Papers by Alexander Romaschin
Journal of Vascular Surgery, 1991
The clinical significance and applicability of interventions aimed at reducing reperfusion injury... more The clinical significance and applicability of interventions aimed at reducing reperfusion injury in postischemic skeletal muscle remain unproven, since long-term muscle salvage has not been demonstrated by most treatment protocols that attenuate early reperfusion injury. We have shown that reperfusion of ischemic skeletal muscle results in an early and prolonged sequestration of white blood cells and activation of the alternative complement cascade. The purpose of this study was to determine if40 minutes of reperfusion with blood depleted of white blood cells and complement proteins, followed by 2 days of normal perfusion, would reduce muscle necrosis after 5 hours ofischemia. The isolated paired canine gracilis muscle model was used. The treatment muscle was initially reperfused with arterial blood that had been spun, washed, passed through a leukocyte removal filter, and resuspended in hydroxyethyl starch (>99.9% removal of white blood cells and the complement proteins factor B and C4). The contralateral control muscle was subjected to unaltered reperfusion. Blood flow (ml/min/100 gm) was measured by timed collection ofgracilis venous blood. Myeloperoxidase activity (absorbance at 655 nm/min/mg tissue protein) in muscle biopsies was used to monitor white blood cell sequestration. After 48 hours of reperfusion in vivo, necrosis was quantified by nitroblue tetrazolium staining. Initial reperfusion with white blood cell and complement depleted blood significantly reduced muscle necrosis (53% -+ 3% vs 29% -+ 8%, p < 0.0025, paired t test). Early blood flow was improved, (p = 0.0025, repeated measure-ANOVA), but subsequent white blood cell sequestration was not altered (p = 0.33, repeated measure-ANOVA). This suggests that a significant amount of white blood cell mediated injury occurs during the first 40 minutes of reperfusion. Preventing early complement activation and white blood cell mediated reperfusion injury is an intervention that is feasible during surgery and may result in clinically significant salvage of postischemic skeletal muscle. (J VAsc SURG 1991;13:58-68.)
Journal of Vascular Surgery, 1990
Prolonged ischemia to skeletal muscle as occurs after an acute arterial occlusion results in alte... more Prolonged ischemia to skeletal muscle as occurs after an acute arterial occlusion results in alterations in adenine nucleotide metabolism. Adenosine triphosphate continues to be used for cellular fimctions, and an ischemia-induced degradation of phosphorylated adenine nucleotides is initiated. In this experiement we demonstrated the time-dependent aspect of adenine nucleotide depletion during ischemia and the production of large quantifies of soluble precursors. In addition, we studied the rate of conversion of xanthine dehydrogenase to xanthine oxidase, a potential source of oxygen-free radicals, after controlled periods of total normothermic ischemia (4 hours and 5 hours) and during the reperfusion phase. During ischemia complete depletion of creatine phosphate occurred in both groups, and adenosine triphosphate fell from 22.1-+ 1.3 to 10.3-+ 1.4 ~mol/gm dry weight after 4 hours and from 21.6 -+ 0.7 to3.9 -+ 0.8 I~mol/gm dry weight after 5 hours (p < 0.05). During reperfusion, creatine phosphokinase resynthesis occurred in both groups, but adenosine triphosphate levels were not significantly increased (p > 0.05). A washout of lipid soluble products of adenine nucleotide metabolism occurred equally in both groups. The relationship between phosphorylated adenine nucleotides as measured by the energy charge potential fell significantly in both groups (p < 0.05), but after the shorter period ofischemia (4 hours it returned to normal during early reperfusion but did not after 5 hours of ischemia. There was 21% -+ 4% necrosis after 4 hours and 51% -+ 8% after 5 hours ofischemic stress when assessed at 48 hours. In conclusion, the degree of adenine nucleotide degeneration as determined primarily by the length of the ischemic period, may be the most important determinant of the ultimate extent of skeletal muscle ischemic necrosis that results from an acute interruption of circulation. (J VAse SURG 1990;12:8-15.) Skeletal muscle ischemia, as occurs after an abrupt cessation of circulation, results in an imbalance in the use and production of high energy phosphate molecules. Energy requirements are reduced in ischemic skeletal muscle since no contractions occur, but hydrolysis of adenosine triphosphate (ATP) does continue in order to meet cellular demands for membrane stabilization and ion Compartmentalization. In our previous work 1 we have shown that in skeletal From the Divisions
