Fab Chaperone-Assisted RNA Crystallography (Fab CARC).

PubMed

Sherman, Eileen; Archer, Jennifer; Ye, Jing-Dong

2016-01-01

Recent discovery of structured RNAs such as ribozymes and riboswitches shows that there is still much to learn about the structure and function of RNAs. Knowledge learned can be employed in both biochemical research and clinical applications. X-ray crystallography gives unparalleled atomic-level structural detail from which functional inferences can be deduced. However, the difficulty in obtaining high-quality crystals and their phasing information make it a very challenging task. RNA crystallography is particularly arduous due to several factors such as RNA's paucity of surface chemical diversity, lability, repetitive anionic backbone, and flexibility, all of which are counterproductive to crystal packing. Here we describe Fab chaperone assisted RNA crystallography (CARC), a systematic technique to increase RNA crystallography success by facilitating crystal packing as well as expediting phase determination through molecular replacement of conserved Fab domains. Major steps described in this chapter include selection of a synthetic Fab library displayed on M13 phage against a structured RNA crystallization target, ELISA for initial choice of binding Fabs, Fab expression followed by protein A affinity then cation exchange chromatography purification, final choice of Fab by binding specificity and affinity as determined by a dot blot assay, and lastly gel filtration purification of a large quantity of chosen Fabs for crystallization.

Immunoscintigraphy of ovarian cancer with indium-111-labeled OV-TL 3 F(ab')2 monoclonal antibody.

PubMed

Massuger, L F; Kenemans, P; Claessens, R A; Verheijen, R H; Schijf, C P; Strijk, S P; Poels, L G; van Hoesel, R G; Corstens, F H

1990-11-01

The safety and diagnostic accuracy of immunoscintigraphy with the indium-111-labeled monoclonal antibody OV-TL 3 F(ab')2(111In-OV-TL 3 F(ab')2) for diagnosis and follow-up of ovarian cancer was prospectively studied in 31 patients. Planar and SPECT scintigraphy were performed up to 4 days after i.v. injection of 140 MBq 111In-OV-TL 3 F(ab')2. Surgical evaluation was possible in 22 out of 31 patients. Imaging results were compared with X-ray computed tomography, ultrasound, and CA 125 serum level using the histologically confirmed surgical findings as a "gold standard." Apart from a transient rash observed in two patients, no other immediate or delayed adverse reactions were observed. Within the surgically evaluated group, ovarian cancer lesions were detected in 16 out of 17 patients (94%). Of 45 distinct tumor deposits found at operation, 67% were detected and localized with immunoscintigraphy while X-ray computed tomography and ultrasound visualized 53% and 23%, respectively.

Radionuclide imaging in myocardial sarcoidosis. Demonstration of myocardial uptake of /sup 99m/Tc pyrophosphate and gallium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Forman, M.B.; Sandler, M.P.; Sacks, G.A.

1983-03-01

A patient had severe congestive cardiomyopathy secondary to myocardial sarcoidosis. The clinical diagnosis was confirmed by radionuclide ventriculography, /sup 201/Tl, /sup 67/Ga, and /sup 99m/Tc pyrophosphate (TcPYP) scintigraphy. Myocardial TcPYP uptake has not been reported previously in sarcoidosis. In this patient, TcPYP was as useful as gallium scanning and thallium imaging in documenting the myocardial process.

FabH Mutations Confer Resistance to FabF-Directed Antibiotics in Staphylococcus aureus

PubMed Central

Parsons, Joshua B.; Yao, Jiangwei; Frank, Matthew W.

2014-01-01

Delineating the mechanisms for genetically acquired antibiotic resistance is a robust approach to target validation and anticipates the evolution of clinical drug resistance. This study defines a spectrum of mutations in fabH that render Staphylococcus aureus resistant to multiple natural products known to inhibit the elongation condensing enzyme (FabF) of bacterial type II fatty acid synthesis. Twenty independently isolated clones resistant to platensimycin, platencin, or thiolactomycin were isolated. All mutants selected against one antibiotic were cross-resistant to the other two antibiotics. Mutations were not detected in fabF, but the resistant strains harbored missense mutations in fabH. The altered amino acids clustered in and around the FabH active-site tunnel. The mutant FabH proteins were catalytically compromised based on the low activities of the purified enzymes, a fatty acid-dependent growth phenotype, and elevated expression of the fabHF operon in the mutant strains. Independent manipulation of fabF and fabH expression levels showed that the FabH/FabF activity ratio was a major determinant of antibiotic sensitivity. Missense mutations that reduce FabH activity are sufficient to confer resistance to multiple antibiotics that bind to the FabF acyl-enzyme intermediate in S. aureus. PMID:25403676

Myocardial 123I-metaiodobenzylguanidine scintigraphy in patients with homozygous and heterozygous parkin mutations.

