statistically less than in the open group (mean, 6.27+/-5.96 vs. 17.18+/-11.60 pg/ml, p=0.025). A statistically significant correlation was identified between IL-6 and CRP values (r=0.79). Using the modified MacNab criteria, the clinical outcomes were 90% satisfactory (9/10) in MED patients and 91.6% satisfactory (11/12) in OD patients at a mean 18.9 months (range 10-25) follow-up. Based on the current data, surgical trauma, as reflected by systemic IL-6 and CRP response, was significantly less following MED than following OD. The difference in the systemic cytokine response may support that the MED procedure is less traumatic. Moreover, our MED patients had achieved satisfactory clinical outcomes as the OD patients at a mean 18.9 months follow-up after surgery.     

Transforming growth factor-beta suppresses macrophage-induced mesangial cell fibronectin expression

BACKGROUND: We have previously shown that macrophages are able to promote prosclerotic responses in rat mesangial cells. Th2-type cytokines, including interleukin-10 (IL-10), IL-13, and IL-4 as well as transforming growth factor-beta (TGF-beta), are known to have suppressive effects on various aspects of macrophage function. In the current study, we investigated the effect of TGF-beta pretreatment on the ability of macrophages to induce fibronectin expression. RESULTS: Conditioned medium from TGF-beta pretreated macrophages (MPCM(TGF)) induced lower fibronectin levels in mesangial cells in both the secreted and cell-associated forms, compared with conditioned medium from standard macrophages (MPCM) (5.5 +/- 0.2 vs. 3.4 +/- 0.3 and 4.05 +/- 0.45 vs. 2.3 +/- 0.2-fold increase over medium alone for MPCM versus MPCM(TGF) in supernatants and cell lysates, respectively). Northern blot analysis demonstrated that fibronectin message was marginally reduced to 0.88 +/- 0.04 (P < 0.03 vs. MPCM, N = 3) of MPCM-induced levels. However, mesangial cell transin mRNA levels induced in response to MPCM(TGF) were 2.29 +/- 0.47-fold greater than those induced by standard MPCM (P = 0.03 vs. MPCM, N = 4). TIMP-1 mRNA levels were also increased in response to MPCM(TGF), but only by 1.43 +/- 0.1-fold (P = 0.02 vs. MPCM, N = 5). Casein-FITC digestion studies confirmed that MPCM(TGF) stimulated more mesangial cell caseinolytic activity than did MPCM. In addition, MPCM-mediated up-regulation of mesangial cell TGF-beta mRNA and protein expression was significantly reduced in response to conditioned medium from macrophages pretreated with TGF-beta. CONCLUSION: This study suggests that TGF-beta is able to regulate negatively the profibrotic effects of macrophages on mesangial cells by both enhancing matrix degradation and reducing synthesis.     

Vasopressin,but not fluid resuscitation,enhances survival in a liver trauma model with uncontrolled and otherwise lethal hemorrhagic shock in pigs

