Monitoring circulating tumor cells in cancer vaccine trials.

The presence of circulating tumor cells (CTC) from various cancers has provided a wealth of information and possibilities. As the role of CTC detection in the treatment assessment of metastatic breast cancer becomes standard, there is interest in applying this tool in cancer vaccine development and clinical trial monitoring. Since we lack a proven immunologic assay that correlates with clinical response, CTC detection, quantification and phenotypic characterization may be a useful surrogate for clinical outcome. The Cancer Vaccine Development Program is involved in the development of HER2/neu peptide based vaccine development for the prevention of recurrence in HER2/neu expressing cancers like breast cancer. The CellSearch System (Veridex, LLC Warren, NJ) has been used by our lab in conjunction with in vivo and/or in vitro immunologic measurements to define a monitoring tool that could predict clinical response. Once validated, this assay could significantly shorten clinical trials and lead to more efficient assessment of potentially promising cancer vaccines.

Involvement of endogenous opiates in glucose-stimulated hyperinsulinism of canine endotoxin shock. Inhibition by naloxone.

Hyperinsulinism has been associated with infection and endotoxin shock in rodents, dogs, and humans. In dogs with Escherichia coli-induced endotoxin shock, this hyperinsulinism was in response to glucose administration. To determine the role of endogenous opiates in endotoxin-induced glucose-stimulated hyperinsulinism, plasma beta-endorphin, Met-enkephalin, Leu-enkephalin, insulin, and glucose concentrations were measured for 6 h in fasted, anesthetized dogs given LD70 of E. coli endotoxin; endotoxin and glucose; endotoxin, glucose, and naloxone (an opiate antagonist); glucose and naloxone; or glucose alone. Plasma endogenous opiate immunoreactivity was elevated in dogs that received endotoxin, regardless of the presence of glucose or naloxone. The elevation of plasma Met-enkephalin and beta-endorphin preceded the onset of hyperinsulinism, but the elevation of plasma Leu-enkephalin did not. Plasma insulin was elevated 100-fold by 360 min in dogs given endotoxin and glucose. The magnitude of this hyperinsulinism was markedly reduced by naloxone, supporting the hypothesis that endogenous opiates are involved in the development of the glucose-stimulated hyperinsulinism associated with endotoxin shock. Interestingly, naloxone, given in conjunction with glucose, appeared to have a stimulatory effect on insulin secretion.

Soluble leukocyte selectin in the analysis of pleural effusions.

STUDY OBJECTIVES: To determine if soluble leukocyte selectin (sL-selectin) levels in serum and pleural fluid (PF) are an inflammatory marker that differentiates pleural effusion transudates from exudates. DESIGN: sL-selectin PF and serum levels were measured in consecutive patients and compared to established criteria. SETTING: A tertiary-care military medical center. PATIENTS: One hundred twenty patients undergoing diagnostic or therapeutic thoracentesis. INTERVENTIONS: PF and serum samples were collected during thoracentesis and analyzed separately for sL-selectin levels. Results were compared with clinical diagnosis and established PF criteria including the criteria of Light et al, cholesterol ratio, total bilirubin ratio, and albumin gradient. MEASUREMENTS AND RESULTS: sL-selectin levels in PF and serum were determined in 109 patients. By clinical diagnosis, mean +/- SD PF sL-selectin levels were 200.2 +/- 124.3 ng/mL in transudates and 496.8 +/- 379.2 ng/mL in exudates (p < 0.001). By the criteria of Light et al, mean PF sL-selectin levels were 195.7 +/- 105.2 ng/mL in transudates and 448.2 +/- 367.6 ng/mL in exudates (p < 0.001). Mean sL-selectin PF to serum ratios were 0.31 +/- 0.17 in transudates and 0.72 +/- 0.31 in exudates (p < 0.001) by clinical criteria, and 0.31 +/- 0.18 in transudates and 0.64 +/- 0.33 in exudates (p < 0.001) by the criteria of Light et al. No significant difference was noted with serum sL-selectin levels between groups. CONCLUSIONS: sL-selectin is an inflammatory marker that differentiates transudates from exudates in pleural effusions and is a sensitive indicator for PF analysis.

Development of sensitive colorimetric capture elisas for Clostridium botulinum neurotoxin serotypes A and B.

