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Background Gaucher disease is characterized by the activation of splenic and hepatic macrophages, accompanied by dramatically increased levels of angiotensin-converting enzyme (ACE). To evaluate the source of the elevated blood ACE, we performed complete ACE phenotyping using blood, spleen and liver samples from patients with Gaucher disease and controls. Methods ACE phenotyping included 1) immunohistochemical staining for ACE; 2) measuring ACE activity with two substrates (HHL and ZPHL); 3) calculating the ratio of the rates of substrate hydrolysis (ZPHL/HHL ratio); 4) assessing the conformational fingerprint of ACE by evaluating the pattern of binding of monoclonal antibodies to 16 different ACE epitopes. Results We show that in patients with Gaucher disease, the dramatically increased levels of ACE originate from activated splenic and/or hepatic macrophages (Gaucher cells), and that both its conformational fingerprint and kinetic characteristics (ZPHL/HHL ratio) differ from controls and from patients with sarcoid granulomas. Furthermore, normal spleen was found to produce high levels of endogenous ACE inhibitors and a novel, tightly-bound 10–30 kDa ACE effector which is deficient in Gaucher spleen. Conclusions The conformation of ACE is tissue-specific. In Gaucher disease, ACE produced by activated splenic macrophages differs from that in hepatic macrophages, as well as from macrophages and dendritic cells in sarcoid granulomas. The observed differences are likely due to altered ACE glycosylation or sialylation in these diseased organs. The conformational differences in ACE may serve as a specific biomarker for Gaucher disease.

Background: Angiotensin-converting enzyme (ACE) inhibitors (ACEI) are widely used in the management of cardiovascular diseases but with significant interindividual variability in the patient’s response. Objectives: To investigate whether interindividual variability in the response to ACE inhibitors is explained by the “ACE phenotype”—for example, variability in plasma ACE concentration, activity, and conformation and/or the degree of ACE inhibition in each individual. Methods: The ACE phenotype was determined in plasma of 14 patients with hypertension treated chronically for 4 weeks with 40 mg enalapril (E) or 20 mg E + 16 mg candesartan (EC) and in 20 patients with hypertension treated acutely with a single dose (20 mg) of E with or without pretreatment with hydrochlorothiazide. The ACE phenotyping included (1) plasma ACE concentration; (2) ACE activity (with 2 substrates: Hip-His-Leu and Z-Phe-His-Leu and calculation of their ratio); (3) detection of ACE inhibitors in patient’s blood (indicator of patient compliance) and the degree of ACE inhibition (ie, adherence); and (4) ACE conformation. Results: Enalapril reduced systolic and diastolic blood pressure in most patients; however, 20% of patients were considered nonresponders. Chronic treatment results in 40% increase in serum ACE concentrations, with the exception of 1 patient. There was a trend toward better response to ACEI among patients who had a higher plasma ACE concentration. Conclusion: Due to the fact that “20% of patients do not respond to ACEI by blood pressure drop,” the initial blood ACE level could not be a predictor of blood pressure reduction in an individual patient. However, ACE phenotyping provides important information about conformational and kinetic changes in ACE of individual patients, and this could be a reason for resistance to ACE inhibitors in some nonresponders.

Background Matrix-metalloproteinases (MMP) and cancer cell invasion are crucial for solid tumour metastasis. Important signalling events triggered by inflammatory cytokines, such as tumour necrosis factor α (TNFα), include Src-kinase-dependent activation of Akt and focal adhesion kinase (FAK) and phosphorylation of caveolin-1. Based on previous studies where we demonstrated amide-type local anaesthetics block TNFα-induced Src activation in malignant cells, we hypothesized that local anaesthetics might also inhibit the activation and/or phosphorylation of Akt, FAK and caveolin-1, thus attenuating MMP release and invasion of malignant cells. Methods NCI-H838 lung adenocarcinoma cells were incubated with ropivacaine or lidocaine (1 nM-100 µM) in absence/presence of TNFα (20 ng ml−1) for 20 min or 4 h, respectively. Activation/phosphorylation of Akt, FAK and caveolin-1 were evaluated by Western blot, and MMP-9 secretion was determined by enzyme-linked immunosorbent assay. Tumour cell migration (electrical wound-healing assay) and invasion were also assessed. Results Ropivacaine (1 nM–100 μM) and lidocaine (1–100 µM) significantly reduced TNFα-induced activation/phosphorylation of Akt, FAK and caveolin-1 in NCI-H838 cells. MMP-9 secretion triggered by TNFα was significantly attenuated by both lidocaine and ropivacaine (half-maximal inhibitory concentration [IC50]=3.29×10−6 M for lidocaine; IC50=1.52×10−10 M for ropivacaine). The TNFα-induced increase in invasion was completely blocked by both lidocaine (10 µM) and ropivacaine (1 µM). Conclusions At clinically relevant concentrations both ropivacaine and lidocaine blocked tumour cell invasion and MMP-9 secretion by attenuating Src-dependent inflammatory signalling events. Although determined entirely in vitro, these findings provide significant insight into the potential mechanism by which local anaesthetics might diminish metastasis.

