Therapeutic plasma exchange in severe sepsis or septic shock

Increases of plasma concentrations of neutrophil myeloperoxidase (MPO) can be used as markers of polymorphonuclear leucocytes (PMN) activation in pathological situations (sepsis, acute lung injury, acute inflammation). To develop an assay for measurement of plasma MPO in horses during the above-mentioned infectious and inflammatory conditions, MPO was purified from equine PMN isolated from blood anticoagulated with citrate. PMN were extracted in a saline milieu (0.2 M Na acetate, 1 M NaCl, pH 4.7) to eliminate most of cellular proteins. Pellets were then extracted in the same buffer containing cationic detergent (1% cetyltrimethyl ammonium bromide). The supernatant was further purified by ion exchange chromatography (Hiload S Sepharose HP column 0.5 x 26 cm, equilibrated with 25 mM Na acetate, 0.2 M NaCl, pH 4.7) with a NaCl gradient (until 1 M). Most of the peroxidase activity of MPO (spectrophotometrically measured by the oxidation of orthodianisidine by hydrogen peroxide) was eluted at 0.65 M NaCl. MPO was further purified by gel filtration chromatography (Sephacryl S 200 column 2.6 x 42 cm with 25 mM Na acetate, 0.2 M NaCl, pH 4.7). MPO (specific activity: 74.3 U/mg) was obtained with a yield of 30% from the detergent extraction supernatant. Electrophoresis (non-reducing conditions) showed 3 bands identified, by comparison with human MPO, (i) the mature tetrameric enzyme (150 kDa) with 2 light and 2 heavy subunits, (ii) the precursor form (88 kDa) and (iii) a form of the heavy subunit without the prosthetic heme group (40 kDa). The mature enzyme and its precursor were glycosylated and possessed peroxidase activity. Equine MPO showed strong similarities with human and bovine MPO, with an absorption peak at 430 nm (Soret peak) characteristic of ferrimyeloperoxidase. Enzymatic activity was pH dependent (optimal value at pH 5.5).     

The effects of intravenous antioxidants in patients with septic shock

Oxidative stress is implicated in septic shock. We investigated the effect of intravenous antioxidant therapy on antioxidant status, lipid peroxidation, hemodynamics and nitrite in patients with septic shock. Thirty patients randomly received either antioxidants (n-acetylcysteine 150 mg/kg for 30 min then 20 mg/kg/h plus bolus doses of 1 g ascorbic acid and 400 mg alpha-tocopherol) or 5% dextrose. Basal vitamin C was low and redox-reactive iron was elevated in all patients. In the 16 patients receiving antioxidants, vitamin C increased (p = .0002) but total antioxidant capacity was unaffected. Lipid peroxides were elevated in all patients but did not increase further in the patients receiving antioxidants. Plasma total nitrite also increased (p = .007) in the antioxidant group. Heart rate increased in patients receiving antioxidants at 60 min (p = .018) and 120 min (p = .004). Cardiac index also increased at 60 min (p = .007) and 120 min (p = .05). Systemic vascular resistance index decreased at 120 min in the antioxidant treated patients (p = .003). The effect of antioxidants on hemodynamic variables has not previously been reported. Antioxidant administration may be a useful adjunct to conventional approaches in the management of septic shock.     

The effects of prostacyclin on gastric intramucosal pH in patients with septic shock

