Amphiphilic, tri-block copolymers provide potent membrane-targeted neuroprotection.
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Animals↗
Biomedical subjects
J D Marks
Publications and source records attributed to J D Marks.
Epitope mapping of neutralizing botulinum neurotoxin A antibodies by phage display.
Single-chain antibodies neutralize activity and bind nonoverlapping epitopes of botulinum A neurotoxin. Two phage display epitope libraries were constructed from the 1.3 kb of binding domain cDNA. The minimal epitopes selected against the single-chain Fv-Fc antibodies correspond to conformational epitopes with amino acid residues 1115 to 1223 (S25), 1131 to 1264 (3D12), and 889 to 1294 (C25).
Animals↗
Lung collapse during low tidal volume ventilation in acute respiratory distress syndrome.
BACKGROUND: Current ventilator management for acute respiratory distress syndrome (ARDS) incorporates low tidal volume (V(T)) ventilation in order to limit ventilator-induced lung injury. Low V(T) ventilation in supine patients, without the use of intermittent hyperinflations, may cause small airway closure, progressive atelectasis, and secretion retention. Use of high positive end-expiratory pressure (PEEP) levels with low V(T) ventilation may not counter this effect, because regional differences in intra-abdominal hydrostatic pressure may diminish the volume-stabilizing effects of PEEP. CASE SUMMARY: A 35-year-old man with abdominal compartment syndrome (intra-abdominal pressure > 48 cm H2O developed ARDS and was treated with V(T) of 4.5 mL/kg and PEEP of 20 cm H2O. Despite aggressive fluid therapy, appropriate airway humidification and tracheal suctioning, the patient developed complete bronchial obstruction, involving the entire right lung and left upper lobe. After bronchoscopy the patient was placed on a higher V(T) (7.0 mL/kg). Intermittent PEEP was instituted at 30 cm H2O for 2 breaths every 3 minutes. This intermittently raised the end-inspiratory plateau pressure from 38 cm H2O to 50 cm H2O. With the same airway humidity and tracheal suctioning practices bronchial obstruction did not reoccur. CONCLUSION: Low V(T) ventilation in ARDS may increase the risk of small airway closure and retained secretions. This adverse effect highlights the importance of pulmonary hygiene measures in ARDS during lung-protective ventilation.
Adult↗
Maturation of vulnerability to excitotoxicity: intracellular mechanisms in cultured postnatal hippocampal neurons.
Neuronal vulnerability to excitotoxicity changes dramatically during postnatal maturation. To study the intracellular mechanisms by which maturation alters vulnerability in single neurons, we developed techniques to maintain hippocampal neurons from postnatal rats in vitro. After establishing their neuronal phenotype with immunohistochemistry and electrophysiology, we determined that these neurons exhibit developmentally regulated vulnerability to excitotoxicity. At 5 days in vitro, NMDA-induced cell death at 24 h increased from 3.6% in 3-day-old rats to >90% in rats older than 21 days. Time-lapse imaging of neuronal morphology following NMDA demonstrated increasingly prevalent and severe injury as a function of postnatal age. Neither high- nor low-affinity calcium dyes demonstrated differences in peak NMDA-induced [Ca(2+)](i) increases between neurons from younger and older animals. However, neurons from older animals were uniformly distinguished from those from younger animals by their subsequent loss of [Ca(2+)](i) homeostasis. Because of the role of mitochondrial Ca(2+) buffering in [Ca(2+)](i) homeostasis, we measured NMDA-induced changes in mitochondrial membrane potential (DeltaPsi) as a function of postnatal age. NMDA markedly dissipated DeltaPsi in neurons from mature rats, but minimally in those from younger rats. These data demonstrate that, in cultures of postnatal hippocampal neurons, (a) vulnerability to excitotoxicity increases as a function of the postnatal age of the animal from which they were harvested, and (b) developmental regulation of vulnerability to NMDA occurs at the level of the mitochondrion.
Aging↗
Targeting of bivalent anti-ErbB2 diabody antibody fragments to tumor cells is independent of the intrinsic antibody affinity.