The calcium binding protein S100B has attracted great attention as a biomarker for a variety of diseases. S100B is mainly expressed in glial cells and functions through intracellular and extracellular signaling pathways. The biological roles of S100B have been closely associated with its concentrations and its physiological states. The released S100B can bind to the receptor of advanced glycation end products and induce the initiation of multiple cell signaling transductions. The regulation of S100B bioactivities has been suggested through phosphoinositide 3 kinase/Akt, p53, mitogen-activated protein kinases, transcriptional factors including nuclear factor-kappaB, and cyclic adenosine monophosphate. The levels of S100B in the blood may function to predict the progress or the prognosis of many kinds of diseases, such as cerebrovascular diseases, neurodegenerative diseases, motor neuron diseases, traumatic brain injury, schizophrenia, depression, diabetes mellitus, myocardial infarction, cancer, and infectious diseases. Given that the activity of S100B has been implicated in the pathological process of these diseases, S100B should not be simply regarded as a biomarker, it may also function as therapeutic target for these diseases. Further elucidation of the roles of S100B may formulate innovative therapeutic strategies for multiple diseases.

Fine epitope mapping of monoclonal antibodies (mAbs) to 16 epitopes on human angiotensin I-converting enzyme (ACE) revealed that the epitopes of all mAbs contained putative glycosylation sites. ACE glycosylation is both cell- and tissue-specific and, therefore, the local conformation of ACE produced by different cells could be also unique. The pattern of ACE binding by a set of mAbs to 16 epitopes of human ACE - "conformational fingerprint of ACE" - is the most sensitive marker of ACE conformation and could be cell- and tissue-specific. The recognition of ACEs by mAbs to ACE was estimated using an immune-capture enzymatic plate precipitation assay. Precipitation patterns of soluble recombinant ACE released from Chinese hamster ovary (CHO)-ACE cells was influenced by conditions that alter ACE glycosylation. This pattern was also strongly cell type specific. Patients with sarcoidosis exhibited conformational fingerprints of tissue ACE (lungs and lymph nodes), as well as blood ACE, which were distinct from controls. Conformational fingerprinting of ACE may detect ACE originated from the cells other than endothelial cells in the blood and when combined with elevated blood ACE levels in patients with sarcoidosis may potentially reflect extrapulmonary sarcoidosis involvement (bone marrow, spleen, liver). If proven true, this would serve as a biomarker of enormous potential clinical significance.

Background: Angiotensin-converting enzyme (ACE), which metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling, as well as in reproductive functions, is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells. ACE also presents as a soluble form in biological fluids, among which seminal fluid being the richest in ACE content - 50-fold more than that in blood. Methods/Principal Findings: We performed conformational fingerprinting of lung and seminal fluid ACEs using a set of monoclonal antibodies (mAbs) to 17 epitopes of human ACE and determined the effects of potential ACE-binding partners on mAbs binding to these two different ACEs. Patterns of mAbs binding to ACEs from lung and from seminal fluid dramatically differed, which reflects difference in the local conformations of these ACEs, likely due to different patterns of ACE glycosylation in the lung endothelial cells and epithelial cells of epididymis/prostate (source of seminal fluid ACE), confirmed by mass-spectrometry of ACEs tryptic digests. Conclusions: Dramatic differences in the local conformations of seminal fluid and lung ACEs, as well as the effects of ACE-binding partners on mAbs binding to these ACEs, suggest different regulation of ACE functions and shedding from epithelial cells in epididymis and prostate and endothelial cells of lung capillaries. The differences in local conformation of ACE could be the base for the generation of mAbs distinguishing tissue-specific ACEs.

Thoracic aortic thrombi are a well-known cause of distal embolic phenomena. There is a paucity of case reports because of the rarity of this condition, and thus clear management guidelines are lacking. The authors present a case of a mobile thoracic aortic thrombus managed by a surgical approach. This report demonstrates how intraoperative transesophageal echocardiography (TEE) proved to be critical in guiding surgical management. The utility of TEE in the diagnosis and management of aortic thrombi is also discussed. In addition, currently reported management strategies for this complex condition are reviewed.

Pulmonary endothelial barrier dysfunction mediated in part by Src-kinase activation plays a crucial role in acute inflammatory disease. Proinflammatory cytokines, such as tumor necrosis factor-α (TNFα), activate Src via phosphatidylinositide 3-kinase/Akt-dependent nitric oxide generation, a process initiated by recruitment of phosphatidylinositide 3-kinase regulatory subunit p85 to TNF-receptor-1. Because amide-linked local anesthetics have well-established anti-inflammatory effects, the authors hypothesized that ropivacaine and lidocaine attenuate inflammatory Src signaling by disrupting the phosphatidylinositide 3-kinase-Akt-nitric oxide pathway, thus blocking Src-dependent neutrophil adhesion and endothelial hyperpermeability.Human lung microvascular endothelial cells, incubated with TNFα in the absence or presence of clinically relevant concentrations of ropivacaine and lidocaine, were analyzed by Western blot, probing for phosphorylated/activated Src, endothelial nitric oxide synthase, Akt, intercellular adhesion molecule-1, and caveolin-1. The effect of ropivacaine on TNFα-induced nitric oxide generation, co-immunoprecipitation of TNF-receptor-1 with p85, neutrophil adhesion, and endothelial barrier disruption were assessed.Ropivacaine and lidocaine attenuated TNFα-induced Src activation (half-maximal inhibitory concentration [IC50] = 8.611 × 10 M for ropivacaine; IC50 = 5.864 × 10 M for lidocaine) and endothelial nitric oxide synthase phosphorylation (IC50 = 7.572 × 10 M for ropivacaine; IC50 = 6.377 × 10 M for lidocaine). Akt activation (n = 7; P = 0.006) and stimulus-dependent binding of TNF-receptor-1 and p85 (n = 6; P = 0.043) were blocked by 1 nM of ropivacaine. TNFα-induced neutrophil adhesion and disruption of endothelial monolayers via Src-dependent intercellular adhesion molecule-1- and caveolin-1-phosphorylation, respectively, were also attenuated.Ropivacaine and lidocaine effectively blocked inflammatory TNFα signaling in endothelial cells by attenuating p85 recruitment to TNF-receptor-1. The resultant decrease in Akt, endothelial nitric oxide synthase, and Src phosphorylation reduced neutrophil adhesion and endothelial hyperpermeability. This novel anti-inflammatory "side-effect" of ropivacaine and lidocaine may provide therapeutic benefit in acute inflammatory disease.

