High Monocyte Count and Expression of S100A9 and S100A12 in Peripheral Blood Mononuclear Cells Are Associated with Poor Outcome in Patients with Metastatic Prostate Cancer.

Increasing evidence indicates calcium-binding S100 protein involvement in inflammation and tumor progression. In this prospective study, we evaluated the mRNA levels of two members of this family, S100A9 and S100A12, in peripheral blood mononuclear cells (PBMCs) in a cohort of 121 prostate cancer patients using RT-PCR. Furthermore, monocyte count was determined by flow cytometry. By stratifying patients into different risk groups, according to TNM stage, Gleason score and PSA concentration at diagnosis, expression of S100A9 and S100A12 was found to be significantly higher in patients with metastases compared to patients without clinically detectable metastases. In line with this, we observed that the protein levels of S100A9 and S100A12 in plasma were higher in patients with advanced disease. Importantly, in patients with metastases at diagnosis, high monocyte count and high levels of S100A9 and S100A12 were significantly associated with short progression free survival (PFS) after androgen deprivation therapy (ADT). High monocyte count and S100A9 levels were also associated with short cancer-specific survival, with monocyte count providing independent prognostic information. These findings indicate that circulating levels of monocytes, as well as S100A9 and S100A12, could be biomarkers for metastatic prostate cancer associated with particularly poor prognosis.

Levels of mannose-binding lectin (MBL) associates with sepsis-related in-hospital mortality in women.

BACKGROUND: Mannose-binding lectin (MBL) mediates the innate immune response either through direct opsonisation of microorganisms or through activation of the complement system. There are conflicting data whether MBL deficiency leads to increased susceptibility to infections or not. The aim of this study was to determine if low levels of mannose-binding lectin (MBL) predict sepsis development, sepsis severity and outcome from severe sepsis or septic shock. METHOD: Patients aged 18 years or more with documented sepsis within 24 h after admission to the intensive care unit were included if they had participated in a health survey and donated blood samples prior to the sepsis event. A subset of these patients had stored plasma also from the acute phase. Two matched referents free of known sepsis were selected for each case. Plasma levels MBL were determined in stored samples from health surveys (baseline) and from ICU admission (acute phase). The association between MBL and sepsis, sepsis severity and in-hospital mortality were determined with 1300 ng/mL as cut-off for low levels. RESULTS: We identified 148 patients (61.5% women) with a first-time sepsis event 6.5 years (median with IQR 7.7) after participation in a health survey, of which 122 also had samples from the acute septic phase. Both high MBL levels in the acute phase (odds ratio [95% confidence interval]) (2.84 [1.20-6.26]), and an increase in MBL levels from baseline to the acute phase (3.76 [1.21-11.72]) were associated with increased risk for in-hospital death in women, but not in men (0.47 [0.11-2.06]). Baseline MBL levels did not predict future sepsis, sepsis severity or in-hospital mortality. CONCLUSIONS: An increase from baseline to the acute phase as well as high levels in the acute phase associated with an unfavourable outcome in women.

Outcome of microdialysis sampling on liver surface and parenchyma.

BACKGROUND: To investigate whether surface microdialysis (µD) sampling in probes covered by a plastic film, as compared to noncovered and to intraparenchymatous probes, would increase the technique's sensitivity for pathophysiologic events occurring in a liver ischemia-reperfusion model. Placement of µD probes in the parenchyma of an organ, as is conventionally done, may cause adverse effects, e.g., bleeding, possibly influencing outcome. METHODS: A transient ischemia-reperfusion model of the liver was used in six anesthetized normoventilated pigs. µD probes were placed in the parenchyma and on the liver surface. Surface probes were either left uncovered or were covered by plastic film. RESULTS: Lactate and glucose levels were significantly higher in plastic film covered probes than in uncovered surface probes throughout the ischemic period. Glycerol levels were significantly higher in plastic film covered probes than in uncovered surface probes at 30 and 45 min into ischemia. CONCLUSIONS: Covering the µD probe increases the sensibility of the µD-technique in monitoring an ischemic insult and reperfusion in the liver. These findings confirm that the principle of surface µD works, possibly replacing need of intraparenchymatous placement of µD probes. Surface µD seemingly allows, noninvasively from an organ's surface, via the extracellular compartment, assessment of intracellular metabolic events. The finding that covered surface µD probes allows detection of local metabolic changes earlier than do intraparenchymatous probes, merit further investigation focusing on µD probe design.

Surface microdialysis sampling: a new approach described in a liver ischaemia model.

