Biomedical subjects
Publications and source records attributed to G Wakabayashi.
Recent clinical trials with experimental immunotherapeutic agents for sepsis have not proven their overall benefit yet although some studies suggested significant effect of these antisepsis therapies. This review article summarizes the results of these trials, their analysis, lessons learned from past failure, and future perspectives for immunotherapies as anti-sepsis treatments.
OBJECTIVE: To investigate the involvement of vasoactive agents, endothelin (ET)-1, and atrial natriuretic peptide (ANP), and the responses of cytokines in patients undergoing total gastrectomy. DESIGN: Prospective study. SETTING: University hospital, Japan. SUBJECTS: 20 patients with advanced gastric cancer who had undergone total gastrectomy with lymph node dissection. INTERVENTIONS: Serum or plasma samples collected on the day before the operation, at the time of skin closure, and on postoperative days 1, 3, 5, and 7. MAIN OUTCOME MEASURES: Concentrations of acute phase reactants, cytokines (interleukin (IL)-1, tumour necrosis factor (TNF) and interleukin (IL)-6), and vasoactive agents (ET-1 and ANP). RESULTS: There were significant increases in concentrations of IL-6 and acute phase reactants postoperatively. ET-1 and ANP concentrations did not change significantly. CONCLUSION: There was no correlation between concentrations of the vasoactive agents ET-1 and ANP, and those of acute phase reactants or cytokines in serum or plasma in patients undergoing total gastrectomy.
The effects of camostat mesilate, a synthetic serine protease inhibitor on gastric microcirculation and active oxygen species generated by leucocytes from the gastric and jugular veins in the early period after thermal injury were assessed. Male Wistar rats were anaesthetized and a 30% full skin-thickness dorsal burn was inflicted. Camostat mesilate (100 mg/kg) was dissolved in distilled water and administered orally to rats 40 min before thermal injury (the camostat group). The control animals (the vehicle group) were administered distilled water orally. Rolling leucocytes as well as Monastral blue B deposits in venules were observed using in vivo microscopy. Active oxygen species were measured by chemiluminescence. Camostat mesilate decreased the total length of gastric erosion, venular deposits of Monastral blue B, and rolling of leucocytes in venules, and relatively increased luminol-dependent chemiluminescence activity generated by zymosan-stimulated leucocytes 15 min after thermal injury. These results suggest that serine proteases are involved in the formation of gastric erosions and gastric microcirculatory disturbance in the early period after thermal injury.
Nuclear matrix protein (NMP), an indicator of apoptosis, was analyzed in patients with multiple organ dysfunction syndrome (MODS). Blood levels of tumor necrosis factor-alpha (TNF-alpha) and nitrite/nitrate (NOx) were also measured in these patients to determine the involvement of these factors in the production of NMP. Forty-six patients with MODS were studied, 21 (45.7%) of whom died. NMP and TNF-alpha were measured by enzyme-linked immunosorbent assay (ELISA). NOx was measured by the Griess's method. Marshall's multiple organ dysfunction score (MOD score) was used as an indicator of organ failure. The severity of organ failure was assessed by use of the acute physiology and chronic health evaluation II score (APACHE II score). The number of organs that failed was found to be significantly correlated with the NMP level. The NMP level was also correlated significantly with MOD score and APACHE II score, as well as with TNF-alpha and NOx levels. NMP and NOx levels in the group that died significantly exceeded those in the surviving group. Results suggest that apoptosis can occur in the presence of MODS, and that its extent increases as the number of failing organs increases. The results also suggest that TNF-alpha and NO are involved in the induction of apoptosis.