BACKGROUND: The authors compared the effects of vasopressin fluid resuscitation on survival in a liver trauma model with uncontrolled and otherwise lethal hemorrhagic shock in pigs. METHODS: A midline laparotomy was performed on 23 domestic pigs, followed by an incision, and subsequent finger fraction across the right medial liver lobe. During hemorrhagic shock, animals were randomly assigned to receive either 0.4 U/kg vasopressin (n = 9), or fluid resuscitation (n = 7), or saline placebo (n = 7), respectively. A continuous infusion of 0.08 U x kg(-1) x min(-1) vasopressin in the vasopressin group, or normal saline was subsequently administered in the fluid resuscitation and saline placebo group, respectively. After 30 min of experimental therapy, bleeding was controlled by surgical intervention, and blood transfusion and rapid fluid infusion were subsequently performed. RESULTS: Maximum mean arterial blood pressure during experimental therapy in the vasopressin-treated animals was significantly higher than in the fluid resuscitation and saline placebo groups (mean +/- SD, 72 +/- 26 vs 38 +/- 16 vs 11 +/- 7 mmHg, respectively; P< 0.05). Subsequently, mean arterial blood pressure remained at approximately 40 mmHg in all vasopressin-treated animals, whereas mean arterial blood pressure in all fluid resuscitation and saline placebo pigs was close to aortic hydrostatic pressure (approximately 15 mmHg) within approximately 20 min of experimental therapy initiation. Total blood loss was significantly higher in the fluid resuscitation pigs compared with vasopressin or saline placebo after 10 min of experimental therapy (65 +/- 6 vs 42 +/- 4 vs 43 +/- 6 ml/kg, respectively; P< 0.05). Seven of seven fluid resuscitation, and seven of seven saline placebo pigs died within approximately 20 min of experimental therapy, while 8 of 9 vasopressin animals survived more than 7 days (P < 0.05). CONCLUSIONS: Vasopressin, but not fluid resuscitation or saline placebo, ensured survival with full recovery in this liver trauma model with uncontrolled and otherwise lethal hemorrhagic shock in pigs.     

Vasopressin, but Not Fluid Resuscitation, Enhances Survival in a Liver Trauma Model with Uncontrolled and Otherwise Lethal Hemorrhagic Shock in Pigs

Background: The authors compared the effects of vasopressin versus fluid resuscitation on survival in a liver trauma model with uncontrolled and otherwise lethal hemorrhagic shock in pigs.

Methods: A midline laparotomy was performed on 23 domestic pigs, followed by an incision, and subsequent finger fraction across the right medial liver lobe. During hemorrhagic shock, animals were randomly assigned to receive either 0.4 U/kg vasopressin (n = 9), or fluid resuscitation (n = 7), or saline placebo (n = 7), respectively. A continuous infusion of 0.08 U [middle dot] kg-1 [middle dot] min-1 vasopressin in the vasopressin group, or normal saline was subsequently administered in the fluid resuscitation and saline placebo group, respectively. After 30 min of experimental therapy, bleeding was controlled by surgical intervention, and blood transfusion and rapid fluid infusion were subsequently performed.

Results: Maximum mean arterial blood pressure during experimental therapy in the vasopressin-treated animals was significantly higher than in the fluid resuscitation and saline placebo groups (mean +/- SD, 72 +/- 26 vs. 38 +/- 16 vs. 11 +/- 7 mmHg, respectively;P < 0.05). Subsequently, mean arterial blood pressure remained at approximately 40 mmHg in all vasopressin-treated animals, whereas mean arterial blood pressure in all fluid resuscitation and saline placebo pigs was close to aortic hydrostatic pressure (~15 mmHg) within approximately 20 min of experimental therapy initiation. Total blood loss was significantly higher in the fluid resuscitation pigs compared with vasopressin or saline placebo after 10 min of experimental therapy (65 +/- 6 vs. 42 +/- 4 vs. 43 +/- 6 ml/kg, respectively;P < 0.05). Seven of seven fluid resuscitation, and seven of seven saline placebo pigs died within approximately 20 min of experimental therapy, while 8 of 9 vasopressin animals survived more than 7 days (P < 0.05).     