OBJECTIVE: To investigate whether infusing prostacyclin (PGI2) in patients with septic shock improves splanchnic oxygenation as assessed by gastric intramucosal pH (pHi). DESIGN: Interventional clinical study. SETTING: Surgical ICU in a university hospital. PATIENTS: 16 consecutive patients with septic shock according to the criteria of the ACCP/SCCM consensus conference all requiring norepinephrine to maintain arterial blood pressure. INTERVENTIONS: All patients received PGI2 (10 ng/kg x min) after no further increase in oxygen delivery could be obtained by volume expansion, red cell transfusion and dobutamine infusion. The results were compared with those before and after conventional resuscitation. The patients received continuous PGI2 infusion for 33-32 days. MEASUREMENTS AND RESULTS: O2 uptake was measured directly in the respiratory gases, pHi was determined by tonometry. Baseline O2 delivery, O2 uptake and pHi were 466 +/- 122 ml/min.m2, 158 +/- 38 ml/min.m2, and 7.29 +/- 0.09, respectively. While O2 uptake remained unchanged, infusing PGI2 increased O2 delivery (from 610 +/- 140 to 682 +/- 155 ml/min.m2, p < 0.01) and pHi (from 7.32 +/- 0.09 to 7.38 +/- 0.08, p < 0.001) beyond the values obtained by conventional resuscitation. While 9 of 11 patients with final pHi > 7.35 survived, all patients with final pHi < 7.35 died (p < 0.01). CONCLUSIONS: Infusing PGI2 in patients with septic shock increases pHi probably by enhancing blood flow to the splanchnic bed and thereby improves splanchnic oxygenation even when conventional resuscitation goals have been achieved.     

Involvement and dual effects of nitric oxide in septic shock

Bacterial endotoxin (LPS) releases many mediators such as interleukins, tumour necrosis factor, oxygen free radicals, toxic eicosanoids, platelet activating factor, and nitric oxide (NO). LPS is a potent inducer of inducible nitric oxide synthase (iNOS). Large amounts of NO (made by iNOS) and peroxynitrite, among other factors, are responsible for the late phase of hypotension, vasoplegia, cellular suffocation, apoptosis, lactic acidosis and multiorgan failure in endotoxic shock. Indeed, experimental and clinical use of NOS inhibitors, which do not differentiate clearly between constitutive endothelial NOS (ceNOS) and iNOS, prevents LPS-induced hypotension. However, many detrimental effects of such NOS inhibitors are also reported, including increases in pulmonary resistance, decreases in cardiac output and organ perfusion, and even an increase in mortality of experimental animals. We believe that, in lungs, NO made by ceNOS plays a protective role against the pneumotoxic effects of LPS-released lipids such as thromboxane, leukotrienes and PAF. This is why selective iNOS inhibitors like aminoguanidine or thiourea derivatives might be preferred over nonselective NOS inhibitors for the treatment of septic shock. However, since iNOS-derived NO seems to have more than just a destructive action, the selective iNOS inhibition may be not as beneficial as expected. Accordingly, inhalation of NO gas or NO-donors in septic shock might be a complementary treatment to the use of NOS inhibitors.     

Treatment of septic shock with immunotherapy

Sepsis is the thirteenth leading cause of death in the United States. Despite increased knowledge about its pathophysiology, availability of powerful antibiotics, and advanced diagnostic and monitoring techniques, mortality rates have not changed significantly over the past 30 years. Immunotherapy may improve outcome in the critically ill with sepsis, although trial results have been disappointing to date.     

High-frequency partial liquid ventilation in respiratory distress syndrome: hemodynamics and gas exchange

Partial liquid ventilation using conventional ventilatory schemes improves lung function in animal models of respiratory failure. We examined the feasibility of high-frequency partial liquid ventilation in the preterm lamb with respiratory distress syndrome and evaluated its effect on pulmonary and systemic hemodynamics. Seventeen lambs were studied in three groups: high-frequency gas ventilation (Gas group), high-frequency partial liquid ventilation (Liquid group), and high-frequency partial liquid ventilation with hypoxia-hypercarbia (Liquid-Hypoxia group). High-frequency partial liquid ventilation increased oxygenation compared with high-frequency gas ventilation over 5 h (arterial oxygen tension 253 +/- 21.3 vs. 17 +/- 1.8 Torr; P < 0.001). Pulmonary vascular resistance decreased 78% (P < 0.001), pulmonary blood flow increased fivefold (P < 0.001), and aortic pressure was maintained (P < 0.01) in the Liquid group, in contrast to progressive hypoxemia, hypercarbia, and shock in the Gas group. Central venous pressure did not change. The Liquid-Hypoxia group was similar to the Gas group. We conclude that high-frequency partial liquid ventilation improves gas exchange and stabilizes pulmonary and systemic hemodynamics compared with high-frequency gas ventilation. The stabilization appears to be due in large part to improvement in gas exchange.     