In immunodeficient mice antitumor single-chain Fv (scFv) molecules penetrate tumors rapidly and have rapid serum clearance, leading to excellent tumor:normal organ ratios. However, the absolute quantity of scFv retained in the tumor is low due to rapid serum clearance and monovalent scFv binding. We previously demonstrated that the presence of an additional binding site prolongs in vitro and in vivo association of scFv-based molecules with tumor cells expressing relevant antigen. The contribution of the intrinsic affinity of each component scFv to the association between a dimeric scFv and its target antigen is largely unknown. Here, we have constructed bivalent diabody molecules from three affinity mutants of the human anti-ErbB2 (HER2/neu) scFv molecule C6.5 by shortening the peptide linker between the heavy (VH) and light (VL) chains variable domains from 15 to 5 amino acids. The shorter linker prevents intramolecular pairing of VH and VL, resulting in intermolecular pairing and creation of a dimeric Mr 50,000 molecule with two antigen-binding sites. The scFv used to create the diabodies span a 133-fold range of affinity for the same epitope of ErbB2 [133 nM (C6G98A), 25 nM (C6.5), and 1 nM (C6ML3-9)] and differ by only one to three amino acids. Diabody binding kinetics were determined by surface plasmon resonance on the immobilized ErbB2 extracellular domain. The association rate constants obtained for each diabody molecule were similar to that of the parental (component) scFv. However, the dissociation rate constants obtained for the bivalent diabodies were up to 15-fold slower. The magnitude of the decrease in the bivalent dissociation rate constant was inversely proportional to the monovalent interaction, ranging from only 3-fold for that of the C6ML3-9 diabody to 15-fold for the C6G98A diabody. This resulted in only a 22-fold difference in bivalent affinity, compared with a 133-fold difference in affinity for the respective scFv. Equilibrium-binding constants obtained by surface plasmon resonance correlated well with the equilibrium-binding constants determined in vitro on ErbB2 overexpressing cells. Biodistribution studies were performed in scid mice bearing established SKOV3 tumors. At 24 h, 3-37-fold more diabody was retained in tumor compared with the parental scFv monomers. This likely results from a higher apparent affinity, because of bivalent binding, and a slower serum clearance. Surprisingly, the differences in affinity between diabodies did not result in differences in quantitative tumor retention or tumor to blood ratios. In fact, the diabody constructed from the lowest affinity scFv exhibited the best tumor-targeting properties. We conclude that, above a threshold affinity, other factors regulate quantitative tumor retention. In addition, straightforward dimerization of a low-affinity scFv leads to significantly greater tumor localization than does exhaustive scFv affinity maturation.
Animals↗
Applying phage antibodies to proteomics: selecting single chain Fv antibodies to antigens blotted on nitrocellulose.
Two-dimensional gel electrophoresis is a powerful tool for identification of proteins that differ between patients with qualitatively or quantitatively different disease states. Further characterization of these protein differences would be greatly facilitated by the availability of antibodies that could be used to detect and quantitate the temporo-spatial pattern and cellular and tissue location of the different proteins. To generate such antibodies, methods were developed which permit the successful selection of monoclonal phage antibodies from phage display libraries against antigens blotted from SDS-PAGE gels onto nitrocellulose. First, it was determined that nitrocellulose and PVDF membranes gave significantly lower levels of background phage binding than two other membranes studied. Next, it was determined that blocking with fish gelatin and binding in the presence of 0.5 M NaCl could reduce nonspecific binding 10,000-fold and result in enrichment ratios greater than 500-fold with antigen concentrations as low as 1 ng/mm(2). When optimized conditions were applied to phage antibody libraries, panels of monoclonal phage antibodies were generated against the proteins ErbB2 and bovine serum albumin electroblotted from SDS-PAGE gels onto nitrocellulose. Antibodies were obtained with as little as 10 to 1 ng of antigen, depending on whether the libraries displayed single or multiple copies of antibody per phage. The antibodies worked as reagents in both ELISA and Western blotting.
Animals↗
Mass spectral analysis of a protein complex using single-chain antibodies selected on a peptide target: applications to functional genomics.