We recently have shown that samples from microdialysis (MD) probes placed on the surface of the heart reflect metabolic events in the myocardium. This new interesting observation challenges us to consider whether surface application of MD applies to other parenchymatous organs and their surfaces. In 13 anesthetized pigs, transient liver ischaemia was achieved by occlusion of arterial and venous inflow to the liver. Two probes on liver surface and two in parenchyma were perfused with a flow rate of 1 µl per min (n = 13). An identical set-up was used for probes with a flow rate of 2 µl per min (n = 9). Samples were collected for every 15-min period during 60 min of baseline, 45 min of ischaemia and 60 min of reperfusion. Lactate, glucose, pyruvate and glycerol were analysed in MD samples. We focused on relative changes in the present study. There was a strong agreement in relative lactate and glucose levels between probes placed on liver surface and those on parenchyma. No significant differences in relative changes in lactate and glucose levels were seen between samples from surface probes and probes in liver parenchyma during equilibration, baseline, ischaemia or reperfusion with a flow rate of 1 µl per min. MD sampling applied on the liver surface is a new application area for the MD technique and may be used to monitor liver metabolism during both physiological and pathophysiological conditions.

Ischaemic preconditioning reduces myocardial calcium overload in coronary-occluded pig hearts shown by continuous in vivo assessment using microdialysis.

During ischaemia, ATP depletion leads to insufficient fuelling for Na(+) /K(+) ATPase, decreased electrochemical potential and increased influx of calcium ions. This study demonstrated a means to assess the effects of ischaemic preconditioning (IP) on the free intracellular Ca(2+) pool during prolonged ischaemia. In a porcine myocardial ischaemia model, microdialysis (MD) was used for sampling of metabolic and injury markers in IP and non-IP (control) groups. (45) Ca(2+) was delivered in microperfusate locally to ischaemic myocardium, with distribution and uptake assessed by (45) Ca(2+) recovery in microdialysate. Cardiomyocytes in vitro were exposed to a Ca(2+) ionophore and tested for (45) Ca(2+) uptake. An accentuated myocardial calcium ion influx (observed as an increased microdialysate (45) Ca(2+) recovery in the extracellular milieu) was noted in control pigs compared with IP pigs during ischaemia. Suspended cardiomyocytes preincubated with a Ca(2+) ionophore to increase the intracellular calcium ion pool and subsequently incubated with (45) Ca(2+) , displayed lower (45) Ca(2+) uptake in cells compared with control cells not exposed to the ionophore, corroborating the idea of a strong relationship between degree of intracellular calcium overload and microdialysate (45) Ca(2+) recovery. The ischaemic insult was differentially verified by metabolic and injury markers. We introduce an in vivo method for serial assessment of myocardial calcium overload during ischaemia, using a MD technique and (45) Ca(2+) inclusion. IP leads to relatively less calcium overload as assessed by this new method, and we interpret this to mean that reduction in calcium overload is an important part of the IP protective effect.

Exogenous carbon monoxide does not affect cell membrane energy availability assessed by sarcolemmal calcium fluxes during myocardial ischaemia-reperfusion in the pig.

Carbon monoxide is thought to be cytoprotective and may hold therapeutic promise for mitigating ischaemic injury. The purpose of this study was to test low-dose carbon monoxide for protective effects in a porcine model of acute myocardial ischaemia and reperfusion. In acute open-thorax experiments in anaesthetised pigs, pretreatment with low-dose carbon monoxide (5% increase in carboxyhaemoglobin) was conducted for 120 min before localised ischaemia (45 min) and reperfusion (60 min) was performed using a coronary snare. Metabolic and injury markers were collected by microdialysis sampling in the ventricular wall. Recovery of radio-marked calcium delivered locally by microperfusate was measured to assess carbon monoxide treatment effects during ischaemia/reperfusion on the intracellular calcium pool. Coronary occlusion and ischaemia/reperfusion were analysed for 16 animals (eight in each group). Changes in glucose, lactate and pyruvate from the ischaemic area were observed during ischaemia and reperfusion interventions, though there was no difference between carbon monoxide-treated and control groups during ischaemia or reperfusion. Similar results were observed for glycerol and microdialysate 45Ca(2+) recovery. These findings show that a relatively low and clinically relevant dose of carbon monoxide did not seem to provide acute protection as indicated by metabolic, energy-related and injury markers in a porcine myocardial ischaemia/reperfusion experimental model. We conclude that protective effects of carbon monoxide related to ischaemia/reperfusion either require higher doses of carbon monoxide or occur later after reperfusion than the immediate time frame studied here. More study is needed to characterise the mechanism and time frame of carbon monoxide-related cytoprotection.