Early detection of melanoma metastases with radioiodinated methylene blue

Melanin synthesised in melanoma cells presents a unique target to which the treatment can be selectively addressed, provided the pigment is recognised by a suitable drug. Methylene blue (MTB) possesses a high affinity for melanin and, therefore, accumulates preferentially in melanoma cells. Since not directly toxic to the tumour, MTB serves as a carrier for radioisotopes and, once taken up by melanoma cells, acts as a selectively localised source of radiation. Hence, radioderivatives of the compound can be used for both diagnosis and therapy of disseminated melanoma. Eleven patients with confirmed metastatic melanoma and one with a recent local recurrence were studied using radioiodinated (iodine-123 or iodine-131) MTB and a gamma camera. Biopsies of cutaneous lesions were taken to determine directly the compound uptake in tumours. This first clinical investigation concerning the diagnostic potential of radioiodinated MTB in patients with disseminated melanoma confirmed the existence of approximately 80% of internal lesions previously identified by routine methods and, additionally, enabled detection of unknown secondaries in 6 of 12 patients studied. There were no false-positive gamma camera images regardless of whether 123I or 131I was used. 131I proved to be more suitable than 123I for detecting melanoma metastases with radioiodinated MTB. Hazy images of the lesions treated with external beam radiation and/or some drugs suggest that initial radio- and chemotherapy might affect MTB uptake in melanoma metastases and reduce the clarity of the scintigrams obtained from a gamma camera. However, small, untreated internal lesions that cannot be visualised easily with the standard diagnostic methods are revealed with 131I-MTB regardless of their localisation. It is concluded that use of radioiodinated MTB in conjunction with gamma camera or positron emission tomographic imaging might prove to be a useful and accessible tool for the detection of early melanoma dissemination.     

Increased circulating thrombomodulin in children with septic shock

OBJECTIVES: To test the hypothesis that children diagnosed with septic shock have increased plasma thrombomodulin values as a manifestation of microcirculatory dysfunction and endothelial injury; to determine whether plasma thrombomodulin concentrations are associated with the extent of multiple organ system failure and mortality. DESIGN: Prospective, cohort study. SETTING: Pediatric intensive care unit. PATIENTS: Twenty-two children with septic shock and ten, healthy, control children. INTERVENTIONS: Blood samples were obtained for plasma thrombomodulin determinations every 6 hrs for 72 hrs in septic shock patients and once in healthy control patients. MEASUREMENTS AND MAIN RESULTS: Thirty-two children (22 septic shock, and 10 healthy controls) were enrolled in the study. Thrombomodulin concentrations were determined by an enzyme-linked immunosorbent assay. Septic shock nonsurvivors had significantly greater mean thrombomodulin concentrations (10.6 +/- 2.2 ng/mL) than septic shock survivors (5.5 +/- 0.6 ng/mL) (p < .05) and healthy control patients (3.4 +/- 0.2 ng/mL) (p < .01). Mean thrombomodulin values increased as the number of organ system failures increased. CONCLUSIONS: Pediatric survivors and nonsurvivors of septic shock have circulating thrombomodulin concentrations 1.5 and 3 times greater than healthy control patients. These findings likely represent sepsis-induced endothelial injury. Patients with multiple organ system failure have circulating thrombomodulin concentrations which are associated with the extent of organ dysfunction. We speculate that measurement of plasma thrombomodulin concentrations in septic shock may be a useful indicator of the severity of endothelial damage and the development of multiple organ system failure.     

Potentiometric sensor for methylene blue based on methylene blue-silicotungstate ion association and its pharmaceutical applications

A methylene blue (MB) poly(vinyl chloride) membrane sensor based on MB-silicotungstate (SLT) ion association as electroactive material was described. The linear response covered the range 1 x 10(-3)-1 x 10(-6) mol x dm(-3) MB solution, with a slope 52.0+/-0.8 mV decade(-1) (pH range 3.0-10.0). The detection limit was 7.65 x 10(-7) mol x dm(-3). The electrode showed stability, good reproducibility and fast response. Interferences from common inorganic cations, some organic base were negligible. These characteristics of the electrode enabled it to be used successfully for the determination of MB in injection. There was a good agreement for the results of MB content in injection between potentiometric method and USP standard procedure.     