Genome projects are identifying an ever-increasing number of genes, accelerating the need for reagents to study the expression of these genes and elucidate the function and cellular location of the gene products. Our goal was to develop a strategy to allow human single-chain variable fragment (scFv) antibodies to be used for these endeavors. A library containing 7x10(9) individual variants was displayed by bacteriophage and selected against a biotinylated peptide corresponding to the C-terminal 15 amino acid residues of Ku86, one component of a heterodimer involved in double-stranded DNA break repair. Four unique scFv antibodies were recovered that not only recognized the selected peptide, but also the intact protein. Three of the scFv antibodies were expressed in soluble form and recognized Ku86 by Western analysis. The affinity of one of the scFv antibodies for Ku86 was 16 nM as measured by BIAcore analysis. scFv immunoprecipitation of Ku86 also isolated the other component of the heterodimer, Ku70, as determined by Western analysis and mass spectrometry. These results demonstrate the utility of scFv antibodies as invaluable reagents for functional genomics.
Antibodies↗
Selection of tumor-specific internalizing human antibodies from phage libraries.
Antibody internalization into the cell is required for many targeted therapeutics, such as immunotoxins, immunoliposomes, antibody-drug conjugates and for targeted delivery of genes or viral DNA into cells. To generate directly tumor-specific internalizing antibodies, a non-immune single chain Fv (scFv) phage antibody library was selected on the breast tumor cell line SKBR3. Internalized phage were recovered from within the cell and used for the next round of selection. After three rounds of selection, 40 % of clones analyzed bound SKBR3 and other tumor cells but did not bind normal human cells. Of the internalizing scFv identified, two (F5 and C1) were identified as binding to ErbB2, and one (H7) to the transferrin receptor. Both F5 and H7 scFv were efficiently endocytosed into SKBR3 cells, both as phage antibodies and as native monomeric scFv. Both antibodies were able to induce additional functional effects besides triggering endocytosis: F5 scFv induces downstream signaling through the ErbB2 receptor and H7 prevents transferrin binding to the transferrin receptor and inhibits cell growth. The results demonstrate the feasibility of selecting internalizing receptor-specific antibodies directly from phage libraries by panning on cells. Such antibodies can be used to target a variety of molecules into the cell to achieve a therapeutic effect. Furthermore, in some instances endocytosis serves as a surrogate marker for other therapeutic biologic effects, such as growth inhibition. Thus, a subset of selected antibodies will have a direct therapeutic effect.
Animals↗
Improved flow and pressure capabilities of the Datex-Ohmeda SmartVent anesthesia ventilator.
STUDY OBJECTIVE: To compare the flow and pressure capabilities of the Datex-Ohmeda SmartVent (Ohmeda 7900, Datex-Ohmeda, Madison, WI) to previous Ohmeda (7810 and 7000, Datex-Ohmeda, Madison, WI) anesthesia ventilators. To determine airway pressure and minute ventilation thresholds for intraoperative use of a critical care ventilator. DESIGN: Three anesthesia ventilators and one critical care ventilator (Siemens Servo 900C, Siemens, Solna, Sweden) were studied in a lung model. Retrospective medical record review. SETTING: Research Laboratory and Critical Care Unit of a Level I Trauma Center. PATIENTS: 145 mechanically ventilated patients treated for acute respiratory failure who underwent 200 surgical procedures. INTERVENTIONS: The effect of increasing pressure on mean inspiratory flow was determined by cycling each ventilator through increasing restrictors. Maximum minute ventilation was measured at low compliance (10-30 mL/cm H2O), positive end-expiratory pressure (PEEP) (0-20 cm H2O), and increased airway resistance (approximately 19 and approximately 36 cm H2O/L/sec) in a mechanical lung model. MEASUREMENTS AND MAIN RESULTS: Flow, volume, and pressure were measured with a pulmonary mechanics monitor (BICORE CP-100, Thermo Respiratory Group, Yorba Linda, CA). Preoperative peak airway pressure and minute ventilation (VE) were extracted from the medical record. Mean inspiratory flow declined with increasing pressure in all anesthesia ventilators. The SmartVent and the 7810 produced greater mean inspiratory flow than did the 7000 ventilator. As compliance progressively decreased, the Siemens, the SmartVent, and the 7810 ventilators maintained VE compared to the 7000 ventilator. The Siemens and the SmartVent maintained VE with PEEP, compared to the 7810 and 7000 ventilators. During increased airway resistance, maximal VE was lower for all ventilators. The SmartVent met the ventilation requirements in 90% of the patients compared to 67% of patients with the 7000 ventilator. CONCLUSION: The improved pressure and flow capabilities of the SmartVent increase the threshold for using a critical care ventilator intraoperatively to a peak airway pressure > 65 cm H2O and/or VE > 18 L/min.