Differential regulation of monocyte/macrophage cytokine production by pressure

BACKGROUND: Cytokine production by macrophages is essential for the inflammatory response. Normal human interstitial tissue pressure is 20 to 30 mm Hg, but generally decreases in acute inflammation. METHODS: We compared the effect of 20 mm Hg increased pressure (approximating normal interstitial tissue pressure) with that of ambient pressure (resembling pressure in inflamed tissues) on tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta production by undifferentiated (monocytic) and PMA (phorbol 12-, myristate 13-acetate)-differentiated (macrophage-like) THP-1 cells with or without lipopolysaccharide (LPS) (10 ng/mL). RESULTS: Pressure stimulated spontaneous macrophage TNF-alpha secretion (30.5 +/- 6.3 vs. 49.1 +/- 2.8 pg/mL, P <.02), but not monocyte TNF-alpha secretion. Pressure did not stimulate IL-1beta release. As expected, LPS increased basal cytokine release. After LPS stimulation, pressure still tended to stimulate macrophage TNF-alpha, but inhibited monocyte TNF-alpha secretion (P <.05). In contrast, pressure inhibited IL-1beta release by both LPS-treated monocytes (986 +/- 134 vs. 595 +/- 226 pg/mL, P <.02) and macrophages (3,112 +/- 229 vs. 979 +/- 61 pg/mL, P <.01). CONCLUSIONS: Extracellular pressure may regulate TNF-alpha and IL-1beta secretion differentially by monocytes and macrophages.     

Interleukin-4 ameliorates crescentic glomerulonephritis in Wistar Kyoto rats

BACKGROUND: Activated macrophages play a central role in crescentic glomerulonephritis. Interleukin-4 (IL-4) down-regulates many macrophage proinflammatory activities. We therefore studied the effect of IL-4 on glomerular injury in a model of crescentic glomerulonephritis in the Wistar Kyoto rat. METHODS: Glomerulonephritis was induced by i.v. administration of rabbit antirat glomerular basement membrane antiserum (nephrotoxic serum, NTS). In experiment 1, IL-4 was given from two hours before NTS until day 6. In experiment 2, rats were treated from day 0 to 7 and were then monitored until killed on day 28. In experiment 3, IL-4 was given from day 4 to 7. RESULTS: Continuous IL-4 treatment (experiment 1) significantly (P = 0.001) reduced proteinuria (3 +/- 1 mg per 24 hr vs. 56 +/- 7), fibrinoid necrosis (0.06 +/- 0.04 quadrants/glomulus vs. 1.2 +/- 0.1), macrophage infiltration (6.7 +/- 2.6 cells/glom vs. 33 +/- 2.5), CD8+ cells (1.5 +/- 0.6 cells/glom vs. 6.2 +/- 1.1), inducible nitric oxide synthase positive cells (0.04 +/- 0.04 cells/glom vs. 3.7 +/- 0.6), proliferating cell nuclear antigen positive cells (3.2 +/- 1 cells/glom vs. 15 +/- 2.3), and glomerular intercellular adhesion molecule-1 expression. Follow-up after seven days of treatment (experiment 2) showed that at four weeks, creatinine clearance was higher in treated rats (1.1 +/- 0.1 ml/min vs. 0.4 +/- 01, P = 0.011), and both glomerular scarring (P = 0.006) and tubular atrophy (P = 0.006) were less. Delayed treatment (experiment 3) reduced proteinuria (41 +/- 5 mg per 24 hr vs. 97 +/- 9, P = 0.004) and fibrinoid necrosis (0.39 +/- 0.05 quadrants/glom vs. 1.6 +/- 0.1, P = 0.004). There was no difference in macrophage infiltration, but inducible nitric oxide synthase positive cells were reduced (0.6 +/- 0.1 cells/glom vs. 1.8 +/- 0.4, P = 0.01) as were ED3+ cells (0.18 +/- 0.06 cells/glom vs. 1.86 +/- 0.21, P = 0.004). CONCLUSION: In this model of crescentic glomerulonephritis, early IL-4 treatment abolished proteinuria and markedly reduced glomerular inflammation. If treatment was stopped after seven days, there was continuing benefit on glomerular and tubulointerstitial scarring and creatinine clearance at four weeks. If treatment was delayed until inflammation was established, there was still a reduction of injury, but without an alteration of macrophage numbers, suggesting that IL-4 may be acting, in part, to reduce macrophage activation.     