Photochemotherapy of experimental colonic tumours with intra-tumorally applied methylene blue

INTRODUCTION: Phototoxicity of intra-tumoral injected methylene blue (MB+) was studied in 48 experimental colonic tumours in comparison with photosan-3, Zn-phthalocyanine and tetrasulphanated ClAl-phthalocyanine. METHODS: In mice. xenotransplanted subcutaneous tumours about 1 cm in diameter were treated photodynamically twice, with different sensitisers. The irradiation was performed at the sensitiser-specific wavelength, and a density of 100 mW/cm2 and a dose of 100 J/cm2. RESULTS: Light alone without sensitiser did not induce any effect in mice tumours. Surprisingly, Al-phthalocyanine could only be used for intratumoral injections because of toxic effects after intravenous applications in nude mice. Using MB+ (1%), 75% of the tumours were destroyed by a single photodynamic treatment (PDT). In addition, toxicity of MB+ was most intense when compared with Zn-phthalocyanine and photosan-3. However, after the second PDT, there was no statistically significant difference among these sensitisers. Dark toxicity of MB+ (1%) could be well demonstrated by sufficient sensitiser incorporation without irradiation, which led to a stationary tumour volume up to 3 weeks after injection. CONCLUSION: Intra-tumoral MB+ PDT is a potential treatment for inducing necrosis in vivo. With regard to tumour tissue, the selectivity of MB+ is high and depends on a precise local injection of the dye.     

Effect of lung volume reduction surgery on gas exchange and pulmonary hemodynamics at rest and during exercise

The present study was an investigation of the regulation of anion secretion across cultured mouse endometrial epithelium by prostaglandin E2 (PGE2) using the short-circuit current (ISC) technique. The cultured endometrial monolayers responded to both apical and basolateral application of PGE2 with a sustained rise in ISC in a concentration-dependent manner. However, the potencies of apical and basolateral addition of PGE2 were different, with apparent EC50 of 200 and 4 nM, respectively. Replacement of Cl- or HCO3- in the bathing solution significantly reduced the ISC responses to both apical and basolateral addition of PGE2; however, the apical response exhibited greater dependence on HCO3- . Pretreatment with diphenylamine 2,2'-dicarboxylic acid, a Cl- channel blocker, significantly reduced both PGE2-induced ISC responses, while pretreatment with amiloride, a Na+ channel blocker, did not exert any effect. Forskolin, an adenylate cyclase activator, and 3-isobutyl-dihydro-testosterone-1-methyl-xanthine, a cAMP phosphodiesterase inhibitor, mimicked the ISC response to PGE2 while MDL12330A, an adenylate cyclase inhibitor, completely abolished the PGE2-induced ISC. The results of the present study indicate that the anion secretion across the mouse endometrial epithelium may be regulated by PGE2 involving a cAMP-dependent mechanism predominantly. The differential responses to apical and basolateral challenge with PGE2 also suggest that PGE2 of different origins may play different roles in uterine function.     

Short-term GnRH-analogue administration in patients with PCOS and its impact on hemodynamics and hormonal profile

PURPOSE: The ovarian response to 14 days of GnRH-analogue (GnRHa) application in patients with polycystic ovary syndrome (PCOS) was evaluated by measuring hemodynamic parameters and hormonal profile. METHODS: Twenty patients with PCOS represented the study group, and 22 patients with normal menstrual cycle (NMC) the reference group. The resistance index (RI) of uteroovarian arterial blood flow and serum level of LH and androstendione were measured on days 4, 13, and 22 of the cycle and after 14 days of GnRHa administration. RESULTS: On day 22 of the cycle the RI in the uterine artery was 0.86 in the PCOS and 0.82 in the NMC group. Throughout the cycle the ovarian arterial blood flow in both ovaries in the PCOS group was similar to that in inactive ovaries in the NMC group. After the 14-day GnRHa administration the RI of uteroovarian blood flow and the serum level of androstenedione remained unchanged in the PCOS group. CONCLUSIONS: RI in the uteroovarian blood flow and serum androstendione level in the PCOS group are not affected by 14-day GnRHa administration.     