Sensitive and specific enzyme-linked immunosorbent assays were developed to detect Clostridium botulinum neurotoxin serotypes A (BoNT A) and B (BoNT B) in assay buffer and human serum. The assay is based upon affinity-purified horse polyclonal antibodies directed against the approximately 50 kDa C-fragments of each toxin. Standard curves were linear over the range of 0.1-10 ng mL. Detection was possible at 0.2 ng mL (20 pg/well) and accurate quantitation at 0.5 ng/mL (50 pg well) in assay buffer and 10% human serum. Variations between triplicates was typically 5-10%. Less than 1% cross reactivity occurred between other serotypes when each assay was performed against serotypes A, B and E. When tested against toxins complexed to their associated nontoxic proteins, interference was absent (BoNT B) or < 25% (BoNT A). These assays demonstrate sensitivity close to that of the mouse bioassay without the use of animals and in a much simpler format than other reported assays of similar sensitivity.

Detection of ricin by colorimetric and chemiluminescence ELISA.

A highly sensitive and specific ELISA was developed to detect ricin in biological fluids. The assay utilizes an affinity-purified goat polyclonal antibody to adsorb ricin from solution. The same antibody (biotinylated) is then used to form a sandwich, and avidin-linked alkaline phosphatase allows color development and measurement of optical density at 405 nm. Our routine assay uses a standard curve over the range of 0-10 ng/ml ricin, with accurate quantitation below 1 ng/ml (100 pg/well) in assay buffer as well as in a 1:10 dilution of human urine or 1:50 dilution of human serum spiked with ricin. Ricin measured in spiked samples demonstrated accuracy typically within 5% of the expected value in all matrices. The coefficient of variation ranged from 3-10% at 10 ng/ml to 8-25% at 2.5 ng/ml. Two variations on the routine assay were also investigated. First, lengthened incubation times and additional time for color development allowed accurate quantitation in serum dilutions as low as 1:2. Second, increased concentrations of biotinylated antibody and avidin-linked enzyme from 1:250 to 1:70 enhanced the sensitivity of the assay 10-fold, achieving a detection limit of at least 100 pg/ml (10 pg/well). The assay was also configured to a format based upon chemiluminescence, which allowed quantitation in the 0.1-1 ng/ml range, but was subject to slightly greater variability than the colorimetric assay.

Effects of inhibitors on chicken polymorphonuclear leukocyte oxygenation activity measured by use of selective chemiluminigenic substrates.

Chicken heterophil polymorphonuclear leukocytes (CPMNLs) have NADPH oxidase activity, but lack myeloperoxidase (MPO). Stimulation of CPMNLs by phorbol 12-myristate 13-acetate or chicken opsonified zymosan results in luminol-dependent chemiluminescence (CL) activity, which is small relative to that of human peroxidase-positive neutrophils (HPMNLs), as well as lucigenin-dependent CL, comparable to HPMNL responses. Inhibitors were used to investigate and characterize the CL activity of CPMNLs. Inhibition constants were calculated, using Dixon inhibition analysis, or were reported as the concentration producing 50% inhibition of the magnitude of CL responses. Azide and cyanide are effective inhibitors of luminol CL in HPMNLs, although these peroxidase inhibitors do not inhibit either luminol or lucigenin CL of CPMNLs. Since these agents also inhibit eosinophil peroxidase, lack of inhibition of CPMNL CL indicates that the small percentages of peroxidase-positive eosinophils in CPMNL preparations are not responsible for the luminol CL observed. Iodoacetate and fluoride, pre-oxidase and pre-peroxidase inhibitors of glycolytic metabolism, effectively inhibit lucigenin and luminol CL activities in CPMNLs. Superoxide dismutase competitively inhibits lucigenin and luminol CL in CPMNLs, but catalase is an ineffective inhibitor. Although luminol is efficiently dioxygenated by a MPO-dependent mechanism in HPMNL, use of peroxidase-deficient CPMNLs indicates that this substrate does not exclusively measure peroxidase activity.

Limited tryptic digestion near the amino terminus of bovine liver rhodanese produces active electrophoretic variants with altered refolding.