Profilin-1 Expression is Associated with High Grade and Stage and Decreased Disease-Free Survival in Renal Cell Carcinoma
Human Pathology, 2014
Clear cell renal cell carcinoma (ccRCC) is associated with high mortality, although individual ou... more Clear cell renal cell carcinoma (ccRCC) is associated with high mortality, although individual outcomes are highly variable. Identification of patients with increased risk of disease progression can guide customizing management plan according to disease severity. Profilin-1 (Pfn1) has been recently identified as overexpressed in metastatic ccRCC compared with primary tumors. We examined Pfn1 expression in a tissue microarray of 384 cases of histologically confirmed primary ccRCC with detailed clinical follow-up. Profilin-1 expression showed both cytoplasmic and nuclear staining patterns. The immunoexpression of Pfn1 was scored in a semiquantitative fashion. There was no significant difference in Pfn1 expression between normal kidney and kidney ccRCC. Our results show that strong cytoplasmic Pfn1 expression is associated with high-grade (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; .001) and high-stage (III-IV) (P = .018) disease. Univariate analysis of the data set showed that higher Pfn1 expression is associated with significantly shorter disease-free survival (hazard ratio 7.36, P = .047) and also lower overall survival. Kaplan-Meier analysis showed that high cytoplasmic expression of Pfn1 was also associated with a statistically significant lower disease-free survival (P = .018). It was also associated with lower overall survival, although this was not statistically significant. Profilin-1 lost its prognostic significance in the multivariate analysis when controlling for grade and stage. Profilin-1 expression was not associated with significant prognostic deference in the subgroup of patients with stage 1 disease. Our results suggest that the evaluation of Pfn1 by immunohistochemistry may help to identify patients with an increased risk of disease progression. We validated our results at the messenger RNA level on an independent patient cohort. Higher messenger RNA expression of Pfn1 is associated with significantly lower survival.
DAILY VARIATION IN ENDOTOXIN LEVELS IS ASSOCIATED WITH INCREASED ORGAN FAILURE IN CRITICALLY ILL PATIENTS
Shock, 2007
High blood levels of endotoxin on admission to the intensive care unit are predictive of adverse ... more High blood levels of endotoxin on admission to the intensive care unit are predictive of adverse outcomes, including organ failure and death. However, the significance of changes in endotoxin levels over time has not been evaluated. We examined whether dynamic daily changes in endotoxin levels resulted in the development of greater organ dysfunction over time in critically ill patients. The study was a retrospective analysis of data from the longitudinal phase of a prospective observational multicenter cohort study of endotoxin levels in patients admitted to the intensive care unit. We analyzed 345 patients. Daily variation in endotoxin levels was assessed by calculating the number of inflections in the curve generated by plotting endotoxin levels against time. The degree of organ dysfunction over time was analyzed using a calculation of the total area under the curve generated by plotting the Multi Organ Dysfunction Score against time. From 1,301 endotoxin activity assay results, patients with dynamic daily variation in endotoxin levels as measured by a greater number of inflections had a greater degree of total organ dysfunction as measured by Multi Organ Dysfunction Score against time (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). The arithmetic mean standard deviation of endotoxin activity assay results increased stepwise in the zero, one, and two inflection groups supporting the association between inflections and variability. Endotoxin activity assay variability was found to be independent of infection status (P = 0.52). Daily dynamic variation in endotoxin levels is a marker of increased severity of illness as measured by burden of total organ dysfunction over time. Further studies are warranted to assess the role of daily variation in endotoxin levels in the pathogenesis and potential therapy of organ failure in the critically ill.