N-acetyl-L-cysteine depresses cardiac performance in patients with septic shock

OBJECTIVE: To investigate the effects of adjunctive therapy with parenteral N-acetyl-L-cysteine in patients with newly diagnosed septic shock. DESIGN: Prospective, randomized, double-blind, placebo-controlled study. SETTING: Multidisciplinary intensive care unit at a university teaching hospital. PATIENTS: Twenty patients (N-acetyl-L-cysteine group [n = 10], placebo group [n = 10]), 15 male and five female, of mean age 64 +/- 15 (SD) yrs and Acute Physiology and Chronic health Evaluation (APACHE) II score 33 +/- 6, with septic shock within 24 hrs of diagnosis. INTERVENTIONS: After a 2-hr stabilization period (time-zero minus 2 hrs to time-zero), patients received either N-acetyl-L-cysteine in 5% dextrose (150 mg/kg in 100 mL over 15 mins, followed by 50 mg/kg in 250 mL over 4 hrs, and then 100 mg/kg/24 hrs in 500 mL for 44 hrs; N-acetyl-L-cysteine group) or the equivalent volume of 5% dextrose (placebo group). MEASUREMENTS AND MAIN RESULTS: Hemodynamic and oxygen transport indices were measured at time-zero minus 2 hrs and time-zero, and at multiple time points thereafter until completion of the trial infusion (time-zero plus 48 hrs). A daily Organ Failure Score was recorded for 14 days. Treatment group demographics and hemodynamic variables did not differ significantly between the two groups at time-zero. Mean (SD), pooled mean arterial pressure (MAP), and cardiac index were 75 +/- 15 mm Hg and 3.9 +/- 1.2 L/min/m2, respectively. Over the next 48 hrs, in the N-acetyl-L-cysteine group, there was a progressive decrease, relative to both time-zero and the placebo group, in MAP, cardiac index, and left ventricular stroke work index (p < .01, repeated-measures analysis of variance). Percentage reductions in these values relative to the placebo group at 48 hrs were 23%, 18%, and 43%, respectively Oxygen transport indices, arterial blood gas analyses, Pao2/Fio2 ratio, and shunt did not differ over time between the groups. There was no difference in either daily Organ Failure Score over time (p > .01, repeated-measures analysis of variance) or hospital mortality rate (90% N-acetyl-L-cysteine group, 50% placebo group) (p > .1, logistic regression) between the two groups. CONCLUSION: Adjunctive therapy with N-acetyl-L-cysteine in newly diagnosed septic shock was associated with a depression in cardiovascular performance, as indicated by progressive reductions in cardiac index, left ventricular stroke work index, and MAP.     

Short-term effects of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor on cholesterol and bile acid synthesis in humans

Competitive inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase improve hypercholesterolemia. However, reports about the effects of these agents on bile acid synthesis, the metabolic pathway of cholesterol, are conflicting. We studied the short-term effect on one of these agents, pravastatin, on bile acid synthesis. Six male volunteers were given 40 mg of pravastatin. Plasma mevalonate level (which reflects cholesterol synthesis) and 7 alpha-hydroxy-4-cholesten-3-one level (which reflects bile acid synthesis) were measured every 2 h for 8 h. These plasma levels were compared to those of the same volunteers without pravastatin. Plasma mevalonate level after 2 h was lower than control (3.0 +/- 1.1 ng/mL vs. 6.7 +/- 2.5, mean +/- SD; P < 0.05). This decrease continued for 8 h (2.5 +/- 0.8 vs. 5.2 +/- 1.5; P < 0.05). On the other hand, plasma 7 alpha-hydroxy-4-cholesten-3-one level did not change until after 6 h; then at 8 h it was lower than control (15.7 +/- 11.8 ng/mL vs. 24.7 +/- 16.9; P < 0.05). According to three-way layout analysis of variance, mevalonate level was influenced by both pravastatin treatment (P < 0.01) and time-course (P < 0.01). On the other hand, the 7 alpha-hydroxy-4-cholesten-3-one level was affected by both individual difference (P < 0.01) and time course (P < 0.01), but pravastatin treatment did not influence this compound. This indicates that bile acid synthesis was influenced by pravastatin, although cholesterol synthesis was inhibited. The short-term inhibition of cholesterol synthesis did not affect bile acid synthesis.     