PubMed

De Rosa, Anna; Pellegrino, Teresa; Pappatà, Sabina; Pellecchia, Maria Teresa; Peluso, Silvio; Saccà, Francesco; Barone, Paolo; Cuocolo, Alberto; De Michele, Giuseppe

2017-02-01

PARK2 is an autosomal recessive parkinsonism caused by parkin gene mutations. Several Parkinson's Disease (PD) cases harbor single parkin mutations, raising a debate about the pathogenic meaning of heterozygous mutations. Here, we evaluate cardiac autonomic innervation in patients with either two or one parkin mutations compared to patients with idiopathic PD (IPD). Myocardial 123 I-metaiodobenzylguanidine (MIBG) scintigraphy was performed in six PD patients with single parkin mutations (HET), four with two mutations (PARK2), and eight with IPD. In comparison to control group, IPD patients showed lower early and late heart-to-mediastinum (H/M) ratios and higher washout rates, whereas HET patients had only lower early H/M ratio, and PARK2 patients were not different for any parameter. At individual level, MIBG findings were abnormal in 7/8 IPD, in 4/6 HET and in 1/4 PARK2 patients. Preserved cardiac 123 I-MIBG uptake confirms that PARK2 pathogenic mechanism, at least partially, differs from that responsible for IPD. HET subjects show intermediate findings, suggesting possible heterogeneity.

Regional heterogeneity in cardiac sympathetic innervation in acute myocardial infarction: relationship with myocardial oedema on magnetic resonance.

PubMed

Gimelli, Alessia; Masci, Pier Giorgio; Liga, Riccardo; Grigoratos, Chrysanthos; Pasanisi, Emilio Maria; Lombardi, Massimo; Marzullo, Paolo

2014-09-01

To assess the relationships between myocardial structure and function on cardiac magnetic resonance (CMR) imaging and sympathetic tone on (123)I-metaiodobenzylguanidine ((123)I-MIBG) scintigraphy early after myocardial infarction (MI). Ten patients underwent (123)I-MIBG and (99m)Tc-tetrofosmin rest cadmium zinc telluride scintigraphy 4 ± 1 days after MI. The segmental left ventricular (LV) relative radiotracer uptake of both (99m)Tc-tetrofosmin and early (123)I-MIBG was calculated. The day after scintigraphy, on CMR imaging, the extent of ischaemia-related oedema and of myocardial fibrosis (late gadolinium enhancement, LGE) was assessed. Accordingly, the extent of oedema and LGE was evaluated for each segment and segmental wall thickening determined. Based on LGE distribution, LV segments were categorized as "infarcted" (56 segments), "adjacent" (66 segments) or "remote" (48 segments). Infarcted segments showed a more depressed systolic wall thickening and greater extent of oedema than adjacent segments (p < 0.001) and remote segments (p < 0.001). Interestingly, while uptake of (99m)Tc-tetrofosmin was significantly depressed only in infarcted segments (p < 0.001 vs. both adjacent and remote segments), uptake of (123)I-MIBG was impaired not only in infarcted segments (p < 0.001 vs. remote) but also in adjacent segments (p = 0.024 vs. remote segments). At the regional level, after correction for (99m)Tc-tetrofosmin and LGE distribution, segmental (123)I-MIBG uptake (p < 0.001) remained an independent predictor of ischaemia-related oedema. After acute MI the regional impairment of sympathetic tone extends beyond the area of altered myocardial perfusion and is associated with myocardial oedema.

Potential diagnostic value of 131I-MIBG myocardial scintigraphy in discrimination between Alzheimer disease and dementia with Lewy bodies.

PubMed

Abbasi, Mehrshad; Ghalandari, Nafise; Farzanefar, Saeed; Aghamollaii, Vajiheh; Ahmadi, Mona; Ganji, Morsaleh; Afarideh, Mohsen; Loloee, Sogol; Naseri, Maryam; Tafakhori, Abbas

2017-12-01

Clinical difficulty to discriminate between the Alzheimer disease (AD) and dementia with Lewy bodies (DLB) has led researchers to focus on highly sensitive functional imaging modalities. The aim of the present study was to assess 131 I-MIBG cardiac imaging to distinguish between AD and DLB. Seventeen patients who were known cases of dementia underwent 131 I-MIBG myocardial scintigraphy to differentiate AD from DLB. Planar and 131 I-MIBG SPECT were obtained 2h after the injection of 1mCi 131 I-MIBG on a dual head gamma camera. The visual assessment of the heart uptake compared with lungs and the quantification based on the heart to mediastinal ratio (HMR) were done. The cardiac receiver operating characteristic (ROC) curve was designed for the optimal HMR cut-off values to predict the diagnoses of the patients. The diagnoses were clinically confirmed during the follow up of 14±8.2 months. Out of 17 patients (13 males; 76.5%), 10 patients had AD (7 males; 70%) and 7 patients had DLB (6 males; 85%). The pooled HMR was 1.74±0.33 in the study population; with 1.95±0.22 in the AD group and 1.43±0.20 in the DLB group to demonstrate significantly different HMR scores between patients with AD and DLB (p value=0.001). The visual interpretation was positive in 10 patients (accuracy of 88.2%). The shortest distance on the ROC curve to the optimal value corresponding to HMR=1.57 identified 10 patients with a high HMR (positive cardiac uptake) and 7 patients with a low HMR (negative cardiac uptake), the accuracy calculated at 88.2%. 131 I-MIBG myocardial scintigraphy is a potential alternative diagnostic modality for discrimination between AD and DLB when 123 I is not available. Copyright © 2017 Elsevier B.V. All rights reserved.