In our previous study we showed that rebamipide attenuated gastric erosions and active oxygen species released only from the gastric mucosa and not from circulating leukocytes after thermal injury. This study was designed to examine whether rebamipide affects the potential of active oxygen generation from circulating leukocytes, and attenuates microcirculatory disturbance caused by thermal injury to skin. Rats were anesthetized and a 30% full skin-thickness dorsal burn was inflicted. Microvascular images and leukocytes were observed using in vivo microscopy. Endothelial damage was assessed by monastral blue B deposits. Active oxygen species were measured by the chemiluminescence method. Rebamipide (100 mg/kg) decreased leukocyte rolling and monastral blue B deposits in venules but did not improve arteriolar contractions 15 min after thermal injury. These results suggest that rebamipide preserves gastric microcirculation possibly through inhibition of leukocyte adhesion and endothelial damage caused by thermal injury to skin.
BACKGROUND: Oxygen-derived free radicals (FRs) are critical mediators of ischemia/reperfusion injury. Inflammatory cytokines have been shown to play important roles in tissue injury. To examine the relationship between FRs and interleukin-1 (IL-1) in hepatic ischemia/reperfusion injury, we used interleukin-1 receptor antagonist (IL-1ra) to block endogenous IL-1 production in a rat model of hepatic ischemia/reperfusion. METHODS: Female SD rats were subjected to 30 min of hepatic ischemia followed by reperfusion. The animals were divided into two groups, control group and IL-1ra-treated group, according to the rinse solution. In both groups, FR production, histological changes, and interactions between leukocytes and endothelial cells were analyzed in the course of reperfusion. RESULTS: In the control group, production of FRs increased significantly after 60 min of reperfusion. After 60 and 180 min of reperfusion, histological examination showed atrophy and degeneration of hepatocytes. Hepatic microcirculation demonstrated a marked increase in the number of leukocytes adherent to endothelial cells and of injured cells after reperfusion. In the IL-1ra-treated group, IL-1ra pretreatment markedly reduced FR production after 60 min of reperfusion, the number of leukocytes adherent to endothelial cells, and tissue injury. CONCLUSION: These data clearly show an important role for IL-1 in the induction of FR production, leukocyte adhesion, and tissue injury after hepatic ischemia/reperfusion injury.
BACKGROUND: Prostaglandin E1 (PGE1) has been reported to have a protective effect in experimental and clinical models of liver damage. The aim of this study was to elucidate the effects of the intraportal infusion of PGE1 on hepatic blood flow and graft viability after orthotopic liver transplantation in pigs. METHODS: First, the hepatic arterial flow (HAF), portal venous flow (PVF), and liver tissue blood flow (LTBF) were measured during the continuous intravenous or intraportal infusion of PGE1. Second, two groups of pigs underwent orthotopic liver transplantation: group A, untreated controls; and group B, animals that received intraportal PGE1 for 2 hr after vascular reconstruction of the allograft. Changes in HAF, PVF, LTBF, and hepatic function were measured. RESULTS: The intraportal infusion of PGE1 significantly increased HAF and had no effect on blood pressure, PVF, or LTBF. In group B, HAF and LTBF increased significantly with time. In group A, HAF remained unchanged and a decrease in LTBF was observed. Group B exhibited a higher arterial ketone body ratio and a greater bile flow compared with group A. A significant elevation in serum glutamic oxaloacetic transaminase concentration was observed in group A, but not in group B. CONCLUSIONS: This study demonstrates that the intraportal infusion of PGE1 improves hepatic allograft blood flow, predominantly through an effect on HAF, and may improve graft viability after orthotopic liver transplantation.