Continuous fluid resuscitation and splenectomy for treatment of uncontrolled hemorrhagic shock after massive splenic injury

BACKGROUND: Using a standardized massive splenic injury (MSI) model of uncontrolled hemorrhagic shock, we studied the effect of continuous fluid resuscitation and splenectomy on the hemodynamic response and survival in rats. METHODS: The animals were randomized into seven groups: group 1 (n = 8), sham-operated; group 2 (n = 8), MSI untreated; group 3 (n = 8), MSI treated with 7.5 mL/kg/h of 7.5% NaCl (hypertonic saline [HTS]) for 1 hour; group 4 (n = 8), MSI treated with 7.5 mL/kg/h hydroxyethyl starch (HES-7.5) for 1 hour; group 5 (n = 8) MSI treated with 35 mL/kg/h Ringer's lactate (RL) solution (RL-35) for 1 hour; group 6 (n = 8) MSI treated with 70 mL/kg/h RL for 1 hour (RL-70); and group 7 (n = 8), MSI treated with 105 mL/kg/h RL for 1 hour (RL-105). In all MSI groups, splenectomy was performed 45 minutes after injury. RESULTS: MSI in untreated group 2 resulted in a fall of mean arterial pressure from 105.9 +/- 10.7 mm Hg to 27.0 +/- 6.7 mm Hg (p < 0.001) in 60 minutes. Mean survival time after splenectomy in this group was 160.7 +/- 29.7 minutes, and total blood loss was 34.8 +/- 4.7% of blood volume. Continuous HTS infusion with splenectomy in group 3 was followed by a total blood loss of 38.7 +/- 4.4% and mean survival time was 176.5 +/- 23.2 minutes. HES-7.5 infusion and splenectomy was followed by a total blood loss of 39.6 +/- 2.5% and survival time was 171.6 +/- 19.5 minutes. Continuous infusion of increasing volumes of RL in groups 5, 6, and 7 was followed by increase in blood loss to 29.0 +/- 4.1%, 50.2 +/- 3.1% (p < 0.001), and 62.7 +/- 7.1% (p < 0.002) of total blood volume, respectively. Mean survival time in groups 5, 6, and 7 was 233.5 +/- 6.5 minutes (p < 0.04), 207.6 +/- 17.0 minutes (p < 0.05), and 158 +/- 26 minutes, respectively. CONCLUSION: Continuous infusion of large-volume RL and splenectomy after massive splenic injury resulted in a significant increase in intra-abdominal bleeding and shortened survival time compared with small-volume RL infusion.     

Do female sex steroids adversely or beneficially affect the depressed immune responses in males after trauma-hemorrhage?

HYPOTHESIS: Administration of female sex steroids in males after trauma-hemorrhage has salutary effects on the depressed immune responses. DESIGN: Randomized laboratory experiment. INTERVENTIONS: Male C3H/HeN mice were subjected to midline laparotomy and hemorrhagic shock (35+/-5 mm Hg for 90 minutes, then resuscitation) or sham operation and received subcutaneous 17beta-estradiol (40 microg/kg body weight) or corn oil vehicle at the beginning of resuscitation. MAIN OUTCOME MEASURES: At 24 hours after hemorrhage, the animals were killed and plasma 17beta-estradiol and IL-6, splenocyte interleukin (IL) 2, IL-3, and IL-10 production as well as splenic and peritoneal macrophage IL-1beta, IL-10, and IL-6 release were measured. RESULTS: Splenocyte IL-2 and IL-3 release were significantly depressed after hemorrhage in vehicle-treated mice (P<.05, analysis of variance). Treatment with 17beta-estradiol after hemorrhage led to the restoration of splenocyte IL-2 and IL-3 release. The depressed proinflammatory cytokine (IL-1 and IL-6) release seen in splenic and peritoneal macrophages was restored in the 17beta-estradiol-treated hemorrhage group. In contrast, the sustained release of the anti-inflammatory cytokine IL-10 by splenocytes and splenic and peritoneal macrophages in vehicle-treated mice after hemorrhage was decreased in 17beta-estradiol-treated mice. The increase in circulating IL-6 levels after hemorrhage was significantly attenuated in 17beta-estradiol-treated mice. Although administration of 17beta-estradiol increased plasma 17beta-estradiol levels by approximately 100% in sham as well as hemorrhage groups, improved immune responses were seen only in posthemorrhage 17beta-estradiol-treated mice. There was no adverse effect of 17beta-estradiol treatment in the sham or posthemorrhage groups. CONCLUSION: Since administration of a single dose of 17beta-estradiol in males after trauma-hemorrhage restores the immune functions and decreases circulating levels of IL-6, hormones with estrogenic properties should be considered as safe and novel therapeutic agents for restoring the immune responsiveness in male trauma victims.     