Comparative in vivo study on the healing qualities of four different presealed vascular prostheses
Journal of Vascular Surgery, 1993
The purpose of this article is to assess the healing qualities of presealed knitted polyester pro... more The purpose of this article is to assess the healing qualities of presealed knitted polyester prostheses. Thoracic aortic replacement was performed with grafts with four different coating materials-collagen (CP), albumin (AP), and two with gelatin (GP1/GP2)-in four groups of 15 pigs each. Two weeks, 6 weeks, and 6 months after operation, five pigs of each group were killed. Healing quality was assessed by morphometric analysis of the remaining coating, the extent of tissue ingrowth, and the thickness of the inner layer. The sealant was rapidly absorbed in all prostheses except for the AP (remaining coating at 2 weeks: GP1 22.1%, GP2 34.7%, and CP 68.0% vs AP 97.1% [p &lt; 0.05]), remaining coating at 6 weeks: GP1/GP2 0% and CP 2.5% vs AP 76.7% (p &lt; .01). At 6 months, remaining coating was only detectable in AP (21.5%). At 2 weeks the extent of tissue ingrowth ranged from 65.7% in GP1 and 75.3% in CP to 80% in GP2 versus 8.9% in AP (p &lt; 0.05). There was a slow increase of tissue ingrowth until the sixth postoperative week (GP1 74.4%, GP2 85.0%, and CP 91.3% versus AP 19.6% [p &lt; 0.01]). Thickness of the internal layer varied from 0.11 to 0.21 mm at 2 weeks in all grafts studied and from 1.02 mm (AP) and 1.28 mm (GP2) to 1.39 mm (GP1), versus 0.41 mm in the CP (p &lt; 0.01) after 6 months of implantation. The type of coating significantly influences the healing properties of knitted polyester prostheses. When used for thoracic aortic replacement in pigs, AP coating clearly results in inferior healing compared with GP1/GP2 or CP impregnation, with digestion of the coating material and tissue ingrowth used as parameters. The thinnest internal layer was found in the CP prostheses, reflecting superior healing properties of this coating in the model studied.
Rupture of an abdominal aortic aneurysm causes priming of phagocytic oxidative burst
Journal of Vascular Surgery, 1997
The purpose of this investigation was to determine whether rupture and repair of an abdominal aor... more The purpose of this investigation was to determine whether rupture and repair of an abdominal aortic aneurysm induced activation of phagocyte oxidant burst, reflecting a systemic inflammatory state, when compared with elective abdominal aortic aneurysm (AAA) repair. Blood samples were harvested from 22 patients with elective AAA and 15 patients with ruptured AAA. Phagocyte oxidant activity was measured in response to a panel of activators with luminol and lucigenin as chemiluminescent substrates. Activity of the complement pathways was measured with plasma levels of C3a des arg. Elective AAA repair resulted in significant elevation in phagocyte count and oxidative activity after surgery in response to maximal dose phorbol myristate acetate (PMA) when compared with the baseline sample. In patients with ruptured AAA the oxidative activity of phagocytes was significantly increased in response to both unopsonized zymosan (899.8 +/- 192 ruptured vs 300 +/- 40 elective, p &lt; 0.01) and maximal dose PMA (8769 +/- 2011 vs 3508 +/- 382, p &lt; 0.01) compared with elective cases at the initial sampling. Phagocyte priming has occurred by way of two distinct pathways: receptor-mediated (unopsonized zymosan, CR3 receptor) and receptor-independent (PMA, protein kinase c). Rupture of an AAA resulted in priming of the phagocyte oxidant capacity before operative repair compared with elective AAA. Phagocyte activation is a critical component of the systemic inflammatory response that may contribute to the high incidence of systemic organ dysfunction and death in this patient group.