Acute Disease↗
Delivery of the alpha-emitting radioisotope bismuth-213 to solid tumors via single-chain Fv and diabody molecules.
Intravenously administered anti-tumor single-chain Fv (scFv) and diabody molecules exhibit rapid clearance kinetics and accumulation in tumors that express their cognate antigen. In an attempt to fit the rate of isotope decay to the timing of delivery and duration of tumor retention, anti-HER2/neu CHX-A" DTPA-C6.5K-A scFv and diabody conjugates were labeled with the alpha-particle emitter (213)Bi (t(1/2) = 47 min). Radioimmunotherapy studies employing 0.64, 0.35, or 0.15 microCi of (213)Bi-labeled C6.5K-A diabody or 1.1, 0.6, or 0. 3 microCi of (213)Bi-labeled C6.5K-A scFv were performed in nude mice bearing early, established SK-OV-3 tumors. Only the 0.3 microCi dose of (213)Bi-labeled C6.5K-A scFv resulted in both acceptable toxicity and a reduction in tumor growth rate. The specificity of the anti-tumor effects was determined by comparing the efficacy of treatment with 0.3 and 0.15 microCi doses of (213)Bi-labeled C6.5K-A scFv and (213)Bi-labeled NM3E2 (an irrelevant scFv) in nude mice bearing large established tumors. The 0.3 microCi dose of (213)Bi on both the C6.5K-A and NM3E2 scFvs resulted in similar anti-tumor effects (p = 0.46) indicating that antigen-specific targeting was not a factor. This suggests that the physical half-life of (213)Bi may be too brief to be effectively paired with systemically-administered diabody or scFv molecules.
Alpha Particles↗
Initial severity of metabolic acidosis predicts the development of acute lung injury in severely traumatized patients.
OBJECTIVES: First, to determine whether the severity of shock, as measured by systemic hypotension and metabolic acidosis, is significantly associated with a higher risk of acute lung injury in patients with severe trauma. Second, to determine whether the volumes of blood and crystalloid solutions administered in the early posttrauma period are independent risk factors for acute lung injury in severely traumatized patients. DESIGN: Prospective observational study. SETTING: Level I urban trauma center in a university hospital. PATIENTS: A total of 102 severely injured, mechanically ventilated trauma patients with an Injury Severity Score > or =16 and aged between 18 and 75 yrs. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Initial clinical and laboratory data were collected in the emergency department, and on a daily basis thereafter during the patient's intensive care unit stay. Of the 102 severely injured patients enrolled, 42 developed acute lung injury (41%) and 60 did not (59%). A total of 93% of the trauma patients who developed acute lung injury during the 17-month study period were included in the study. Initial base deficit was significantly lower in patients who developed acute lung injury than in those who did not (-8.8+/-4.5 vs. -5.6+/-5.1, p<.01). The difference in systolic blood pressure between the two groups was not significant. CONCLUSIONS: In this group of severely injured trauma patients, the degree of metabolic acidosis at the time of admission identified those patients with the highest probability of developing acute lung injury. In addition, the volume of crystalloid solution administered during the first 24 hrs was significantly greater in patients who later developed acute lung injury. Finally, there was a significantly higher morbidity in patients who developed acute lung injury, whereas mortality did not differ between the two groups.
APACHE↗
The selective toxicity of 1-methyl-4-phenylpyridinium to dopaminergic neurons: the role of mitochondrial complex I and reactive oxygen species revisited.
