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Bacterial lipoprotein induces endotoxin (LPS) independent tolerance to systemic inflammatory response syndrome
S8
Surgical Forum Abstracts
Montreal, Quebec. Correspondence: Nicolas V. Christou, MD, PhD, FRCS(C), Professor of Surgery, Royal Victoria Hospital, 687 Pine Ave. W., Room C5-53, Montreal, Quebec, Canada H3A 1A1; Phone: 514-843-1531; Fax: 514-843-1430. Introduction: In vivo regulation of polymorphonuclear neutrophil (PMN) chemoattractant receptors and their functional significance remain poorly understood. We hypothesized that (1) alteration in receptors for both PMN specific (e.g. Interleukin-8) & non-specific (e.g. C5a) chemoattractants occurs in humans in vivo, and (2) this regulates the ability of PMN’s to physically respond to chemoattractants. Methods: We evaluated chemoattractant receptor expression (C5a receptor (C5aR), Interleukin-8 RA (IL-8RA), & B (IL-8RB) with immunofluorescent monoclonal antibodies & flow cytometry) and in vitro chemotaxis to C5a & IL-8 (Boyden chambers), in three functionally separate groups of human PMN: (1) circulating PMN (venipuncture), (2) exudate PMN (collected with skin blister skin window method), both from healthy controls (n ⫽ 10), and (3) circulating PMN from patients with sepsis (n ⫽ 6, APACHE II mean 26.6, range 15–38). Data presented as mean ⫾ SD; ANOVA and paired t-test used for analysis. Results: Compared to circulating control PMN, septic PMN had a reduction in C5aR and chemotaxis to C5a; whereas exudate PMN had an elevation in C5aR, but no significant increase in chemotaxis to C5a (Figure 1). Conversely, exudate PMN displayed reduced IL-8RA, IL8RB, & chemotaxis to IL-8; and septic PMN displayed reduced IL-8RA & IL-8RB, and no change in chemotaxis to IL-8 (Figure 2).
J Am Coll Surg
P38 was assayed 30mins after BLP, LPS or LPS ⫹ BLP stimulation in tolerised and non-tolerised macrophages by western immunoblotting. Results: BLP tolerance resulted in 0% mortality compared to controls (60%: p ⬍ 0.05). BLP tolerance also protected against LPS mortality, 30% vs 80% (control): p ⬍ 0.05 and BLP ⫹ LPS lethality, 0% vs 60% (control): p ⬍ 0.05. Although LPS tolerance protects against its own lethality, it did not protect against BLP or BLP ⫹ LPS mortality. Pre-treatment of macrophages with BLP significantly attenuated LPS, BLP and LPS ⫹ BLP stimulation-induced TNF-␣ production and supressed the p38 dependent signal transduction pathway. Conclusions: BLP tolerance protects against its own lethal effects as well as endotoxin. This effect appears to be mediated through supression of p38 and TNF-␣.
Cardiothoracic Surgery I: Innovations/Neonatal-Congenital Laser versus mechanical transmyocardial revascularization: do you need a light? Keith A Horvath, MD, Noam Belkind, Rodney Greene, BS, Bonnie Kane, David D McPherson, MD, David A Fullerton, MD. Feinberg Cardiovascular Research Institute, Northwestern Univ. Med. School, 251 E. Chicago Avenue, Wesley 1030, Chicago, IL 60611, USA, (312) 908-3121 Introduction: The success of transmyocardial laser revascularization in treating patients with severe coronary artery disease has lead to a renewed effort to achieve the same benefit by mechanical means. While both methods create a similar transmural channel the importance of laser light in creating these channels is unknown. The purpose of this study was to determine whether mechanical revascularization improves myocardial function and increases angiogenesis as compared to laser revascularization in chronically ischemic myocardium.
Conclusions: Simultaneous evaluation of chemoattractant receptors & chemotaxis in three distinct groups of human PMN demonstrated: (1) PMN chemoattractant receptor expression is capable of dynamic alteration in vivo; (2) selective reduction of chemoattractant receptors provides a mechanism to explain reduced PMN delivery in septic patients; and (3) changes in PMN chemotaxis correlate with corresponding changes in chemoattractant receptors, but receptor alteration does not necessarily alter chemotaxis. These results suggest that change in receptor expression is necessary, but not sufficient to alter PMN chemotaxis.