Detection of myocardial ischaemia using surface microdialysis on the beating heart.

Microdialysis (MD) can be used to study metabolism of the beating heart. We investigated whether microdialysis results obtained from epicardial (surface) sampling reflect acute changes in the same way as myocardial sampling from within the substance of the ventricular wall. In anaesthetized open-thorax pigs a coronary snare was placed. One microdialysis probe was placed with the sampling membrane intramyocardially (myocardial), and a second probe was placed with the sampling membrane epicardially (surface), both in the area which was made ischaemic. Ten minutes collection intervals were used for microdialysis samples. Samples from 19 pigs were analysed for lactate, glucose, pyruvate and glycerol during equilibration, baseline, ischaemia and reperfusion periods. For both probes (surface and myocardial), a total of 475 paired simultaneous samples were analysed. Results from analyses showed no differences in relative changes for glucose, lactate and glycerol during baseline, ischaemia and reperfusion. Surface microdialysis sampling is a new application of the microdialysis technique that shows promise and should be further studied.

Effects of some modulators on purine nucleoside phosphorylase activity in myocardial tissue.

Purine nucleoside phosphorylase (PNP) in mammalian tissue is an enzyme responsible for formation of purine bases in DNA. It is also believed that PNP is crucial under energy-deprived conditions for the cell to metabolise adenosine during ATP degradation. This work describes a new method for determination of PNP activity in myocardial tissue using a commercially available substrate, 2-amino-6-mercapto-7-methylpurine riboside (MESG). The method involves the photometric assessment of the reaction between PNP (extracted from myocardial tissue) and MESG. Quantification as well as temperature- and pH-dependency for myocardial PNP activity is described. Also, the effect of some modulators has been studied. We have established the presence of PNP activity in pig myocardial tissue. Further, the results indicate a pH tolerance under slightly acid conditions and a calcium ion dependence of the enzyme.

Carbon monoxide concentration in donated blood: relation to cigarette smoking and other sources.

BACKGROUND: Carbon monoxide (CO) is normally present in the human body due to endogenous production of CO. CO can also be inhaled by exposure to external sources such as cigarette smoke, car exhaust, and fire. The purpose of this study was to investigate CO concentrations in blood from 410 blood donors at the blood center in Umeå, Sweden. To further evaluate the effects of cigarette smoking on CO concentrations, the elimination time for CO was examined in six volunteer smokers after a smoked cigarette. STUDY DESIGN AND METHODS: Blood samples from whole blood donors were obtained during the blood center's routine operation. In connection with blood donations, demographic and behavioral data were collected from the donors. The CO concentration was determined using gas chromatography. RESULTS: The majority of blood donors had approximately the same CO concentration (mean, 84.5 micromol/L). In 6 percent of the samples, the concentrations were higher than 130 micromol per L. The highest CO concentration was 561 micromol per L. The main source for these high CO concentrations appeared to be cigarette smoking. In the volunteer smokers, the elimination time after a smoked cigarette varied significantly, with elimination half-lives from 4.7 to 8.4 hours. CONCLUSION: These results show that blood bank red blood cell bags may have CO concentrations above the physiologic level. The time interval between cigarette smoking and blood donation seems to be a particularly important factor for elevated CO concentrations.

Optimised sample handling in association with use of the CMA 600 analyser.

A large degree of variability for batched analysis of serially collected microdialysis samples measured with the CMA 600 analyser has been described. This study was designed to identify sources of variability related to sample handling. Standard concentrations of four solutes were placed in microdialysis vials and then stored and analysed at intervals. Results were analysed for variability related to vial and cap type, duration and temperature of storage, centrifugation and re-analysis. The main results were that centrifugation of samples reduced variability. When a batch of 24 samples was analysed, the use of crimp caps reduced evaporation. Samples in glass vials with crimp caps could be stored in a refrigerator for up to 14 days without large variability in concentration compared to plastic vials which demonstrated variability already when stored for more than 1 day. We conclude that variability in microdialysis results can occur in relation to storage and analysis routines if routines are not optimised concerning evaporation. Centrifugation before analyses, glass vials with crimp caps even during frozen storage, and attention to minimal times for samples to be uncapped during analysis all contribute to minimise variability in the handling and analysis of microdialysis samples.

Does carbon monoxide treatment alter cytokine levels after endotoxin infusion in pigs? A randomized controlled study.