Background Angiotensin I-converting enzyme (ACE) has two functional N- and C-domain active centers that display differences in the metabolism of biologically-active peptides including the hemoregulatory tetrapeptide, Ac-SDKP, hydrolysed preferentially by the N domain active center. Elevated Ac-SDKP concentrations are associated with reduced tissue fibrosis. Results We identified a patient of African descent exhibiting unusual blood ACE kinetics with reduced relative hydrolysis of two synthetic ACE substrates (ZPHL/HHL ratio) suggestive of the ACE N domain center inactivation. Inhibition of blood ACE activity by anti-catalytic mAbs and ACE inhibitors and conformational fingerprint of blood ACE suggested overall conformational changes in the ACE molecule and sequencing identified Ser333Trp substitution in the N domain of ACE. In silico analysis demonstrated S333W localized in the S1 pocket of the active site of the N domain with the bulky Trp adversely affecting binding of ACE substrates due to steric hindrance. Expression of mutant ACE (S333W) in CHO cells confirmed altered kinetic properties of mutant ACE and conformational changes in the N domain. Further, the S333W mutant displayed decreased ability (5-fold) to cleave the physiological substrate AcSDKP compared to wild-type ACE. Conclusions and Significance A novel Ser333Trp ACE mutation results in dramatic changes in ACE kinetic properties and lowered clearance of Ac-SDKP. Individuals with this mutation (likely with significantly increased levels of the hemoregulatory tetrapeptide in blood and tissues), may confer protection against fibrosis.

Retrospective analysis of patients undergoing cancer surgery suggests the use of regional anesthesia may reduce cancer recurrence and improve survival. Amide-linked local anesthetics have antiinflammatory properties, although the mechanism of action in this regard is unclear. As inflammatory processes involving Src tyrosine protein kinase and intercellular adhesion molecule-1 are important in tumor growth and metastasis, we hypothesized that amide-linked local anesthetics may inhibit inflammatory Src-signaling involved in migration of adenocarcinoma cells.NCI-H838 lung cancer cells were incubated with tumor necrosis factor-α in absence/presence of ropivacaine, lidocaine, or chloroprocaine (1 nM-100 μM). Cell migration and total cell lysate Src-activation and intercellular adhesion molecule-1 phosphorylation were assessed. The role of voltage-gated sodium-channels in the mechanism of local anesthetic effects was also evaluated.Ropivacaine treatment (100 μM) of H838 cells for 20 min decreased basal Src activity by 62% (P=0.003), and both ropivacaine and lidocaine coadministered with tumor necrosis factor-α statistically significantly decreased Src-activation and intercellular adhesion molecule-1 phosphorylation, whereas chloroprocaine had no such effect. Migration of these cells at 4 h was inhibited by 26% (P=0.005) in presence of 1 μM ropivacaine and 21% by 1 μM lidocaine (P=0.004). These effects of ropivacaine and lidocaine were independent of voltage-gated sodium-channel inhibition.This study indicates that amide-, but not ester-linked, local anesthetics may provide beneficial antimetastatic effects. The observed inhibition of NCI-H838 cell migration by lidocaine and ropivacaine was associated with the inhibition of tumor necrosis factor-α-induced Src-activation and intercellular adhesion molecule-1 phosphorylation, providing the first evidence of a molecular mechanism that appears to be independent of their known role as sodium-channel blockers.

Background: The pattern of binding of monoclonal antibodies (mAbs) to 16 epitopes on human angiotensin I-converting enzyme (ACE) comprise a conformational ACE fingerprint and is a sensitive marker of subtle protein conformational changes. Hypothesis: Toxic substances in the blood of patients with uremia due to End Stage Renal Disease (ESRD) can induce local conformational changes in the ACE protein globule and alter the efficacy of ACE inhibitors. Methodology/Principal Findings: The recognition of ACE by 16 mAbs to the epitopes on the N and C domains of ACE was estimated using an immune-capture enzymatic plate precipitation assay. The precipitation pattern of blood ACE by a set of mAbs was substantially influenced by the presence of ACE inhibitors with the most dramatic local conformational change noted in the N-domain region recognized by mAb 1G12. The ‘‘short’’ ACE inhibitor enalaprilat (tripeptide analog) and ‘‘long’’ inhibitor teprotide (nonapeptide) produced strikingly different mAb 1G12 binding with enalaprilat strongly increasing mAb 1G12 binding and teprotide decreasing binding. Reduction in S-S bonds via glutathione and dithiothreitol treatment increased 1G12 binding to blood ACE in a manner comparable to enalaprilat. Some patients with uremia due to ESRD exhibited significantly increased mAb 1G12 binding to blood ACE and increased ACE activity towards angiotensin I accompanied by reduced ACE inhibition by inhibitory mAbs and ACE inhibitors. Conclusions/Significance: The estimation of relative mAb 1G12 binding to blood ACE detects a subpopulation of ESRD patients with conformationally changed ACE, which activity is less suppressible by ACE inhibitors. This parameter may potentially serve as a biomarker for those patients who may need higher concentrations of ACE inhibitors upon antihypertensive therapy.