Clinical study on 6 cases of urosepsis associated with septic shock

A linear lesion created at the right atrial isthmus by radiofrequency current application can successfully eliminate common atrial flutter (AF). The mechanism of unsuccessful cases has not yet been well delineated. This study sought to investigate the cause of unsuccessful cases of radiofrequency catheter ablation of AF. Sixty-six patients with refractory common AF were referred for radiofrequency catheter ablation. Radiofrequency current was applied to the right atrial isthmus between the inferior vena cava and tricuspid annulus or between the coronary sinus orifice and tricuspid annulus. In 5 (8%) of the 66 patients, a morphological change of the flutter wave was observed in the 12-lead ECG concomitant with the change of the atrial excitation sequence during the delivery of radiofrequency energy without the termination of atrial flutter. In 8 (12%) patients, the morphology of the new AF wave, which was provoked electrically after the termination of the original AF, was different, and the average flutter cycle length also differed in 3 cases (2%). The results of radiofrequency application could be misinterpreted as unsuccessful when the occurrence of another, different type of AF has been overlooked following the elimination of the original AF during the radiofrequency catheter ablation procedure. It is possible that the flutter circuit can take an alternative pathway despite the complete conduction block at the right atrial isthmus.     

Sensitive enzymatic assay for erythrocyte creatine with production of methylene blue

We developed a new, highly sensitive enzymatic method for quantifying creatine in erythrocytes, which comprises creatine amidinohydrolase, sarcosine oxidase, and peroxidase. In the present method, an N-methylcarbamoyl derivative of methylene blue, 10-N-methylcarbamoyl-3,7-bis(dimethylamino)phenothiazine (MCDP), was used as a sensitive chromogenic compound. Potassium ferrocyanide was used to prevent nonspecific oxidation of MCDP. The enzymatic method exhibited good analytical performance: precision, within-run CVs <1.0% and between-day CVs <2.0%; average analytical recovery, 99.3% +/- 1.8%; detection limit, 1.0 micromol/L in hemolysate; and linearity, at least up to 500 micromol/L as creatine concentration in hemolysate. Excellent agreement was observed between the present method (y) and HPLC (x), y = 1.029x - 0.002 micromol/g hemoglobin, r = 0.9998, S(y/x) = 0.053 micromol/g hemoglobin (n = 110). No significant interference was produced by various compounds, including guanidino compounds, amino acids, and reducing materials. The reference intervals (mean +/- 2 SD) for erythrocyte creatine obtained from 60 males and 60 females were (in micromol/g hemoglobin) 1.18 +/- 0.52 (0.66-1.70) for males and 1.35 +/- 0.49 (0.86-1.84) for females. Using this method, we documented changes in erythrocyte creatine in patients with various hemolytic conditions, including hemolytic anemia, liver cirrhosis, renal insufficiency, and chronic renal failure treated with hemodialysis with or without the administration of erythropoietin. We conclude that the use of MCDP allows sensitive measurement of erythrocyte creatine and that MCDP with potassium ferrocyanide can improve the sensitivity of assays that use peroxidase for detection of H2O2.     

Effects of prior arm exercise on pulmonary gas exchange kinetics during high-intensity leg exercise in humans

The activation of MAPKs is controlled by the balance between MAPK kinase and MAPK phosphatase activities. The latter is mediated by a subset of phosphatases with dual specificity (VH-1 family). Here, we describe a new member of this family encoded by the puckered gene of Drosophila. Mutations in this gene lead to cytoskeletal defects that result in a failure in dorsal closure related to those associated with mutations in basket, the Drosophila JNK homolog. We show that puckered mutations result in the hyperactivation of DJNK, and that overexpression of puc mimics basket mutant phenotypes. We also show that puckered expression is itself a consequence of the activity of the JNK pathway and that during dorsal closure, JNK signaling has a dual role: to activate an effector, encoded by decapentaplegic, and an element of negative feedback regulation encoded by puckered.