1-Methyl-4-phenylpyridinium (MPP(+)) is selectively toxic to dopaminergic neurons and has been studied extensively as an etiologic model of Parkinson's disease (PD) because mitochondrial dysfunction is implicated in both MPP(+) toxicity and the pathogenesis of PD. MPP(+) can inhibit mitochondrial complex I activity, and its toxicity has been attributed to the subsequent mitochondrial depolarization and generation of reactive oxygen species. However, MPP(+) toxicity has also been noted to be greater than predicted by its effect on complex I inhibition or reactive oxygen species generation. Therefore, we examined the effects of MPP(+) on survival, mitochondrial membrane potential (DeltaPsim), and superoxide and reduced glutathione levels in individual dopaminergic and nondopaminergic mesencephalic neurons. MPP(+) (5 microM) selectively induced death in fetal rat dopaminergic neurons and caused a small decrease in their DeltaPsim. In contrast, the specific complex I inhibitor rotenone, at a dose (20 nM) that was less toxic than MPP(+) to dopaminergic neurons, depolarized DeltaPsim to a greater extent than MPP(+). In addition, neither rotenone nor MPP(+) increased superoxide in dopaminergic neurons, and MPP(+) failed to alter levels of reduced glutathione. Therefore, we conclude that increased superoxide and loss of DeltaPsim may not represent primary events in MPP(+) toxicity, and complex I inhibition alone is not sufficient to explain the selective toxicity of MPP(+) to dopaminergic neurons. Clarifying the effects of MPP(+) on energy metabolism may provide insight into the mechanism of dopaminergic neuronal degeneration in PD.
1-Methyl-4-phenylpyridinium↗
The treatment of acidosis in acute lung injury with tris-hydroxymethyl aminomethane (THAM).
Mechanical hyperventilation of acidemic patients with acute lung injury (ALI) requires the use of high volumes and pressures that may worsen lung injury. However, permissive hypercapnia in the presence of shock, metabolic acidosis, and multi-organ system dysfunction may compromise normal cellular function. Tris-hydroxymethyl aminomethane (THAM) may be an effective method to control acidosis in this circumstance. Protonated THAM is excreted by the kidneys, so that carbon dioxide production is not raised. In an uncontrolled study, we administered THAM to 10 patients with acidosis (mean pH = 7.14) and ALI (mean lung injury score = 3.28) in whom adequate control of arterial pH could not be maintained during either eucapnic ventilation or permissive hypercapnia ventilation. THAM was given at a mean dose of 0.55 mmol/kg/h. Administration of THAM was associated with significant improvements in arterial pH and base deficit, and a decrease in arterial carbon dioxide tension that could not be fully accounted for by ventilation. Although further studies are needed to confirm these observations, THAM appears to be an effective alternative to sodium bicarbonate for treating acidosis during ALI.
Acidosis↗
Phage libraries for generation of anti-botulinum scFv antibodies.
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Animals↗
Measuring intra-esophageal pressure to assess transmural pulmonary arterial occlusion pressure in patients with acute lung injury: a case series and review.
BACKGROUND: Positive end-expiratory pressure (PEEP) may interfere with accurate assessment of cardiac function. PEEP may decrease left ventricular volume by lowering the transmural gradient between ventricular and pleural surface pressure (P(PL)) around the heart while raising the absolute pulmonary arterial occlusion pressure (PAOP). Clinical formulas used to predict the transmural PAOP (PAOP(TM)) require subtracting 25-50% of the PEEP level from the PAOP. However, both PAOP and P(PL) are influenced by transmitted PEEP and transmitted intra-abdominal pressure (IAP). We compared PAOP(TM) calculated by measuring intra-esophageal pressure (P(ES)) with PAOP(TM) estimated by clinical formulas. METHODS: Twenty-two P(ES) measurements were made with a bedside pulmonary mechanics monitor (BICORE CP-100) on 11 patients with acute lung injury who had an elevated PAOP (mean +/- standard deviation) of 21.1 +/- 6.2 mm Hg and PEEP of 13.0 +/- 3.8 mm Hg. Paired comparisons were made with the Wilcoxon signed-rank test and multiple comparisons were made using one-way analysis of variance (ANOVA) and the Student-Newman-Keuls test. Pearson product-moment correlation coefficients were calculated. A MEDLINE literature search was done to survey the reported range of PEEP transmitted to P(PL). RESULTS: P(ES) (14.6 +/- 5.0 mm Hg) exceeded PEEP; 9 of 11 patients had clinical evidence of increased IAP. PAOP(TM) predicted by clinical formulas were 13.5-17.7 mm Hg, whereas PAOP(TM) calculated by P(ES) was 6.2 +/- 3.6 mm Hg (p < 0.05). Linear regression revealed a moderate correlation between PAOP and PEEP (r = 0.49, p = 0.02). In contrast, there was a strong correlation between PAOP and P(ES) (r = 0.83, p < 0.0001). A review of data derived from the literature did not show a consistent pattern of PEEP transmission. CONCLUSION: PAOP(TM) calculated by P(ES) may reflect transmitted IAP to the pleural surface. Using P(ES) to calculate PAOP(TM) may provide a more accurate assessment of hemodynamic status than predicting PAOP(TM) using clinical formulas based solely on estimated PEEP transmission.