BACKGROUND: Angiotensin I-converting enzyme (ACE) metabolizes a range of peptidic substrates and plays a key role in blood pressure regulation and vascular remodeling. Thus, elevated ACE levels may be associated with an increased risk for different cardiovascular or respiratory diseases. Previously, a striking familial elevation in blood ACE was explained by mutations in the ACE juxtamembrane region that enhanced the cleavage-secretion process. Recently, we found a family whose affected members had a 6-fold increase in blood ACE and a Tyr465Asp (Y465D) substitution, distal to the stalk region, in the N domain of ACE. METHODOLOGY/PRINCIPAL FINDINGS: HEK and CHO cells expressing mutant (Tyr465Asp) ACE demonstrate a 3- and 8-fold increase, respectively, in the rate of ACE shedding compared to wild-type ACE. Conformational fingerprinting of mutant ACE demonstrated dramatic changes in ACE conformation in several different epitopes of ACE. Cell ELISA carried out on CHO-ACE cells also demonstrated significant changes in local ACE conformation, particularly proximal to the stalk region. However, the cleavage site of the mutant ACE - between Arg1203 and Ser1204 - was the same as that of WT ACE. The Y465D substitution is localized in the interface of the N-domain dimer (from the crystal structure) and abolishes a hydrogen bond between Tyr465 in one monomer and Asp462 in another. Conclusions/Significance The Y465D substitution results in dramatic increase in the rate of ACE shedding and is associated with significant local conformational changes in ACE. These changes could result in increased ACE dimerization and accessibility of the stalk region or the entire sACE, thus increasing the rate of cleavage by the putative ACE secretase (sheddase).

Background Angiotensin-converting enzyme (ACE; Kininase II; CD143) hydrolyzes small peptides such as angiotensin I, bradykinin, substance P, LH-RH and several others and thus plays a key role in blood pressure regulation and vascular remodeling. Complete absence of ACE in humans leads to renal tubular dysgenesis (RTD), a severe disorder of renal tubule development characterized by persistent fetal anuria and perinatal death. Methodology/Principal Findings Patient with RTD in Lisbon, Portugal, maintained by peritoneal dialysis since birth, was found to have a homozygous substitution of Arg for Glu at position 1069 in the C-terminal domain of ACE (Q1069R) resulting in absence of plasma ACE activity; both parents and a brother who are heterozygous carriers of this mutation had exactly half-normal plasma ACE activity compared to healthy individuals. We hypothesized that the Q1069R substitution impaired ACE trafficking to the cell surface and led to accumulation of catalytically inactive ACE in the cell cytoplasm. CHO cells expressing wild-type (WT) vs. Q1069R-ACE demonstrated the mutant accumulates intracellularly and also that it is significantly degraded by intracellular proteases. Q1069R-ACE retained catalytic and immunological characteristics of WT-ACE N domain whereas it had 10–20% of the nativity of the WT-ACE C domain. A combination of chemical (sodium butyrate) or pharmacological (ACE inhibitor) chaperones with proteasome inhibitors (MG 132 or bortezomib) significantly restored trafficking of Q1069R-ACE to the cell surface and increased ACE activity in the cell culture media 4-fold. Conclusions/Significance Homozygous Q1069R substitution results in an ACE trafficking and processing defect which can be rescued, at least in cell culture, by a combination of chaperones and proteasome inhibitors. Further studies are required to determine whether similar treatment of individuals with this ACE mutation would provide therapeutic benefits such as concentration of primary urine.

Background Angiotensin-converting enzyme (ACE) metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling. Elevated ACE levels may be associated with an increased risk for different cardiovascular or respiratory diseases, including asthma. Previously, a molecular mechanism underlying a 5-fold familial increase of blood ACE was discovered: Pro1199Leu substitution enhanced the cleavage-secretion process. Carriers of this mutation were Caucasians from Europe (mostly Dutch) or had European roots. Methodology/Principal Findings We have found a family of African-American descent whose affected members' blood ACE level was increased 13-fold over normal. In affected family members, codon TGG coding for Trp1197 was substituted in one allele by TGA (stop codon). As a result, half of ACE expressed in these individuals had a length of 1196 amino acids and lacked a transmembrane anchor. This ACE mutant is not trafficked to the cell membrane and is directly secreted out of cells; this mechanism apparently accounts for the high serum ACE level seen in affected individuals. A haplotype of the mutant ACE allele was determined based on 12 polymorphisms, which may help to identify other carriers of this mutation. Some but not all carriers of this mutation demonstrated airflow obstruction, and some but not all have hypertension. Conclusions/Significance We have identified a novel Trp1197Stop mutation that results in dramatic elevation of serum ACE. Since blood ACE elevation is often taken as a marker of disease activity (sarcoidosis and Gaucher diseases), it is important for clinicians and medical scientists to be aware of alternative genetic causes of elevated blood ACE that are not apparently linked to disease.