The inflammatory cytokines interleukin (IL) 1 and tumor necrosis factor (TNF) may play an important role in hepatic ischemia-reperfusion (I/R) injury. To study the role of IL-1 in hepatic I-R injury, we investigated the effect of pretreatment with IL-1 receptor antagonist (IL-1ra) on the production of IL-1, TNF, histological findings in the liver, and the survival rate for 7 days. Rats were subjected to 90 min of partial liver warm ischemia by clamping the vessels of the left and middle lobes. In the IL-1ra-treated group, IL-1ra was given 5 min before liver ischemia was induced. IL-1alpha and TNF levels were determined in blood and liver at 0, 30, 90, and 180 min after reperfusion. In a second experiment to determine the effect of IL-1ra pretreatment on survival rate, after 90 min of partial liver ischemia, the right lateral and caudate lobes were excised, leaving only the ischemic lobes. In both groups, IL-1alpha was undetectable in blood, but increased in liver tissue. TNF increased in both blood and liver tissue as reperfusion time increased. Histological evidence of tissue injury was minimal in the IL-1ra-treated group. Furthermore, in the IL-1ra-treated group, the production of TNF decreased in both blood and liver tissue compared with the nontreated group. Survival rates in the IL-1ra-treated and nontreated group were 80% and 30%, respectively. The data demonstrated that the production of IL-1 and TNF increases in hepatic I-R injury and that pretreatment with IL-1ra protects the liver from ischemic insult, indicating an important role for IL-1 in I-R injury.
BACKGROUND: Although high levels of interleukin-8 (IL-8) have been found in patients with sepsis and a monoclonal antibody (MoAb) against IL-8 has been successfully used in some animal models of inflammation, no specific therapeutic agent against IL-8 has been tested for the treatment of sepsis. We studied the effects of a MoAb against IL-8 in the treatment of endotoxic shock with a prospective randomized rabbit endotoxic shock model. METHODS: Twenty New Zealand white rabbits were anesthetized and divided into four groups: normal, anti-IL-8, control-Ab, and lipopolysaccharide (LPS). Anti-IL-8 and control-Ab groups received a MoAb (immunoglobulin G, 3 mg/kg) 5 minutes before the LPS injection. All groups, except the normal group, received a continuous 20-minute infusion of LPS (500 micrograms/kg). The normal group received NaCl (0.9%) rather than LPS. RESULTS: The 7-day survival rates were 100% for normal group, 80% for anti-IL-8 group, 40% for control-Ab group, and 0% for LPS group. Compared with the LPS group, anti-IL-8 rabbits had a smaller decrease in mean arterial blood pressure (p < 0.05) and increased urinary volume (p < 0.05). Anti-IL-8 rabbits had lower plasmatic levels of IL-1 beta, less free radical production (p < 0.05), and a higher survival rate (p < 0.01). CONCLUSIONS: IL-8 plays a significant role in endotoxic shock, and IL-8 blockage results in attenuation of the hypotensive and tachypneic effects of LPS, reduced free radical production, and an increased survival rate after lethal endotoxic shock.
Microcirculatory disturbance is a crucial step in development of gastric mucosal lesions. In this study we investigated the influence of nitric oxide (NO) on the irregular constriction of gastric venules observed in rats subjected to thermal injury. Male Wistar rats were anesthetized and a 30% full skin thickness dorsal burn was inflicted. Microvascular images in the submucosal layer and basal region of the gastric mucosa were obtained through an intravital microscope. Distribution of constitutive-type nitric oxide synthase (cNOS) was observed by the immunofluorescence method. Venule walls were basically smooth in the control group (no injury), whereas irregular constrictions tended to be observed in the 5-h group (5 h after thermal injury). Venule diameter was significantly smaller in the 5-h group. In the control group, cNOS-associated fluorescence was found on the venule endothelium, whereas it was almost negligible after thermal injury. A NO donor, 3-morpholinosydnonimine hydrochloride, simultaneously diminished the thermal injury-associated venule constriction and irregularity of venule walls. Therefore, the present study suggests that venule constriction, which is observed 5 h after thermal injury, is related to decreased NO production by endothelial cNOS and is a cause of irregularity of the venule walls.
The effect of ulinastatin, a protease inhibitor, on hematopoiesis was studied. Ulinastatin was found to increase the number of colony forming units of mouse myelocytes in culture, suggesting that it might promote hematopoiesis.