Journal of Vascular Surgery, 1999
Complement c5a receptor antagonist attenuates multiple organ injury in a model of ruptured abdominal aortic aneurysm
Journal of Vascular Surgery, 2004
Abdominal aortic aneurysm (AAA) rupture is associated with a systemic inflammatory response syndr... more Abdominal aortic aneurysm (AAA) rupture is associated with a systemic inflammatory response syndrome, characterized by increased microvascular permeability and neutrophil sequestration, leading to multiorgan dysfunction. We examined the role of a novel complement factor 5a (C5aR) receptor antagonist, the cyclic peptide AcF-(OpdChaWR), in attenuation of pathologic complement activation and tissue injury in a model of AAA rupture. Anesthetized rats were randomized to sham (control) or shock and clamp (s+c) groups. Animals in the s+c group underwent 1 hour of hemorrhagic shock (mean arterial blood pressure &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; or =50 mm Hg), followed by 45 minutes of supramesenteric aortic clamping, then 2 hours of resuscitated reperfusion. Animals in the s+c group were randomized to receive an intravenous bolus of C5aR antagonist at 1 mg/kg or saline solution control at the end of hemorrhagic shock. Intestinal and pulmonary permeability to iodine 125-labeled albumin was measured as an indicator of microvascular permeability. Tissue myeloperoxidase activity, proinflammatory cytokine tissue necrosis factor-alpha (TNF-alpha) protein and mRNA, and C5aR mRNA levels were measured as indicators of neutrophil sequestration and inflammatory signaling, respectively. Lung permeability index was significantly increased in the s+c group compared with the sham group (4.43 +/- 0.96 vs 1.30 +/- 0.17; P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;.01), and prevented with treatment with C5aR antagonist (1.74 +/- 0.50; P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;.03). Lung myeloperoxidase activity was significantly increased in the the s+c group compared with the sham group (2.41 +/- 0.34 U/mg vs 1.03 +/- 0.29 U/mg; P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;.009), and significantly attenuated with treatment with C5aR antagonist (1.11 +/- 0.09 U/mg; P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;.006). Lung TNF-alpha protein levels were significantly elevated in both s+c groups, whereas lung TNF-alpha mRNA expression was significantly downregulated in both s+c groups compared with the sham group. Intestinal permeability index was significantly increased in animals in the s+c groups during reperfusion, compared with sham (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt;.001), which was attenuated in early reperfusion with treatment with C5a receptor antagonist. Data represent mean +/- SEM, group comparisons with analysis of variance and post hoc Scheffé test. These results indicate that a potent antagonist of C5a receptor protects the rat intestine and lung from neutrophil-associated injury in a model of AAA rupture. These data suggest that complement-mediated inflammation can be modulated at the C5a receptor level, independent of proinflammatory TNF-alpha production, and prevent acute local and remote organ injury.
Journal of Vascular Surgery, 1987
Extensive skeletal muscle necrosis may occur after prolonged ischemia to the lower extremity, wit... more Extensive skeletal muscle necrosis may occur after prolonged ischemia to the lower extremity, with serious consequences both locally and systemically. The extent of necrosis is a combination of cellular damage that occurs during both the period of ischemia and the period of reperfusion. The purpose of this study was to reduce the extent of reperfusion-induced muscle necrosis by therapeutic interventions administered only during the initial period of reperfusion. Indeed, the pretreatment of patients who have an acute arterial occlusion is rarely possible and only interventions applicable to the reperfusion phase would be clinically relevant. By perfusing the isolated gracilis muscle in a controlled manner with reduced oxygen concentrations alone and in combination with free radical scavengers, we were able to reduce the extent of muscle necrosis. By means of controlled oxygen delivery alone, muscle necrosis was reduced from 87% + 8% in the control muscle to 67% -+ 9% (p < 0.05) in the treated muscle. The combination of reduced oxygen delivery and free radical scavengers reduced necrosis from 78% +-8% in the control muscle to 53% -+ 7% (p < 0.01) on the experimental side. We conclude that controlled oxygen delivery and free radical scavengers can reduce skeletal muscle necrosis occurring after prolonged normothermic ischemia. (J Vase SURG 1987;5
The Value of Serum Biomarkers in Prediction Models of Outcome After Mild Traumatic Brain Injury
The Journal of Trauma: Injury, Infection, and Critical Care, 2011