Angiotensin I-converting enzyme (ACE; CD143) has two homologous enzymatically active domains (N and C) and plays a crucial role in blood pressure regulation and vascular remodeling. A wide spectrum of monoclonal antibodies (mAbs) to different epitopes on the N and C domains of human ACE have been used to study different aspects of ACE biology. In this study, we characterized a set of nine mAbs, developed against the C domain of human ACE, which recognize the denatured forms of ACE and thus are suitable for the detection and quantification of somatic ACE (sACE) and testicular ACE (tACE) using Western blotting and immunohistochemistry on paraffin-embedded human tissues. The epitopes for these mAbs were defined using species cross-reactivity, phage display library screening, Western blotting and ACE mutagenesis. Most of the mAbs recognized common/overlapping region(s) on both somatic and testicular forms of human ACE, whereas mAb 4E10 was relatively specific for the testicular isoform and mAb 5B9 mainly recognized the glycan attached to Asn 731. This set of mAbs is useful for identifying even subtle changes in human ACE conformation because of denaturation. These mAbs are also sensitive tools for the detection of human sACE and tACE in biological fluids and tissues using proteomic approaches. Their high reactivity in paraffin-embedded tissues provides opportunities to study changes in the pattern of ACE expression and glycosylation (particularly with mAb 5B9) in different tissues and cells.

Angiotensin I-converting enzyme (ACE, CD143) has two homologous domains, each having a functional active site. Fine epitope mapping of 8 mAbs to the C-terminal domain of human ACE was carried out using plate precipitation assays, mAbs' cross-reactivity with ACE from different species, site-directed mutagenesis, and antigen- and cell-based ELISAs. Almost all epitopes contained potential glycosylation sites. Therefore, these mAbs could be used to distinguish different glycoforms of ACE expressed in different tissues or cell lines. mAbs 1B8 and 3F10 were especially sensitive to the composition of the N-glycan attached to Asn 731; mAbs 2H9 and 3F11 detected the glycosylation status of the glycan attached to Asn 685 and perhaps Asn1162; and mAb 1E10 and 4E3 recognized the glycan on Asn 666. The epitope of mAb 1E10 is located at the N-terminal end of the C domain, close to the unique 36 amino acid residues of testicular ACE (tACE). Moreover, it binds preferentially to tACE on the surface of human spermatozoa and thus may find application as an immunocontraceptive drug. mAb 4E3 was the best mAb for quantification of ACE-expressing somatic cells by flow cytometry. In contrast to the other mAbs, binding of mAb 2B11 was not markedly influenced by ACE glycosylation or by the cell culture conditions or cell types, making this mAb a suitable reference antibody. Epitope mapping of these C-domain mAbs, particularly those that compete with N-domain mAbs, enabled us to propose a model of the two-domain somatic ACE that might explain the interdomain cooperativity. Our findings demonstrated that mAbs directed to conformational epitopes on the C-terminal domain of human ACE are very useful for the detection of testicular and somatic ACE, quantification using flow cytometry and ELISA assays, and for the study of different aspects of ACE biology.

In hepatitis C virus (HCV)-infected patients receiving pegylated interferon (PEG-IFN)/ribavirin (RBV) combination therapy, anemia is a well-known side effect. The purpose of this study was to describe the time course and extent of hemoglobin (Hb) changes and the erythropoietic response to PEG-IFN/RBV-induced anemia.In this multicenter, observational, 8-wk study, laboratory parameters were measured weekly for 8 wk or until early withdrawal. Primary endpoints included changes in Hb and serum erythropoietin (sEPO) from baseline to week 8; other measures were changes in reticulocytes and RBV dose. The predictive value of baseline factors for maximum Hb decline was assessed.In the 97 evaluable patients, mean Hb decreased from 14.4 +/- 1.4 g/dl (baseline) to 11.9 +/- 1.3 g/dl (week 8). Twenty-one percent of patients withdrew before week 8. The estimated erythropoietic response was lower than that seen in two historic control populations of iron deficiency anemia patients. Mean RBV dose decreased from 986 +/- 190 mg/day (baseline) to 913 +/- 228 mg/day (week 8). Fifty-seven out of 77 (74%) patients who completed the study maintained their initial prescribed RBV dose. Patients maintained on the initial dose of RBV who had a higher baseline Hb and viral load showed a trend toward larger Hb declines. Platelets and white blood cells (WBCs) also declined during the study.HCV-infected patients receiving PEG-IFN/RBV therapy have reductions in Hb, platelets, and WBCs, possibly due to bone marrow suppression. They also have diminished endogenous sEPO production for their degree of anemia.

In humans, the sole endogenous source of carbon monoxide is heme degradation. We report the development of prolonged carboxyhemoglobinemia in a critically ill mechanically ventilated patient who required massive transfusion because of retroperitoneal hemorrhage secondary to pheochromocytoma. After the transfusion of 27 U of packed red blood cells, the maximum carboxyhemoglobin level was 6.4%. Although ventilation was controlled with a fraction of inspired oxygen of 0.35- 0.5 and external drainage of blood occurred, the concentration of carboxyhemoglobin remained at 1.7%-5.6% for days. Red blood cells for transfusion may be contaminated with carbon monoxide and may have carboxyhemoglobin levels of up to 12%; this may also have contributed to carboxyhemoglobinemia in our patient. If significantly increased concentrations of carboxyhemoglobin develop, therapy to decrease the concentration of carboxyhemoglobin (such as fraction of inspired oxygen of 1.0 and/or minute ventilation or hyperbaric oxygen) or removal of the source should be considered.