OBJECTIVE: To detect cytokine gene expression in unfractionated peripheral blood by reverse transcriptase-polymerase chain reaction (RT-PCR). DESIGN: Prospective study. SETTING: University hospital, Japan. SUBJECTS: 3 healthy volunteers and 3 severely infected patients. INTERVENTIONS: Peripheral blood was obtained and total RNA was extracted from 0.5 ml unfractionated whole blood with a 4 M guanidinium isothiocyanate mixture, 0.2 M sodium acetate, phenol, and chloroform. The mRNA was reverse transcripted, and interleukin-1 beta (IL-1beta) and tumour necrosis factor (TNF) cDNA were selectively amplified by synthetic primers with PCR. MAIN OUTCOME MEASURES: Establishment of cytokine gene expression in unfractionated peripheral blood. RESULTS: About 10 microg of total RNA was obtained from a 0.5 ml sample of blood. IL-1beta and TNF mRNA were not detected in blood from healthy volunteers, though they were detected in patients with severe infection. CONCLUSION: This method avoids artefactual gene activation and may be applicable to monitoring cytokine gene expression in various pathophysiological states.
We measured the plasma levels of anti-inflammatory cytokines, including interleukin 1 receptor antagonist (IL-1ra), IL-4 and IL-10; inflammatory cytokines, including IL-2, IL-6, IL-8 and tumor necrosis factor receptor I and II (TNFR I and TNFR II); and endotoxin in 11 patients with septic shock associated with gram-negative bacteria and 12 patients with sepsis not associated with shock. The plasma levels of IL-1ra and IL-10 were elevated in the septic shock group compared with the sepsis group. TNFR I and TNFR II levels tend to be higher in the septic shock group. The plasma level of TRNF-alpha was significantly correlated with levels of IL-1ra, IL-4, IL-10, TNFR I, and TNFR II. The elevated levels of the anti-inflammatory cytokines, TNFR I, and TNFR II, appeared to reflect an attempt to suppress the shock syndrome.
Although small intestine is frequently injured in endotoxin shock, the exact pathological sequence has not been fully understood. The major objective of this study is to elucidate the role of interleukin (IL)-1 in endotoxin-induced microcirculatory disturbance of rat small intestine. Mucosal and submucosal microvessels of the rat ileum were observed by intravital microscope with a high speed video camera system and the attenuating effect of E5090, an inhibitor of IL-1 generation, on endotoxin-induced intestinal microcirculatory disturbances was investigated. Endotoxin infusion produced significant mucosal damage, but before these morphological changes became significant, microvascular stasis in villi, decreased red blood cell velocity, and increased leukocyte adherence to venular walls were observed in intestinal microcirculatory beds 30 min after endotoxin administration. Intestinal IL-1alpha levels were also significantly increased at that time. Endotoxin treatment enhanced chemiluminescence activity from neurophils and rapidly mobilized CD18 on leukocytes. E5090, which suppressed the IL-1 production in intestinal mucosa, attenuated the microcirculatory disturbances induced by endotoxin, and significantly reduced the subsequent mucosal damage. E5090 also attenuated the increased chemiluminescence activity and CD18 expression on leukocytes. In conclusion, the production of IL-1alpha is enhanced in the intestinal mucosa during endotoxin infusion. IL-1 may be an important mediator of microcirculatory changes, including decreased red blood cell velocity and increased leukocyte sticking and its activation, leading to the mucosal damage.
STUDY OBJECTIVE: To determine the effects of steroids on the lung accumulation of polymorphonuclear leukocytes (PMNs) and monocytes in rabbits with endotoxemia. DESIGN: A prospective, randomized, controlled animal trial. SETTING: Surgical research laboratory, Keio University School of Medicine. SUBJECTS: Twenty-four female Japanese white rabbits. INTERVENTIONS: Rabbits with endotoxemia were pretreated with steroids sufficient to inhibit the production of tumor necrosis factor (TNF) and to prevent the fall of BP. MEASUREMENTS AND RESULTS: The circulating leukocyte counts and the leukocyte accumulation in the lung were evaluated. Endotoxin caused a rapid decrease in circulating PMNs and monocytes followed by an 8-fold greater accumulation of PMNs (p < 0.001) and a 6.5-fold greater accumulation of monocytes (p < 0.05). Steroids failed to inhibit this initial drop of PMNs and monocytes. However, steroids inhibited the PMN accumulation in the lung by 50% (p < 0.05), without inhibiting the monocyte accumulation in the lung. CONCLUSIONS: These findings suggest that the lung accumulation of PMNs is dependent on TNF or other inflammatory mediators that are inhibited by steroids while the lung accumulation of monocyte is not.