Comparative effect of oral pulse and intravenous calcitriol treatment in hemodialysis patients: the effect on serum IL-1 and IL-6 levels and bone mineral density

INTRODUCTION: Increased serum levels of bone-resorptive cytokines such as interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) have been implicated for changes in bone remodeling in hemodialysis patients. In this prospective randomized study, we aimed to compare the effect of oral and intravenous (IV) pulse calcitriol on serum levels of IL-1 beta and IL-6. PATIENTS AND METHODS: Twenty-eight hemodialysis patients were included and consecutively randomized to receive either oral (n = 14, M/F = 7/7, mean age 42 +/- 15 years) or IV pulse (n = 14, M/F = 6/8, mean age 38 +/- 14 years) calcitriol treatment. No difference was found between groups for age, sex distribution, primary renal disease, mean time on hemodialysis and baseline biochemical parameters including serum levels of IL-1 beta and IL-6. RESULTS: The percent fall of intact parathyroid hormone (iPTH) was significantly less with oral compared to IV calcitriol between 0 and the 3rd month (32 +/- 21 vs. 56 +/- 28%, p = 0.03). However, the percent fall in iPTH at the 6th month of the therapy was not different in the oral group compared to the IV group (57 +/- 22 vs. 73 +/- 24%, p = 0.12). The increase in bone mineral densities was higher in the IV group than the oral group. Oral and IV calcitriol caused a significant fall in IL-1 beta (p = 0.02 and p = 0.03, respectively) and IL-6 levels (p = 0.02 and p < 0.001, respectively) at the 6th month of treatment. The percent fall in serum IL-6 levels at the 6th month was significantly greater in the IV compared to the oral group (61 +/- 18 vs. 36 +/- 33%, p = 0.04), while the percent changes in serum IL-1 beta levels were similar. CONCLUSION: IV calcitriol therapy has a greater suppression of PTH at the 3rd month of the therapy. Despite no difference in serum PTH levels at the 6th month, IV therapy has a greater increase in bone mineral densities and a greater decrease in serum IL-6 levels. These findings suggest IV calcitriol treatment has a superior effect on bone remodeling by influencing the levels of bone-resorptive cytokines as compared to the oral therapy group, beyond its suppressive effect on iPTH.     

Relation of inflammatory cytokines to atrial fibrillation after off-pump coronary artery bypass grafting.