IMPLICATIONS The elimination of potassium in patients with end-stage kidney failure is limited. An increase in potassium concentrations can lead to lethal arrhythmias. In the described case, a large potassium concentration was treated during a liver transplantation using a new technical approach.

To describe the difficulties that can be encountered during mechanical ventilation of severe status asthmaticus and to discuss the safety of permissive hypercapnia as a ventilatory strategy and the role and limitations of inhalation anesthesia in the treatment of refractory cases.Case series and review of literature.Intensive care unit of a tertiary care hospital.Two patients with severe status asthmaticus.Administration of inhalational anesthetics.Both patients had respiratory failure secondary to status asthmaticus requiring mechanical ventilation and permissive hypercapnia. They also received inhalational anesthetics because of refractory bronchoconstriction. Levels of PaCO(2) in each case were among the highest and most prolonged elevations (150 mm Hg for several hours) reported to date. In one case, life-threatening difficulties with ventilation were encountered related to the use of an anesthesia ventilator. Although they had complications related to the severity of their illnesses, both were treated to recovery.Mechanical ventilation in severe status asthmaticus can be challenging. Permissive hypercapnia is a relatively safe strategy in the ventilatory management of asthma. High levels of hypercapnia and associated severe acidosis are well tolerated in the absence of contraindications (i.e., preexisting intracranial hypertension). Inhalation anesthesia may be useful in the treatment of refractory cases of asthma but should be used carefully because it may be hazardous owing to poor flow capabilities of most anesthesia ventilators.

Nonsteroidal antiinflammatory drugs(NSAIDs) are known as clinically effective agents for treatment of inflammatory diseases. Inhibition of cyclooxygenase has been thought to be a major facet of the pharmacological mechanism of NSAIDs. However, it is difficult to ascribe the antiinflammatory effects of NSAIDs solely to the inhibition of prostaglandin synthesis. Human neutrophil elastase (HNElastase; HNE, EC 3.4.21.37) has been known as a causative factor in inflammatory diseases. To investigate the specific relationship between HNElastase inhibition and specificity of molecular structure of several NSAIDs, HNElastase was purified by Ultrogel AcA54 gel filtration, CM-Sephadex ion exchange, and HPLC (with TSK 250 column) chromatography. HNElastase was inhibited by aspirin and salicylate in a competitive manner and by naproxen, ketoprofen, phenylbutazone, and oxyphenbutazone in a partial competative manner, but not by ibuprofen and tolmetin. HNElastase-phenylbutazone-complex showed strong Raman shifts at 200, 440, 1124, 1194, 1384, 1506, and 1768 cm(-1). The Raman bands 1194, 1384, and 1768 cm(-1) may represent evidences of the conformational change at -N=N-phi radical, pyrazol ring, and -C=O radical of the elastase-drug complex, respectively. Phenylbutazone might be bound to HNElastase by ionic and hydrophobic interaction, and masked the active site. Inhibition of HNElastase could be another mechanism of action of NSAIDs besides cyclooxygenase inhibition in the treatment of inflammatory diseases. Different inhibition characteristics of HNE-lastase by NSAIDs such as aspirin, phenylbutazone-like drugs and ineffective drugs could be important points for drawing the criteria for appropriate drugs in clinical application.

Cerebral venous sinus thrombosis (CVST) can present with a headache similar to that after a dural puncture. We report on a patient who developed postural headache after epidural anesthesia for delivery. The headache became more intense during the following 6 days, and the patient had a tonic clonic seizure. A magnetic resonance angiogram demonstrated CSVT, and anticoagulation therapy was started, with resolution of the symptoms over 2 wk. Any postdural-puncture headache that loses its positional character, becomes persistent, or does not improve with a properly performed blood patch should raise the suspicion of CVST.

In this review some of the major mechanistic pathways by which tumor cells are thought to invade host tissues are discussed. Tumor invasion has been conceived to be the result of pathological, close-range interactions between malignant cells and host stroma. The sequence of events that characterize invasion can be summarized as follows: (a) Tumor cell clusters break from the confinement of the primary tumor. Loss of intercellular junctions (desmosomes), alterations in the chemical composition and physical properties of the cell surface coat (loss of fibronectin and heparan sulfate; excessive amounts of hyaluronate), and loosening of cell-substrate interactions (loss of hemidesmosomes, fibronectin, and heparan sulfate), are among the most frequently listed causes of tumor cell shedding. (b) Increased proteolytic activities at the invasion front cause focal alterations in the surrounding extracellular matrix, thereby changing its physical properties. Collagenases and cathepsins, as well as elastase and other neutral proteinases are the enzymes most frequently associated with matrix destruction and invasion. In some tissues this process is effectively regulated by inhibitors of matrix-degrading, proteolytic enzymes. (c) Tumor cells migrate into the altered matrix, possibly moving as aggregates along guidance tracks provided by host structures (blood vessels, lymphatics, nerves) or matrix macromolecules (collagen and fibronectin tracks). Migration seems to be preceded by increased swelling of glycosaminoglycan (i.e., hyaluronate) in the matrix, ahead of the migrating cell population. Various host cell types (mast cells, fibroblasts, endothelial cells, macrophages, etc.) may participate in these events.