We assessed the plasma levels of nitrite/nitrate (NOx) and soluble Fas antigen (sFas) in patients with multiple organ failure (MOF). The NOx levels showed high levels in MOF patients with sepsis and without infection. A significant difference was not seen between the MOF patients complicated by sepsis and uncomplicated by infection, although the NOx levels in the former group tended to be higher. There was a significant correlation between NOx levels and sFas levels in MOF patients. Both sFas and NOx levels were high during MOF, and rapidly decreased when MOF was relieved. These findings suggest that sFas and NOx contribute to the development of MOF.
Microcirculatory disturbances in the gastric mucosa was investigated in rats after thermal injury using intravital video microscopy. Mucosal blood flow decreased significantly 5 hrs after thermal injury and increased neutrophil-endothelial cell interaction was observed at the same time. Zymosan stimulated-free radical production from peripheral blood was also increased and acute gastric mucosal lesion (AGML) appeared 5 hrs after thermal injury. These data suggest that neutrophil-endothelial cell interaction and increased free radical production induce AGML formation and that microcirculatory disturbance is one of the main reasons for organ failure after surgical insults. The role of inflammatory cytokines in hepatic microcirculatory disturbance was also studied in endotoxin-injected rats using IL-1 receptor antagonist (IL-1Ra) or TNF binding protein (TNFbp). Pretreatment with IL-1Ra or TNFbp significantly improved hepatic microcirculatory disturbance and reduced both the number of leukocytes adhered to the sinusoidal wall and the number of injured cells as well. These data suggest that inflammatory cytokines play a crucial role in microcirculatory disturbance after surgical insults by promoting neutrophil-endothelial cell interaction and systemic excessive inflammation.
The function of vascular endothelial cells is to adjust blood vessel tonus, which contributes to maintaining homeostasis within blood vessels. However, inflammatory cytokines are produced in response to invasion by stimulating vascular endothelial cells and sometimes lead to shock or multiple organ failure. In the present study, we assessed cytokines in sepsis and septic shock, and various factors that are said to have a damaging effect on vascular endothelium. Endotoxin was measured by endotoxin-specific methods. Tumor necrosis factor-alpha (TNF-alpha), interleukin 6 (IL-6), and interleukin 8 (IL-8) were measured by enzyme-linked immunosorbent assay (ELISA). Endothelin-I was measured by radioimmunoassay (RIA). Nitric oxide was measured as metabolites of nitrite and nitrate oxides (NOx) by a method based on the Griess method. Thromboxane B2 (TXB2) and 6-keto-prostaglandin F1 alpha (PGF 1 alpha) were both measured by RIA. All of the factors except endotoxin were significantly higher in the septic shock group than in the non-shock group and significantly higher in the non-survivor group than in the survivor group. Significant correlations were also found between endothelin-1 and NOx and between TXB2 and PG1 alpha. Significant correlations were also found between TNF-alpha and IL-6, endothelin-1, NOx and TXB2, but no significant correlations were detected between any of them and endotoxin. In serious diseases such as septic shock, the vascular endothelial constricting factors, endothelin and TXB2, and the blood vessel relaxing factors NOx and PGF1 alpha increase almost simultaneously. This suggests that the body's regulating mechanisms are disrupted in these serious conditions. The results of this study also suggest that inflammatory cytokines may be involved in stimulating the production of these factors.