OBJECTIVE: It has been observed that a systemic inflammatory response after on-pump coronary artery bypass grafting (CABG) participates in the pathogenesis of postoperative atrial fibrillation (AF). In patients undergoing off-pump CABG, it is plausible that inflammation is associated with the development of postoperative AF. The present study examined relation of proinflammatory cytokines, which play an important role in the upstream of inflammatory cascade, to the development of AF after off-pump CABG. METHODS: The present study included 39 patients undergoing off-pump CABG. Tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, and IL-8, were measured by enzyme-linked immunosorbent assay, on anesthetic induction, after sternotomy before anastomoses, at the completion of anastomoses, 3 and 6h thereafter, and on postoperative days (POD) 1-4. C-reactive protein (CRP) was also measured by turbidimetric immunoassay, preoperatively, and on POD 1, 2, 3, 6, 9, and 13. RESULTS: Eleven patients (28%) developed postoperative AF. Patients with postoperative AF were older (70+/-6.4 years vs 60+/-8.8 years, P=0.001); however, there was no difference in other pre- and perioperative variables. TNF-alpha level did not change during the study period. However, IL-8 and CRP levels significantly increased after the surgery, although there was no significant difference between the two groups. IL-6 level also increased after the surgery with its peak at 6h after the completion of anastomoses. IL-6 levels of 3 and 6h after anastomoses were significantly higher in patients with postoperative AF (360+/-143 pg/ml vs 230+/-94 pg/ml, P=0.0047, 435+/-175 pg/ml vs 247+/-102 pg/ml, P=0.0005, respectively). Logistic regression analysis indicated that the highest quartile of IL-6 level immediately after the surgery (odds ratio 7.63; 95% CI, 1.06-54.9; P=0.04) and age (odds ratio 1.18; 95% CI, 1.01-1.39; P=0.04) independently predict postoperative AF. Furthermore, the maximum level of IL-6 immediately after the surgery significantly correlated to age and intraoperative blood loss (r=0.04, P=0.01, and r=0.47, P=0.04, respectively). CONCLUSIONS: Advanced age was a major risk factor for postoperative AF. Furthermore, inflammatory response induced by surgical trauma was also associated with the development of AF after off-pump CABG.     

Modified rapid deployment hemostat bandage reduces blood loss and mortality in coagulopathic pigs with severe liver injury

BACKGROUND: Hemostasis can be difficult to achieve after blunt abdominal trauma, especially if the patient is coagulopathic. The U.S. Food and Drug Administration has recently approved a hemostatic dressing for treating bleeding after extremity trauma (RDH bandage; Marine Polymer Technologies, Cambridge, MA). It has not been evaluated for internal bleeding after trauma. We redesigned this dressing for internal use, and then tested whether this modified bandage (Miami-modified Rapid Deployment Hemostat) could achieve hemostasis when used as an adjunct to standard laparotomy pad packing in a pig model of severe liver injury with coagulopathy. METHODS: Anesthetized swine (35-45 kg) received an isovolemic 45% blood volume replacement with refrigerated Hextend (6% hetastarch). Core body temperature was maintained at 33-34 degrees C with intra-abdominal ice packs. A coagulopathic condition was documented by thromboelastography. At this point a severe liver injury was induced by the avulsion of the left lateral hepatic lobe, then the pigs were randomized to treatment with either standard abdominal packing (control) or packing plus Miami-modified Rapid Deployment Hemostat. Two series of experiments were conducted. In series one (n = 14), the abdomen was closed and the animals were observed with no resuscitation. After one hour, the abdomen was opened, the packing was removed and the presence of bleeding was noted. In series two (n = 10), the abdomen was closed and the animal resuscitated with one unit of blood plus as much lactated Ringers intravenous fluid (IVF) as required to maintain a mean arterial pressure (MAP) > 70 mm Hg. After one hour, the packing was removed, the abdomen closed, and data were collected for an additional two hours. RESULTS: Series one: 6/7 animals in the control group had continued bleeding at one hour; 1/7 animals in the treatment group had active bleeding (p = 0.0291). Series two: With control vs. Miami-modified Rapid Deployment Hemostat, the three-hour survival was zero vs. 80% (p = 0.0476). The total blood loss was 1.2 +/- 0.1 vs. 0.3 +/- 0.1 mL/kg/min (p = 0.001) and the IVF requirement was 1.6 +/- 0.3 vs. 0.6 +/- 0.3 mL/kg/min (p = 0.026). CONCLUSIONS: The Miami-modified Rapid Deployment Hemostat bandage significantly reduced mortality, blood loss, and fluid requirements when used as an adjunct to standard abdominal packing following severe liver injury in coagulopathic pigs [corrected].