The cause of exercise intolerance in congestive heart failure is unclear. Hemodynamic and ventilatory responses were measured during symptomatic maximal upright bicycle exercise in 28 patients with chronic severe left ventricular failure who achieved a maximal oxygen uptake of only 12 +/- 4 ml/min/kg (+/- standard deviation). All patients reached anaerobic metabolism as the respiratory exchange ratio rose and arterial pH fell significantly. Pulmonary capillary wedge pressure increased from 20 +/- 10 mm Hg at rest to 38 +/- 9 mm Hg at peak exercise and cardiac index increased from 2.51 +/- 0.73 to 4.54 +/- 1.65 liters/min/m2 (both p less than 0.001). Systemic vascular resistance decreased, but pulmonary vascular resistance did not change during exercise. Despite the marked pulmonary venous hypertension at peak exercise, blood gases were unchanged (PaO2, 96 +/- 15 mm Hg; PaCO2, 35 +/- 7 mm Hg). Systemic arterial oxygen content increased from 16 +/- 2 to 17 +/- 2 vol% (p less than 0.01). Changes in pulmonary capillary wedge pressure did not correlate with changes in arterial oxygen content. Results were similar whether patients were limited by dyspnea or fatigue. Thus, exercise intolerance in patients with severe left ventricular failure is associated with marked elevation of pulmonary capillary wedge pressure and anaerobic metabolism without hypoxemia or altered carbon dioxide tension. These findings suggest that exercise ability in congestive heart failure is more dependent on cardiac output than on ventilatory consequences of pulmonary congestion.

The pathophysiologic role of high levels of circulating catecholamines in patients with congestive heart failure remains unclear. To assess the hemodynamic contribution of circulating catecholamines, metyrosine (alpha-methyl-p-tyrosine), an inhibitor of catecholamine synthesis, was administered to nine patients with acutely decompensated chronic congestive heart failure. Baseline left ventricular ejection fraction averaged 23.3 +/- 9.9%, whereas cardiac output averaged 3.69 +/- 1.03 liters/min, with a pulmonary wedge pressure of 27.4 +/- 8.5 mm Hg. After 48 h of metyrosine administration, plasma norepinephrine concentration decreased from 919.4 +/- 810.6 to 335.4 +/- 143.1 pg/ml (p less than 0.05). Plasma epinephrine concentration averaged 176.4 +/- 166.0 pg/ml at baseline, and was unchanged during metyrosine administration. Despite the significant decrease in circulating norepinephrine, no significant hemodynamic changes were observed during metyrosine administration. These results suggest that high levels of circulating norepinephrine may be more a marker of severe congestive heart failure than an important contributor to the underlying pathophysiology at this advanced stage of the disease process.

Fibroblasts from normal adult forearm skin and neonatal foreskin were cultured and examined for their ability to synthesize and secrete elastase and neutral cathepsin. All of the cultures examined produced detectable amounts of elastase using insoluble elastin as substrate. An enzyme was also found that hydrolyzed the synthetic elastin substrate, N-succinyl-(Ala)3-p-nitroanilide, but did not degrade insoluble elastin. In addition, activity against the synthetic cathepsin substrate N-benzoyl-DL-phenylalanine-naphthyl ester was found. Inhibitor profiles indicate that the elastin and N-succinyl-(Ala)3-p-nitroanilide degrading activities are due to metalloproteinases. Degradation of N-benzoyl-DL-phenylalanine-naphthyl ester can be inhibited by phenylmethylsulfonyl fluoride. These proteinases were usually found associated with the cell layer. Although activities of the measured proteinases were detected in all cultures, increased or decreased enzyme activities were not predictably related to passage number or length of serum starvation. Degree of confluence also affected proteinase activities. Separation of the dermal-epidermal junction can be produced by the injection of these proteinases into intact mouse skin.

High-density (HD) lipoprotein cholesterol levels have been inversely associated with the incidence of coronary heart disease. The clinical features were reviewed and the prevalence of clin...

We consecutively and prospectively studied 219 critically ill patients to evaluate the accuracy of the physical examination in assessing ETT position and the appropriateness of taking routine chest x-ray films after intubation in the ICU. As a result of x-ray findings, 14 percent of the patients required ETT repositioning, and 5 percent had main-stem intubations. Endobronchial intubation was more common in females than in males, and frequently occurred after emergency intubations. Sixty percent of the main-stem intubations occurred despite the presence of equal breath sounds on examination. Techniques to minimize the risk of tube malposition, such as cuff ballottement in the suprasternal notch and referencing the ETT centimeter markings, were not completely reliable. This study confirms the unreliability of the physical examination to assess ETT position. Chest x-ray films after intubation are indicated to verify tube position, particularly after emergency intubations. Other techniques such as use of a lighted stylet require evaluation to determine whether they are more cost-effective in verifying ETT placement in patients who have no other indication for postintubation x-ray films.

We conducted a randomized prospective trial in 19 disease-free soft tissue sarcoma patients with doxorubicin-induced cardiomyopathy identified by ECG radionuclide angiography at rest and during exercise to determine the efficacy of the free radical scavenger, N-Acetyl Cysteine (NAC), in reversing the drug's cardiotoxic effect. Of the 19 patients, 11 received oral NAC (5.5 gm/m2 daily for 30 days) and eight patients served as controls. Patients were stratified for age less than greater than 45 years, time from final dose of doxorubicin to randomization less than greater than 8 months, and history of treatment with mediastinal irradiation. The two groups were well-matched for all parameters. Cumulative mean doxorubicin dose (523 mg/m2 and 532 mg/m2) and range 500-600 mg/m2 was comparable. Left ventricular (LV) ejection fraction before randomization was not significantly different between the two groups either at rest (39 +/- 10% control, 38 +/- 13% NAC) or during exercise (38 +/- 12% control, 35 +/- 11% NAC). Neither rest nor exercise ejection fraction values changed significantly in either group between prerandomization and 1-month postrandomization studies. Late studies performed in seven NAC patients 3-5 months after randomization revealed no difference in LV ejection fraction compared to 1-month postrandomization values. Clinical course in patients with overt congestive heart failure was similar in both groups. LV function did not return to normal in any patient in either group. We conclude that N-Acetyl Cysteine has no effect in reversing long standing doxorubicin-induced cardiomyopathy.