To determine, using a civilian model of mild traumatic brain injury (TBI), the added value of bio... more To determine, using a civilian model of mild traumatic brain injury (TBI), the added value of biomarker sampling upon prognostication of outcome at 1 week and 6 weeks postinjury. The Galveston Orientation and Amnesia test was administered, and blood samples for serum protein S100B and neuron-specific enolase (NSE) were collected from 141 emergency department patients within 4 hours of a suspected mild TBI (mTBI). The Rivermead Post-Concussion Symptoms Questionnaire (RPQ) was administered via telephone 3 days postinjury. Patients were assessed by a physician at 1 week (n = 113; 80%) and 6 weeks (n = 95; 67%) postinjury. Neurocognitive and postural stability measures were also administered at these follow-ups. Levels of S100B and NSE were found to be abnormally elevated in 49% and 65% of patients with TBI, respectively. Sixty-eight percent and 38% of the patients were considered impaired at 1 week and 6 weeks postinjury, respectively. Stepwise logistic regression modeling identified admission Galveston Orientation and Amnesia test score, S100B level, and RPQ score at day 3 postinjury to be predictive of poor outcome at 1 week postinjury (c-statistic 0.877); female gender, loss of consciousness, NSE level, and RPQ score at day 3 postinjury were predictive of poor outcome at 6 weeks postinjury (c-statistic 0.895). The discriminative power of the biomarkers alone was limited. Biomarkers, in conjunction with other readily available determinants of outcome assessed in the acute period after injury, add value in the early prognostication of patients with mTBI. Our findings are consistent with the notion that S100B and NSE point to biological mechanisms underlying poor outcome after mTBI.
Selective inducible nitric oxide synthase (iNOS) inhibition attenuates remote acute lung injury in a model of ruptured abdominal aortic aneurysm1
Journal of Surgical Research, 2004
Abdominal aortic aneurysm rupture is associated with a systemic inflammatory response syndrome an... more Abdominal aortic aneurysm rupture is associated with a systemic inflammatory response syndrome and acute lung injury. Using a selective inducible nitric oxide synthase (iNOS) inhibitor, N(6)-(iminoethyl)-lysine (L-NIL), we explored the role of iNOS in the early pro-inflammatory signaling and acute lung injury in experimental abdominal aortic aneurysm rupture. Anesthetized rats were randomized to sham control or shock and clamp (s + c) groups, which underwent one hour of hemorrhagic shock, followed by 45 minutes of supramesenteric aortic clamping, and then two hours resuscitated reperfusion. Animals in s + c were randomized to receive intravenous L-NIL at 50 microg/kg/h or saline at the start of reperfusion. Pulmonary permeability to (125)I-labeled albumin, myeloperoxidase (MPO) activity, cytokine levels, and semi-quantitative RT-PCR for mRNA were indicators of microvascular permeability, leuco-sequestration, and pro-inflammatory signaling, respectively. Lung permeability index were significantly increased in s + c compared to sham (4.43 +/- 0.96 versus 1.30 +/- 0.17, P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.01), and attenuated by L-NIL treatment (2.14 +/- 0.70, P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). Lung tissue MPO activity was significantly increased in s + c compared to sham (2.80 +/- 0.32 versus 1.03 +/- 0.29, P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.002), and attenuated by L-NIL treatment (1.50 +/- 0.20, P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.007). Lung tissue iNOS activity was significantly increased in s + c compared to sham animals (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05), and attenuated by L-NIL treatment (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). Lung tissue iNOS mRNA was upregulated 8-fold in s + c compared to sham (P &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;lt; 0.05). Data represents mean +/- standard error mean, comparisons with ANOVA. These data suggest that in our model of ruptured abdominal aortic aneurysm iNOS plays a crucial role in reperfusion lung injury. Selective inhibition of iNOS during early reperfusion prevents neutrophil mediated acute lung injury.
Quantitation of postischemic skeletal muscle necrosis: Histochemical and radioisotope techniques
Journal of Surgical Research, 1988
Skeletal muscle necrosis will result from prolonged periods of ischemia. The purpose of this stud... more Skeletal muscle necrosis will result from prolonged periods of ischemia. The purpose of this study was to develop a method to estimate the extent of necrosis using nitroblue tetrazolium staining and technetium scanning. The bilateral canine gracilis muscle preparation with total ...