When the enzyme rhodanese was partially digested by immobilized trypsin, it retained greater than 50% of its original activity although less than 10% of the undigested enzyme remained. The predominant daughter species were two 31-kDa polypeptides whose amino termini corresponded to either residue 44 or 45 of the enzyme's sequence. Following digestion, charged species were isolated by ion exchange chromatography. Denaturing electrophoresis revealed that a 4-kDa peptide remained associated with the 31-kDa fragment. This 4-kDa peptide appears to correspond to the amino-terminal 45 residues of rhodanese. Further proteolysis gave a 2.5-kDa peptide that dissociated under non-denaturing conditions without apparent change in migration of the 31-kDa fragment on SDS gels. Refolding of undigested, urea-denatured rhodanese restored much of its activity. Similar treatment of rhodanese following limited tryptic digestion resulted in no regain of activity. Refolding of a mixture of intact and digested rhodanese resulted in regain of activity appropriate for the amount of intact rhodanese in the sample, indicating that clipped rhodanese does not inhibit refolding of intact rhodanese. It is concluded that portions of the amino terminus of rhodanese are important in the enzyme's folding, but are not essential for the enzyme's sulfurtransferase activity.

Elimination of matrix-based interferences to a fluorescent nitrite/nitrate assay by a simple filtration procedure.

Pathophysiological levels of oxygen radical metabolites have been studied as indicators of trauma caused by burn insult. The 2, 3-diaminonaphthalene assay is routinely used in the determination of nitrite/nitrate levels in biological fluids and cellular extracts as one indicator of nitric oxide activity. Several laboratories, including ours, have noted matrix-based interferences resulting in decreased assay sensitivity during nitrite/nitrate analysis. We evaluated filtration using Millipore Ultrafree-MC 10,000 NMWL filters for the ability to eliminate matrix-based interferences from human serum and tissue culture medium, thereby restoring assay sensitivity.

Immunological evidence for a conformational difference between recombinant bovine rhodanese and rhodanese purified from bovine liver.

Rhodanese has been utilized as a model enzyme for the study of protein structure-function relationships. The enzyme has recently been cloned and the recombinant enzyme is now available for investigation. However, prior to use in structure-function studies, the recombinant enzyme must be shown to have the same structure and activity as the bovine liver enzyme used in the previous studies. An immunological study of the conformations of these enzyme conformers is described. Three antibodies (two monoclonal and one polyclonal, site-directed antibody) were shown to detect distinct and nonoverlapping epitopes. The epitopes of the monoclonal antirhodanese antibodies (R207 and MAB11) were mapped to the same CNBr digest fragment of the amino terminal domain of rhodanese, and the epitope of the site-directed antibody prepared against the interdomain tether sequence of rhodanese (PAT-T1) was mapped to that region of rhodanese (residues 142-156). The rhodanese conformers were studied by monitoring the accessibility of the epitopes recognized by each antibody in each conformer using an indirect ELISA. None of the antibodies could detect its epitope on the purified liver enzyme. Two of the antibodies (R207 and PAT-T1) could also not detect their epitopes on the recombinant enzyme. However, MAB11 did detect a conformational difference between the natural and recombinant rhodanese conformers, indicating the conformational difference is localized in the first 73 amino acids of rhodanese. This difference presumably reflects the difference in the histories of the two enzymes and may be due to differences in enzyme folding, differences in the purification procedures, and differences in storage conditions--all of which could influence the final conformation of the enzyme.

Detection of time-dependent and oxidatively induced antigens of bovine liver rhodanese with monoclonal antibodies.

Rhodanese has been extensively utilized as a model protein for the study of enzyme structure-function relationships. An immunological study of conformational changes occurring in rhodanese as a result of oxidation or thermal inactivation was conducted. Three monoclonal antibodies (MABs) to rhodanese were produced. Each MAB recognized a unique epitope as demonstrated by binding of the MABs to different proteolytic fragments of rhodanese. While none of the MABs significantly bound native, soluble, sulfur-substituted bovine rhodanese, as indicated in indirect enzyme-linked immunosorbent assay experiments, each MAB was immunoadsorbed from solution by soluble rhodanese as a function of the time rhodanese was incubated at 37 degrees C. Thus, as rhodanese was thermally inactivated, conformational changes resulted in the expression of three new epitopes. Catalytic conformers demonstrated different rates of thermally induced antigen expression. Each MAB also recognized epitopes expressed when rhodanese was immobilized on microtiter plates at 37 degrees C. Two conformers resulting from oxidation of rhodanese by hydrogen peroxide were identified immunologically. All MABs recognized rhodanese that was oxidized with peroxide and allowed to undergo a secondary cyanide-dependent reaction which also resulted in a fluorescence shift and increased proteolytic susceptibility. Only one MAB was capable of recognizing an epitope expressed when rhodanese was oxidized with peroxide and then separated from the reactants to prevent induction of the secondary conformational changes.

Oxygenation activities of chicken polymorphonuclear leukocytes investigated by selective chemiluminigenic probes.