The amount of cross-sectional area (XSA) narrowing in each 5 mm long segment of each of the four major epicardial coronary arteries was determined in each of five patients with type III hyperlipoproteinemia (HLP) and symptomatic, fatal atherosclerotic coronary disease (CAD). Four had angina pectoris; two had acute myocardial infarcts which healed, and two died suddenly. Of the four major epicardial coronary arteries, all four were narrowed 76% to 100% in XSA by atherosclerotic plaques in two patients, three were narrowed to this degree in two patients, and two were so narrowed in one patient. Three patients had severe narrowing of the left main coronary artery. The percent of 5 mm long segments of coronary artery narrowed to various degrees was as follows: 96% to 100%, 0 to 37 (mean 14); 76% to 95%, 14 to 61 (mean 35); 51% to 75%, 9 to 41 (mean 24); 26% to 50%, 0 to 42 (mean 16), and 0% to 25%, 0 to 27 (mean 11). Utilizing a scoring system of 1 to 4 for the four categories of narrowing (1 = 0% to 25%, 2 = 26% to 50%, 3 = 51% to 75% and 4 = 76% to 100% XSA narrowing), scores per 5 mm segment for each patient ranged from 2.5 to 3.9 (mean 3.1). Thus these five type III HLP patients had severe diffuse coronary narrowing by atherosclerotic plaques.

Radionuclide cineangiography was used to evaluate 32 patients who sustained long-term remission of soft tissue sarcoma after adjuvant therapy with a cumulative doxorubicin dose from 480 to 550 mg/m body surface area. Left ventricular ejection fraction at rest was below normal (less than 45%) in eight of 32 patients. The abnormal response of ejection fraction to exercise identified an additional 12 patients with diminished left ventricular functional reserve. Ejection fraction determined at rest or during exercise did not differ 1 to 9 months) and those studied 30 months (range, 21 to 43 months) after completing doxorubicin treatment. Sequential studies in 13 patients, done 6 to 15 months after initial post-doxorubicin evaluation also showed persistent depression of average ejection fraction at rest and with exercise, with the continued deterioration of left ventricular function in six patients. Left ventricular dysfunction, evident in over half of asymptomatic patients even long after "acceptable" cumulative doses of doxorubicin, may persist for years.

Acute lung injury (ALI) is associated with high mortality due to the lack of effective therapeutic strategies. Mechanical ventilation itself can cause ventilator-induced lung injury. Pulmonary vascular barrier function, regulated in part by Src kinase-dependent phosphorylation of caveolin-1 and intercellular adhesion molecule-1 (ICAM-1), plays a crucial role in the development of protein-/neutrophil-rich pulmonary edema, the hallmark of ALI. Amide-linked local anesthetics, such as ropivacaine, have anti-inflammatory properties in experimental ALI. We hypothesized ropivacaine may attenuate inflammation in a “double-hit” model of ALI triggered by bacterial endotoxin plus hyperinflation via inhibition of Src-dependent signaling. C57BL/6 (WT) and ICAM-1 −/− mice were exposed to either nebulized normal saline (NS) or lipopolysaccharide (LPS, 10 mg) for 1 hour. An intravenous bolus of 0.33 mg/kg ropivacaine or vehicle was followed by mechanical ventilation with normal (7 ml/kg, NTV) or high tidal volume (28 ml/kg, HTV) for 2 hours. Measures of ALI (excess lung water (ELW), extravascular plasma equivalents, permeability index, myeloperoxidase activity) were assessed and lungs were homogenized for Western blot analysis of phosphorylated and total Src, ICAM-1 and caveolin-1. Additional experiments evaluated effects of ropivacaine on LPS-induced phosphorylation/expression of Src, ICAM-1 and caveolin-1 in human lung microvascular endothelial cells (HLMVEC). WT mice treated with LPS alone showed a 49% increase in ELW compared to control animals (p = 0.001), which was attenuated by ropivacaine (p = 0.001). HTV ventilation alone increased measures of ALI even more than LPS, an effect which was not altered by ropivacaine. LPS plus hyperinflation (“double-hit”) increased all ALI parameters (ELW, EVPE, permeability index, MPO activity) by 3–4 fold compared to control, which were again decreased by ropivacaine. Western blot analyses of lung homogenates as well as HLMVEC treated in culture with LPS alone showed a reduction in Src activation/expression, as well as ICAM-1 expression and caveolin-1 phosphorylation. In ICAM-1 −/− mice, neither addition of LPS to HTV ventilation alone nor ropivacaine had an effect on the development of ALI. Ropivacaine may be a promising therapeutic agent for treating the cause of pulmonary edema by blocking inflammatory Src signaling, ICAM-1 expression, leukocyte infiltration, and vascular hyperpermeability.