Decreased postoperative myocardial fatty acid oxidation
Journal of Surgical Research, 1988
Myocardial substrate preferences following cardioplegic arrest for coronary bypass surgery have n... more Myocardial substrate preferences following cardioplegic arrest for coronary bypass surgery have not been established. Fatty acids are believed to be the major fuel source for aerobic metabolism. Following cardioplegic arrest arterial fatty acid levels are elevated and myocardial fatty acid accumulation without oxidation may contribute to reperfusion injury. Perioperative fatty acid metabolism was evaluated in 18 patients undergoing elective coronary bypass surgery who were randomized to receive either blood (n = 11) or crystalloid (n = 7) cardioplegia. Palmitate labeled with 14carbon was infused perioperatively and arterial and coronary sinus blood samples were obtained to calculate myocardial fatty acid extraction and oxidation before and after cardioplegic arrest. Lactate and glycerol were released from the heart during both blood and crystalloid cardioplegia, suggesting ischemic glycolysis and lipolysis. Myocardial oxygen consumption was depressed and the myocardial consumptions of lactate, glucose, and fatty acids were minimal during the first 60 min after aortic clamp removal in both groups despite high arterial concentrations. Fatty acid oxidation was minimal after blood cardioplegia and was not found after crystalloid cardioplegia. Fatty acids were extracted by the heart, but were not aerobically metabolized following cardioplegic arrest. Myocardial fatty acid accumulation without oxidation may have been deleterious. The inability of the heart to oxidize exogenous fatty acids may reflect altered myocardial exogenous substrate preferences during reperfusion following coronary bypass surgery.
Protein Expression Profiling of Endometrial Malignancies Reveals a New Tumor Marker: Chaperonin 10
Journal of Proteome Research, 2004
Endometrial carcinoma is a common malignancy in women, being exceeded in incidence only by that o... more Endometrial carcinoma is a common malignancy in women, being exceeded in incidence only by that of breast, lung, and colorectal cancers. At present, no serum tumor markers are available for the monitoring of endometrial carcinoma patients, and patients with recurrent disease are detected only following the development of symptoms or abnormalities in imaging assessments. Similarly, no screening tools are available for endometrial carcinoma. Protein profiling by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has proven to be a sensitive and fast method of analysis for small proteins or peptides to yield specific biomarkers. In this study, a variety of normal and malignant endometrial tissue samples were fractionated and analyzed by SELDI-TOF MS (SELDI is a version of MALDI utilizing protein &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;chips&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;). A number of proteins displayed differential expression in malignant endometrial tissues. One of the prominent proteins fractionated by weak cation exchange chromatography and displaying enhanced expression in these malignant tissues was identified as chaperonin 10. The increased expression of chaperonin 10 in malignant endometrial tissues was further confirmed by parallel Western blot and immunohistochemistry analyses.
Verification of Endometrial Tissue Biomarkers Previously Discovered Using Mass Spectrometry-Based Proteomics by Means of Immunohistochemistry in a Tissue Microarray Format
Journal of Proteome Research, 2007
Verification of candidate protein biomarkers is a necessary step in moving from the initial disco... more Verification of candidate protein biomarkers is a necessary step in moving from the initial discovery to application. Here, we report results of a verification exercise involving six candidate endometrial cancer biomarkers previously discovered using mass-tagging and multidimensional liquid chromatography/tandem mass spectrometry (DeSouza L., et al. J. Proteome Res. 2005, 4, 377-386) on a cohort of 148 patient samples by means of immunohistochemistry on a tissue microarray format. A panel of the three best-performing biomarkers, chaperonin 10, pyruvate kinase M2, and alpha-1-antitrypsin, achieved a sensitivity of 0.85, specificity of 0.93, predictive value of 0.90, and positive predictive value of 0.88 in discriminating malignant from benign endometrium. The ruggedness of this panel of biomarkers was verified in a 2/3-training-set-1/3-test-set cross-validation analysis by randomly splitting the cohort in 10 ways. The roles of chaperonin 10 and pyruvate kinase M2 in tumorigenesis confirm them as credible cancer biomarkers.