Bacterial lipoprotein induces endotoxin (LPS) independent tolerance to systemic inflammatory response syndrome Majella Doyle AFRCSI, Jiang Huai Wang PhD, H. Paul Redmond FRCSI. Department of Academic Surgery, Cork University Hospital Wilton, Cork, Ireland. Tel. 35321901290 fax. 35321344230. Introduction: Sublethal doses of endotoxin (LPS) induce tolerance through a p38-dependent kinase pathway and protect against the lethal effects of a further LPS challenge. Tolerance is characterised by a diminished production of tumour necrosis factor-␣ (TNF-␣). The role of bacterial lipoprotein (BLP), an ubiquitous bacterial cell wall component, in tolerance induction has not been investigated. We hypothesised that BLP, could induce tolerance against itself and endotoxin and that this tolerance is characterised by reduced p38 kinase activity and TNF-␣ production. Methods: Male MF-1 mice were randomised into three groups (n ⫽ 30/group). Group 1 received a single BLP tolerant dose (IP, 10mg/kg). Group 2 received LPS (IP, 10mg/kg) tolerance. Following induction of tolerance, all mice were challenged with high doses of LPS (IP, 45mg/ kg), BLP (IP, 45mg/kg) or a combination of LPS ⫹ BLP (IP, 30 ⫹ 30mg/kg). Group 3 received only high doses of the above agents. Mortality was assessed over 48hrs. TNF-␣ was assayed from peritoneal macrophage culture supernatants, 6hrs after BLP, LPS or LPS ⫹ BLP stimulation ⫾ 24hr pre-treatment with BLP or medium, by ELISA.
Methods: In a model of chronic ischemia by Ameroid occlusion of the circumflex artery, 18 domestic pigs were randomized to: Group I treatment with revascularization by an excimer laser, Group II - mechanically revascularization using the same fiber that delivers the excimer laser light, or Group III - no treatment. Segmental contraction was assessed at rest and with dobutamine stress echocardiography. Six weeks after revascularization the animals were restudied at rest and with stress. Angiogenesis was assessed by counting blood vessels identified by factor VIII immunohistochemistry. Results: Compared to the mechanical group, transmyocardial revascularization by an excimer laser completely restored resting function in chronically ischemic myocardium. Control animals did not demonstrate significant recovery of function.
Laser Mechanical
Baseline Resting Function
Postop Resting Function
p value
0.38 ⫾ 0.07cm 0.49 ⫾ 0.08cm
0.67 ⫾ 0.11cm 0.53 ⫾ 0.05cm
⬍0.05 ns
Laser treatment of the ischemic zone led to an increase in the number of blood vessels per 200⫻ high powered field vs. mechanical treatment. Blood Vessels/hpf Ischemic Non-ischemic Zone Zone Laser Mechanical
31 ⫾ 2 17 ⫾ 3
16 ⫾ 2 15 ⫾ 3
p value ⬍0.05 ns
Control animals demonstrated no increase in blood vessels/hpf.
Conclusions: The addition of laser light to transmyocardial revascularization leads to improvement in function and increased angiogenesis in chronically ischemic myocardium. These data indicate that laser revascularization is more effective than transmyocardial revascularization by mechanical means.
Surgical Forum Abstracts
Montreal, Quebec. Correspondence: Nicolas V. Christou, MD, PhD, FRCS(C), Professor of Surgery, Royal Victoria Hospital, 687 Pine Ave. W., Room C5-53, Montreal, Quebec, Canada H3A 1A1; Phone: 514-843-1531; Fax: 514-843-1430. Introduction: In vivo regulation of polymorphonuclear neutrophil (PMN) chemoattractant receptors and their functional significance remain poorly understood. We hypothesized that (1) alteration in receptors for both PMN specific (e.g. Interleukin-8) & non-specific (e.g. C5a) chemoattractants occurs in humans in vivo, and (2) this regulates the ability of PMN’s to physically respond to chemoattractants. Methods: We evaluated chemoattractant receptor expression (C5a receptor (C5aR), Interleukin-8 RA (IL-8RA), & B (IL-8RB) with immunofluorescent monoclonal antibodies & flow cytometry) and in vitro chemotaxis to C5a & IL-8 (Boyden chambers), in three functionally separate groups of human PMN: (1) circulating PMN (venipuncture), (2) exudate PMN (collected with skin blister skin window method), both from healthy controls (n ⫽ 10), and (3) circulating PMN from patients with sepsis (n ⫽ 6, APACHE II mean 26.6, range 15–38). Data presented as mean ⫾ SD; ANOVA and paired t-test used for analysis. Results: Compared to circulating control PMN, septic PMN had a reduction in C5aR and chemotaxis to C5a; whereas exudate PMN had an elevation in C5aR, but no significant increase in chemotaxis to C5a (Figure 1). Conversely, exudate PMN displayed reduced IL-8RA, IL8RB, & chemotaxis to IL-8; and septic PMN displayed reduced IL-8RA & IL-8RB, and no change in chemotaxis to IL-8 (Figure 2).
J Am Coll Surg
P38 was assayed 30mins after BLP, LPS or LPS ⫹ BLP stimulation in tolerised and non-tolerised macrophages by western immunoblotting. Results: BLP tolerance resulted in 0% mortality compared to controls (60%: p ⬍ 0.05). BLP tolerance also protected against LPS mortality, 30% vs 80% (control): p ⬍ 0.05 and BLP ⫹ LPS lethality, 0% vs 60% (control): p ⬍ 0.05. Although LPS tolerance protects against its own lethality, it did not protect against BLP or BLP ⫹ LPS mortality. Pre-treatment of macrophages with BLP significantly attenuated LPS, BLP and LPS ⫹ BLP stimulation-induced TNF-␣ production and supressed the p38 dependent signal transduction pathway. Conclusions: BLP tolerance protects against its own lethal effects as well as endotoxin. This effect appears to be mediated through supression of p38 and TNF-␣.