Adult↗
Targeted gene delivery to mammalian cells by filamentous bacteriophage.
We report that prokaryotic viruses can be re-engineered to infect eukaryotic cells resulting in expression of a reporter gene inserted into the bacteriophage genome. Phage capable of binding mammalian cells expressing the growth factor receptor ErbB2 and undergoing receptor-mediated endocytosis were isolated by selection of a phage antibody library on breast tumor cells and recovery of infectious phage from within the cell. As determined by immunofluorescence, F5 phage were efficiently endocytosed into 100 % of ErbB2 expressing SKBR3 cells. To achieve reporter gene expression, F5 phage were engineered to package the green fluorescent protein (GFP) reporter gene driven by the CMV promoter. These phage when applied to cells underwent ErbB2-mediated endocytosis leading to GFP expression. GFP expression occurred only in cells overexpressing ErbB2, was dose-dependent reaching, 4 % of cells after 60 hours and was detected with phage titers as low as 2.0 x 10(7) cfu/ml (500 phage/cell). The results demonstrate that bacterial viruses displaying the appropriate antibody can bind to mammalian receptors and utilize the endocytic pathway to infect eukaryotic cells, resulting in expression of a reporter gene inserted into the viral genome. This represents a novel method to discover targeting molecules capable of delivering a gene intracellularly into the correct trafficking pathway for gene expression by directly screening phage antibodies. This should significantly facilitate the identification of appropriate targets and targeting molecules for gene therapy or other applications where delivery into the cytosol is required. This approach can be adapted to directly select, rather than screen, phage antibodies for targeted gene expression. The results also demonstrate the potential of phage antibodies as an in vitro or in vivo targeted gene delivery vehicle.
Bacteriophages↗
Toward selection of internalizing antibodies from phage libraries.
Antibodies which bind cell surface receptors in a manner whereby they are endocytosed are useful molecules for the delivery of drugs, toxins, or DNA into the cytosol of mammalian cells for therapeutic applications. Traditionally, internalizing antibodies have been identified by screening hybridomas. For this work, we studied a human scFv (C6.5) which binds ErbB2 to determine the feasibility of directly selecting internalizing antibodies from phage libraries and to identify the most efficient display format. Using wild-type C6.5 scFv displayed monovalently on a phagemid, we demonstrate that anti-ErbB2 phage antibodies can undergo receptor-mediated endocytosis. Using affinity mutants and dimeric diabodies of C6.5 displayed as either single copies on a phagemid or multiple copies on phage, we define the role of affinity, valency, and display format on phage endocytosis and identify the factors that lead to the greatest enrichment for internalization. Phage displaying bivalent diabodies or multiple copies of scFv were more efficiently endocytosed than phage displaying monomeric scFv and recovery of infectious phage was increased by preincubation of cells with chloroquine. Measurement of phage recovery from within the cytosol as a function of applied phage titer indicates that it is possible to select for endocytosable antibodies, even at the low concentrations that would exist for a single phage antibody member in a library of 10(9).
Antibodies, Bispecific↗
Treatment of infected left ventricular assist device using antibiotic-impregnated beads.
There is no well-established therapy for treating infections of heart-assist or artificial heart devices, a serious problem with life-threatening consequences. We used a promising new approach in which antibiotic-impregnated polymethylmethacrylate beads were placed around an implanted left ventricular assist device to control an external blood pump infection in a bridge-to-transplant patient. In this case report, we describe the potential of antimicrobial-impregnated polymethylmethacrylate beads for in situ control of infections involving external surfaces of cardiovascular devices.
Combined Modality Therapy↗