The redox metabolism of myeloperoxidase-deficient rooster (chicken) polymorphonuclear leukocytes (PMNL) was analyzed by differential chemiluminigenic probes. Chicken complement-opsonified zymosan, a phagocytosable particulate stimulus, and phorbol myristate acetate, a chemical stimulus, were used to activate the PMNL respiratory burst. The two probes used were luminol (5-amino-2,3-dihydro-1,4-phthalazinedione), a general probe of oxidase-peroxidase activities, and lucigenin (dimethylbiacridinium binitrate), a selective probe of oxidase activity. Rooster PMNLs yielded dimethylbiacridinum binitrate-dependent chemiluminescence (CL) comparable to those of myeloperoxidase-containing human PMNLs after stimulation with opsonified zymosan and to a lesser extent with phorbol myristate acetate. However, the luminol-dependent CL of opsonified zymosan or phorbol myristate acetate-stimulated rooster PMNLs were approximately two orders of magnitude lower than responses observed with human PMNLs. At physiologic pH, luminol is a highly sensitive, but not specific, probe of myeloperoxidase activity. Rooster erythrocytes yielded no CL with any of the probe-stimulus combinations described. Rooster PMNL viability and oxygen were required for CL. No strong correlation could be drawn between CL responses and eosinophil leukocyte concentration. The major conclusion is that rooster PMNLs, which do not have myeloperoxidase, present a significant and reproducible oxidative burst to chemical and particulate stimuli. Although lacking in peroxidase, rooster PMNLs can still present small luminol-dependent responses.

Mucosal protection against sulphide: importance of the enzyme rhodanese.

BACKGROUND: Hydrogen sulphide (H(2)S) is a potent toxin normally present in the colonic lumen which may play a role in ulcerative colitis (UC). Two enzymes, thiol methyltransferase (TMT) and rhodanese (RHOD), are thought to be responsible for sulphide removal but supportive evidence is lacking. AIMS: To determine the distribution of TMT and RHOD in different sites throughout the gastrointestinal tract and their efficacy as detoxifiers of H(2)S. METHODS: Enzyme activities were measured in normal tissue resected from patients with cancer. TMT and RHOD activities were determined using their conventional substrates, 2-mercaptoethanol and sodium thiosulphate, respectively. For measurement of H(2)S metabolism, sodium sulphide was used in the absence of dithiothreitol. Thiopurine methyltransferase (TPMT), which in common with TMT methylates sulphydryl groups but is not thought to act on H(2)S, was also examined. RESULTS: TMT, RHOD, and TPMT activities using their conventional substrates were found throughout the gastrointestinal tract with highest activity in the colonic mucosa. When H(2)S was given as substrate, no reaction product was found with TMT or TPMT but RHOD was extremely active (Km 8.8 mM, Vmax 14.6 nmol/mg/min). Incubation of colonic homogenates with a specific RHOD antibody prevented the metabolism of H(2)S, indicating that RHOD is responsible for detoxifying H(2)S. A purified preparation of RHOD also detoxified H(2)S. CONCLUSIONS: RHOD, located in the submucosa and crypts of the colon, is the principal enzyme involved in H(2)S detoxication. TMT does not participate in the detoxication of H(2)S.

Staphylococcal food poisoning associated with an Easter egg hunt.

Staphylococcal contamination of intact, hard-boiled eggs resulted in the food poisoning of an estimated 300 children out of 850 who had participated in an Easter egg hunt. Enterotoxigenic staphylococci that were isolated from the Easter eggs matched that obtained from an infected cook who prepared the eggs three to five days before the hunt and which he left unrefrigerated. Experimental studies demonstrated that heated eggs can absorb 2 mL of contaminated cool water through intact eggshells. When water was inoculated with pathogenic staphylococci at even low contamination levels, rapid growth and enterotoxin production within cooked eggs could be easily duplicated. This is the first large outbreak of its type; safeguards can and should be employed to prevent future ones.

Intravenous ketorolac tromethamine worsens platelet function during knee arthroscopy under spinal anesthesia.