Journal of Proteome Research, 2007
Candidate biomarker proteins, including chaperonin 10 and pyruvate kinase, previously discovered ... more Candidate biomarker proteins, including chaperonin 10 and pyruvate kinase, previously discovered and identified using mass-tagging reagents with multidimensional liquid chromatography and tandem mass spectrometry (DeSouza, L.; et al. J. Proteome Res. 2005, 4, 377-386) have been identified in serum-free media of cultured endometrial cancer (KLE and HEC-1-A) and cervical cancer (HeLa) cells. These and other cancer-associated proteins were released by the cultured cells within 24 h of growth. A total of 203 proteins from the KLE cells, 86 from HEC-1-A, and 161 from HeLa are reported.
Search for Cancer Markers from Endometrial Tissues Using Differentially Labeled Tags iTRAQ and cICAT with Multidimensional Liquid Chromatography and Tandem Mass Spectrometry
Journal of Proteome Research, 2005
A total of nine potential markers for endometrial cancer (EmCa) have been discovered and identifi... more A total of nine potential markers for endometrial cancer (EmCa) have been discovered and identified from endometrial tissue homogenates using a combination of differentially labeled tags, iTRAQ and cICAT, with multidimensional liquid chromatography and tandem mass spectrometry. The tissues were snap frozen in liquid nitrogen within 15-20 min after devitalization. Samples for proteomic analysis were treated with protease inhibitors before processing. Marker proteins that were overexpressed in EmCa are chaperonin 10, pyruvate kinase M1 or M2 isozyme, calgizzarin, heterogeneous nuclear ribonucleoprotein D0, macrophage migratory inhibitory factor, and polymeric immunoglobulin receptor precursor; those that were underexpressed are alpha-1-antitrypsin precursor, creatine kinase B, and transgelin. The chaperonin 10 result confirms our earlier observation of overexpression in EmCa tissues using surface-enhanced laser desorption/ionization mass spectrometry, verified by Western analysis and immunohistochemistry [Yang, E. C. C. et al. J. Proteome Res. 2004, 3, 636-643]. Pyruvate kinase was observed to be overexpressed using both iTRAQ and cICAT labeling. All nine markers have been found to be associated with various forms of cancer. A panel of these plus other markers may confer sufficient selectivity for diagnosing and screening of EmCa. The use of cICAT led to identification of a higher proportion of lower-abundance signaling proteins; conversely, iTRAQ resulted in a higher percentage of the more abundant ribosomal proteins and transcription factors.
Journal of Proteome Research, 2008
While iTRAQ analyses have proved invaluable for the discovery of potential cancer markers, two ou... more While iTRAQ analyses have proved invaluable for the discovery of potential cancer markers, two outstanding issues that remained were its ineffectiveness to consistently detect specific proteins of interest in a complex sample and to determine the absolute abundance of those proteins. These have been addressed by availability of the mTRAQ reagents (Applied Biosystems, Inc., Foster City, CA) a nonisobaric variant of iTRAQ. We have applied this newly emerging technique to quantify one of our potential markers for endometrial cancer, viz. pyruvate kinase M1/M2. The mTRAQ methodolgy relies on multiple reaction monitoring (MRM) to target tryptic peptides from the protein of interest, thus, ensuring maximal opportunity for detection, while the nonisobaric tags enable specific quantification of each version of the labeled peptides through unique MRM transitions conferred by the labels. Known amounts of synthetic peptides tagged with one of the two available mTRAQ labels, when used as quantification standards in a mixture with the oppositely labeled tryptically digested sample, permit determination of the absolute amounts of the corresponding protein in the sample. The ability to label the sample and reference peptides with either one of the two possible combinations is an inherent advantage of this method, as it provides a means for verification of the reported ratios. In this study, we determined that the amount of pyruvate kinase present in the homogenate from a biopsied EmCa tissue sample was 85 nmol/g of total proteins, while the equivalent concentration in the nonmalignant controls was 21-26 nmol/g of total proteins. This approximately 4-fold higher amount of pyruvate kinase in the cancer sample was further confirmed not only by a direct comparison between the cancer sample and one of the nonmalignant controls, but also independently by an enzyme-linked immunosorbant assay (ELISA). Additionally, the 4-fold higher level of pyruvate kinase amount in the cancer homogenate reported in this study is considerably higher than the 2-fold higher ratio reported across 20 cancer samples in the discovery phase with the iTRAQ technique, suggesting that there exists a possibility that the dynamic range of ratios determined by the iTRAQ technique may have been compressed.