Cardiothoracic Surgery I: Innovations/Neonatal-Congenital Laser versus mechanical transmyocardial revascularization: do you need a light? Keith A Horvath, MD, Noam Belkind, Rodney Greene, BS, Bonnie Kane, David D McPherson, MD, David A Fullerton, MD. Feinberg Cardiovascular Research Institute, Northwestern Univ. Med. School, 251 E. Chicago Avenue, Wesley 1030, Chicago, IL 60611, USA, (312) 908-3121 Introduction: The success of transmyocardial laser revascularization in treating patients with severe coronary artery disease has lead to a renewed effort to achieve the same benefit by mechanical means. While both methods create a similar transmural channel the importance of laser light in creating these channels is unknown. The purpose of this study was to determine whether mechanical revascularization improves myocardial function and increases angiogenesis as compared to laser revascularization in chronically ischemic myocardium.
Conclusions: Simultaneous evaluation of chemoattractant receptors & chemotaxis in three distinct groups of human PMN demonstrated: (1) PMN chemoattractant receptor expression is capable of dynamic alteration in vivo; (2) selective reduction of chemoattractant receptors provides a mechanism to explain reduced PMN delivery in septic patients; and (3) changes in PMN chemotaxis correlate with corresponding changes in chemoattractant receptors, but receptor alteration does not necessarily alter chemotaxis. These results suggest that change in receptor expression is necessary, but not sufficient to alter PMN chemotaxis.
Bacterial lipoprotein induces endotoxin (LPS) independent tolerance to systemic inflammatory response syndrome Majella Doyle AFRCSI, Jiang Huai Wang PhD, H. Paul Redmond FRCSI. Department of Academic Surgery, Cork University Hospital Wilton, Cork, Ireland. Tel. 35321901290 fax. 35321344230. Introduction: Sublethal doses of endotoxin (LPS) induce tolerance through a p38-dependent kinase pathway and protect against the lethal effects of a further LPS challenge. Tolerance is characterised by a diminished production of tumour necrosis factor-␣ (TNF-␣). The role of bacterial lipoprotein (BLP), an ubiquitous bacterial cell wall component, in tolerance induction has not been investigated. We hypothesised that BLP, could induce tolerance against itself and endotoxin and that this tolerance is characterised by reduced p38 kinase activity and TNF-␣ production. Methods: Male MF-1 mice were randomised into three groups (n ⫽ 30/group). Group 1 received a single BLP tolerant dose (IP, 10mg/kg). Group 2 received LPS (IP, 10mg/kg) tolerance. Following induction of tolerance, all mice were challenged with high doses of LPS (IP, 45mg/ kg), BLP (IP, 45mg/kg) or a combination of LPS ⫹ BLP (IP, 30 ⫹ 30mg/kg). Group 3 received only high doses of the above agents. Mortality was assessed over 48hrs. TNF-␣ was assayed from peritoneal macrophage culture supernatants, 6hrs after BLP, LPS or LPS ⫹ BLP stimulation ⫾ 24hr pre-treatment with BLP or medium, by ELISA.
Methods: In a model of chronic ischemia by Ameroid occlusion of the circumflex artery, 18 domestic pigs were randomized to: Group I treatment with revascularization by an excimer laser, Group II - mechanically revascularization using the same fiber that delivers the excimer laser light, or Group III - no treatment. Segmental contraction was assessed at rest and with dobutamine stress echocardiography. Six weeks after revascularization the animals were restudied at rest and with stress. Angiogenesis was assessed by counting blood vessels identified by factor VIII immunohistochemistry. Results: Compared to the mechanical group, transmyocardial revascularization by an excimer laser completely restored resting function in chronically ischemic myocardium. Control animals did not demonstrate significant recovery of function.
Laser Mechanical
Baseline Resting Function
Postop Resting Function
p value
0.38 ⫾ 0.07cm 0.49 ⫾ 0.08cm
0.67 ⫾ 0.11cm 0.53 ⫾ 0.05cm
⬍0.05 ns
Laser treatment of the ischemic zone led to an increase in the number of blood vessels per 200⫻ high powered field vs. mechanical treatment. Blood Vessels/hpf Ischemic Non-ischemic Zone Zone Laser Mechanical
31 ⫾ 2 17 ⫾ 3
16 ⫾ 2 15 ⫾ 3
p value ⬍0.05 ns
Control animals demonstrated no increase in blood vessels/hpf.
Conclusions: The addition of laser light to transmyocardial revascularization leads to improvement in function and increased angiogenesis in chronically ischemic myocardium. These data indicate that laser revascularization is more effective than transmyocardial revascularization by mechanical means.