BACKGROUND: Carbon monoxide (CO) has recently been suggested to have anti-inflammatory properties, but data seem to be contradictory and species-specific. Thus, in studies on macrophages and mice, pretreatment with CO attenuated the inflammatory response after endotoxin exposure. On the other hand, human studies showed no effect of CO on the inflammatory response. Anti-inflammatory efficacy of CO has been shown at concentrations above 10% carboxyhaemoglobin. This study was undertaken to elucidate the possible anti-inflammatory effects of CO at lower CO concentrations. METHODS: Effects of CO administration on cytokine (TNF-alpha, IL-6, IL-1beta and IL-10) release were investigated in a porcine model in which a systemic inflammatory response syndrome was induced by endotoxin infusion. Endotoxin was infused in 20 anaesthetized and normoventilated pigs. Ten animals were targeted with inhaled CO to maintain 5% COHb, and 10 animals were controls. RESULTS: In the control group, mean pulmonary artery pressure increased from a baseline value of 17 mmHg (mean, n = 10) to 42 mmHg (mean, n = 10) following 1 hour of endotoxin infusion. Similar mean pulmonary artery pressure values were found in animals exposed to carbon monoxide. Plasma levels of all of the measured cytokines increased in response to the endotoxin infusion. The largest increase was observed in TNF-alpha, which peaked after 1.5 hours at 9398 pg/ml in the control group and at 13395 pg/ml in the carbon monoxide-exposed group. A similar peak was found for IL-10 while the IL-6 concentration was maximal after 2.5 hours. IL-1beta concentrations increased continuously during the experiment. There were no significant differences between carbon monoxide-exposed animals and controls in any of the measured cytokines. CONCLUSION: Our conclusion is that 5% COHb does not modify the cytokine response following endotoxin infusion in pigs.

Parallel up-regulation of FGF-2 and hyaluronan during development of cardiac hypertrophy in rat.

The importance of glycosaminoglycan hyaluronan (HA) and its receptor CD44 in cell proliferation is becoming increasingly evident. Expression of the genes coding for hyaluronan synthase 1 (HAS1), HAS2, HAS3, CD44, fibroblast growth factor-2 (FGF-2), and FGF receptor-1 (FGFR-1) and the histological evidence for increases of HA and CD44 were investigated in an experimental rat model of cardiac hypertrophy. The abdominal aorta was ligated to induce cardiac hypertrophy, and mRNAs prepared from heart tissue were analyzed after 1, 6, and 42 days. The total concentration of HA was quantified, and HA and CD44 were studied histochemically. The expression of HAS1, HAS2, CD44, and FGF-2 was considerably up-regulated at days 1 and 6 and returned to basal levels after 42 days. FGFR-1 was up-regulated at day 1 but at basal levels once more at days 6 and 42. The concentration of HA significantly increased in aorta-ligated rats. Histochemical analysis showed increased expression of CD44 in hypertrophied myocardium mainly in and around the coronary arteries. These results agree well with other studies of tissue growth (malignancies and wound healing). The increase of HA, its synthases, and receptor in parallel with FGF-2 and its receptor illustrates their complicated interplay in the development of cardiac hypertrophy. The up-regulation of both HAS1 and HAS2 indicates the importance of HA production in the hypertrophic process and the possibility that HA is needed for two different purposes for the heart to be able to adapt to the increased afterload caused by aortic ligature.

Circulatory effects and kinetics following acute administration of carbon monoxide in a porcine model.

Carbon monoxide is produced in the endothelial cells and has possible vasodilator activity through three different pathways. The aim of this study was to demonstrate circulatory effects after administration of saturated carbon monoxide blood and to describe the pharmacokinetics of carbon monoxide. Six pigs were anesthetized and 150 ml blood was removed. This blood was bubbled with carbon monoxide until the carboxyhemoglobin (COHb) levels were 90-99%. A specific amount of this blood was then injected back to the animal. At predetermined times; arterial and mixed venous blood was drawn and analyzed for carbon monoxide. Systemic and pulmonary vascular resistance index (SVRi and PVRi) were measured and exhaled air was sampled and measured for carbon monoxide. Blood samples were gathered over 300 minutes along with measurements of invasive pressures, heart rate, cardiac output, oxygen saturation (SpO2), Hb, temperature and blood gases. We conclude that this type of exposure to carbon monoxide appears to have little or no effect on general vasomotor tone and, after correcting for basal levels of carbon monoxide, elimination occurs through the lungs as predicted by a single compartment model. The half-life of carbon monoxide was determined to be 60.5 minutes (SEM 4.7).