Journal of Molecular and Cellular Cardiology, 1987
Free radical reactions have been suggested to be important events in the mechanism of myocardial ... more Free radical reactions have been suggested to be important events in the mechanism of myocardial injury during ischemia and reperfusion. Most of the in vivo evidence implicating free radical mediated events in the etiology of myocardial injury has been based on intervention studies which document the efficacy of oxygen free radical scavengers in improving function or tissue salvage. We have assayed free fatty acid oxidation products (hydroxy conjugated dienes) derived from myocardial phospholipid as chemical markers of oxidative injury. Biopsies, harvested from canine myocardium subjected to cardiopulmonary bypass with no aortic crossclamp (control), from pre-ischemic, end ischemic (at the end of 45 min of global normothermic ischemia induced by aortic crossclamp) and at 5, 10, 15, 30 and 60 mins of reperfusion were analyzed for hydroxy conjugated dienes using HPLC with structural confirmation by GC-MS. All biopsies were taken from non-necrotic myocardium as indicated by gross tetrazolium staining of myocardial cross sections. Trace levels of hydroxy conjugated dienes could be detected in the preischemic biopsies or control biopsies harvested from hearts subjected to cardiopulmonary bypass with no ischemia. At the end of 45 min ischemia, however, a significant increase in 18:2 and 20:4 hydroxy isomers was detected and confirmed by GC-MS (P less than 0.05 vs. control). After 5 mins of reperfusion a further significant increase in hydroxy conjugated dienes was noted with 18:2, 20:4 and 22:6 isomers being identified (P less than 0.01 vs. end ischemia). By 30 min of reperfusion the concentration of phospholipid oxidation products had returned to pre-ischemic levels. This study presents the first chemically rigorous in vivo evidence for the formation of products of phospholipid oxidation (hydroxy conjugated dienes) during myocardial ischemia and reperfusion and supports the concept of oxygen free radical mediated lipid peroxidation. This study emphasizes the formation of phospholipid oxidation products during ischemia and particularly during the early phase of reperfusion and illustrates the transient nature of these products in myocardial phospholipid.
Diagnostic and Prognostic Implications of Endotoxemia in Critical Illness: Results of the MEDIC Study
The Journal of Infectious Diseases, 2004
A novel assay for endotoxin, based on the ability of antigen-antibody complexes to prime neutroph... more A novel assay for endotoxin, based on the ability of antigen-antibody complexes to prime neutrophils for an augmented respiratory burst response, was studied in a cohort study of 857 patients admitted to an intensive-care unit (ICU). On the day of ICU admission, 57.2% of patients had either intermediate (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;or=0.40 endotoxin activity [EA] units) or high (&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;gt;or=0.60 units) EA levels. Gram-negative infection was present in 1.4% of patients with low EA levels, 4.9% with intermediate levels, and 6.9% with high levels; EA had a sensitivity of 85.3% and a specificity of 44.0% for the diagnosis of gram-negative infection. Rates of severe sepsis were 4.9%, 9.2%, and 13.2%, and ICU mortality was 10.9%, 13.2%, and 16.8% for patients with low, intermediate, and high EA levels, respectively. Stepwise logistic regression analysis showed that elevated Acute Physiology and Chronic Health Evaluation II score, gram-negative infection, and emergency admission status were independent predictors of EA.
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Papers by Alexander Romaschin