Ketorolac prolongs bleeding time and inhibits platelet aggregation and platelet thromboxane production in healthy, awake volunteers. However, platelet function was recently shown not to worsen after ketorolac was given during general anesthesia. The purpose of this study was to investigate platelet function changes during a standardized spinal anesthetic and surgery, as well as after a single intraoperative dose of intravenous (IV) ketorolac. The study comprised 30 ASA physical status I patients undergoing spinal anesthesia for knee arthroscopy. Subjects were randomized to receive either ketorolac 60 mg IV 15 min after skin incision or placebo IV. Platelet function testing consisted of an Ivy bleeding time, platelet aggregometry with adenosine diphosphate (ADP) and collagen, thromboelastography (TEG), and serum thromboxane B2 (TxB2) assays. Platelet function testing was performed: 1) 15 min prior to the performance of spinal anesthesia; 2) 10 min after surgical skin incision; and 3) 45 min after administration of study drug. The placebo group demonstrated no changes in any platelet function variable during spinal anesthesia and surgery relative to preoperative values. The ketorolac group, however, demonstrated a significant increase in bleeding time from postincision to poststudy drug data points (213 +/- 60s to 275 +/- 85s, mean +/- SD; P < 0.01). Further, platelet aggregometry to collagen was diminished in the ketorolac group from preoperative to poststudy drug data points (90.8% +/- 7.6% to 60.5% +/- 32.5%; P < 0.01). Platelet aggregometry with ADP, however, was unchanged in the ketorolac group. Platelet TxB2 production decreased dramatically in the ketorolac group from preoperative to poststudy drug data points (157.2 +/- 129.4 to 0.3 +/- 0.3 ng/mL; P < 0.01). Platelet function does not appear to be accentuated during spinal anesthesia as it is during general anesthesia. Unlike during general anesthesia, platelet function during spinal anesthesia is impaired, with respect to bleeding time and platelet aggregometry to collagen, by a single intraoperative dose of IV ketorolac.

Intravenous ketorolac tromethamine does not worsen platelet function during knee arthroscopy under general anesthesia.

Ketorolac (KT) prolongs bleeding time and inhibits platelet aggregation and platelet thromboxane production in healthy, awake volunteers. However, platelet function may be accentuated during the stress of general anesthesia (GA) and surgery. The purpose of this study was to investigate platelet function changes during a standard GA technique and surgery, as well as after a single intraoperative dose of intravenous (i.v.) KT. The study comprised 30 ASA physical status I patients undergoing GA for knee arthroscopy. Subjects were randomized to receive either KT 60 mg IV 15 min after skin incision or placebo i.v. Platelet function testing consisted of an Ivy bleeding time (BT), platelet aggregometry (PA) with adenosine diphosphate (ADP) and collagen, thromboelastography (TEG), and serum thromboxane B2 assays (TxB2). Platelet function testing was performed: 1) 15 min prior to the induction of GA, 2) 10 min after skin incision, and 3) 45 min after administration of study drug. BT decreased significantly in the placebo group from 263 +/- 133 s (mean +/- SD) preoperatively to 207 +/- 89 s postincision. BT did not change in the KT group. PA was unchanged after IV KT. TEG data was unchanged in both groups during anesthesia and surgery. TxB2 levels decreased markedly in the KT group from 106.9 +/- 96.2 ng/mL preoperatively to 0.4 +/- 1.2 ng/mL poststudy drug, P = 0.002. Platelet function appears to be accentuated during GA and surgery as evaluated by BT in the placebo group. Further, platelet function by BT, PA, and TEG was not inhibited after i.v. KT despite near complete abolition of TxB2 production.

The importance of the N-terminal segment for DnaJ-mediated folding of rhodanese while bound to ribosomes as peptidyl-tRNA.

Two lines of evidence indicate the importance of the N-terminal portion of rhodanese for correct folding of the nascent ribosome-bound polypeptide. A mutant gene lacking the codons for amino acids 1-23 of the wild-type protein is expressed very efficiently by coupled transcription/translation on Escherichia coli ribosomes; however, the mutant protein that is released from the ribosomes is enzymatically inactive. The mutant protein does not undergo the reaction that is promoted by the bacterial chaperone, DnaJ, which appears to be essential for folding of ribosome-bound rhodanese into the native conformation. The effect of DnaJ is monitored by fluorescence from coumarin cotranslationally incorporated at the N terminus of nascent rhodanese. Secondly, a synthetic peptide corresponding to the N-terminal 17 amino acids of the wild-type protein interferes with the synthesis of wild-type rhodanese but has much less effect on the synthesis of the N-terminal deletion mutant. The N-terminal peptide inhibits the effect of DnaJ on the nascent wild-type rhodanese and blocks the chaperone-mediated release and activation of ribosome-bound full-length rhodanese polypeptides that accumulate during in vitro synthesis. The results lead to the hypothesis that the N-terminal segment of rhodanese is required for its chaperone-dependent folding on the ribosome.