Utilization of nuclear medicine scintigraphy in Taiwan, 1997-2009.

PubMed

Hung, Mao-Chin; Hsieh, Wanhua Annie; Chang, Peter Wushou; Hwang, Jeng-Jong

2011-12-01

To analyze the utilization of nuclear medicine scintigraphy in the Taiwanese population within the national health-care system between 1997 and 2009. Based on the Taiwan's National Health Insurance Research Database of 1997-2009, a retrospective population-based analysis was conducted. Descriptive statistics and regression analysis were employed to analyze the frequencies and longitudinal trends in the utilization of diagnostic nuclear medicine procedures during the period. In addition, correlation analysis was applied to determine the correlated factors in the utility of nuclear medicine scintigraphy. The annual total nuclear medicine scintigraphy was estimated to be 256,389 on average in 1997-2009 and 11.7 per 1,000 population over the period. The frequency had increased by 67% over the years, from 8.2 per 1,000 population in 1997 to 13.7 per 1,000 population in 2009. The most frequently performed procedures were whole-body bone scans (33.4% of total) and myocardial perfusion scans (29.4% of total), with 4,615 and 5,620 increments per year, respectively. Most patients were in the age group of 41-65 years old when taking examinations. In addition, male subjects were slightly more than female patients (51.5 vs. 48.5%). Furthermore, the frequencies of whole-body bone scans and PET scans were proportional to the incidences of cancers (correlation coefficients were 0.96 and 0.94, respectively). The utilization of nuclear medicine scintigraphy with the National Health Insurance system in Taiwan has been changed considerably in the past 13 years. Both whole-body bone scan and myocardial perfusion scan were performed most often with significantly increases. The trend of nuclear medicine scintigraphy may have potential impact on making health-care policy in Taiwan.

DNA double-strand breaks in blood lymphocytes induced by two-day 99mTc-MIBI myocardial perfusion scintigraphy.

PubMed

Rief, Matthias; Hartmann, Lisa; Geisel, Dominik; Richter, Felicitas; Brenner, Winfried; Dewey, Marc

2018-07-01

To investigate DNA double-strand breaks (DSBs) in blood lymphocytes induced by two-day 99m Tc-MIBI myocardial perfusion scintigraphy (MPS) using y-H2AX immunofluorescence microscopy and to correlate the results with 99m Tc activity in blood samples. Eleven patients who underwent two-day MPS were included. DSB blood sampling was performed before and 5min, 1h and 24h after the first and second radiotracer injections. 99m Tc activity was measured in each blood sample. For immunofluorescence microscopy, distinct foci representing DSBs were quantified in lymphocytes after staining for the phosphorylated histone variant y-H2AX. The 99m Tc-MIBI activity measured on days one and two was similar (254±25 and 258±27 MBq; p=0.594). Compared with baseline DSB foci (0.09±0.05/cell), a significant increase was found at 5min (0.19±0.04/cell) and 1h (0.18±0.04/cell) after the first injection and at 5min and 1h after the second injection (0.21±0.03 and 0.19±0.04/cell, respectively; p=0.003 for both). At 24h after the first and second injections, the number of DSB foci had returned to baseline (0.06±0.02 and 0.12±0.05/cell, respectively). 99m Tc activity levels in peripheral blood samples correlated well with DSB counts (r=0.451). DSB counts reflect 99m Tc-MIBI activity after injection for two-day MPS, and might allow individual monitoring of biological effects of cardiac nuclear imaging. • Myocardial perfusion scintigraphy using 99m Tc induces time-dependent double-strand breaks (DSBs) • γ-H2AX immunofluorescence microscopy shows DSB as an early response to radiotracer injection • Activity measurements of 99m Tc correlate well with detected DSB • DSB foci induced by 99m Tc return to baseline 24h after radiotracer injection.

Fab(nimotuzumab)-HYNIC-99mTc: Antibody Fragmentation for Molecular Imaging Agents.

PubMed

Calzada, Victoria; García, María Fernanda; Alonso-Martínez, Luis Michel; Camachoc, Ximena; Goicochea, Enzo; Fernández, Marcelo; Castillo, Abmel Xiques; Díaz-Miqueli, Arlhee; Iznaga-Escobar, Normando; Montaña, René Leyva; Alonso, Omar; Gambini, Juan Pablo; Cabral, Pablo

2016-01-01

Finally, fast blood clearance nimotuzumab is a humanized monoclonal antibody that recognise, with high specific affinity, the epidermal growth factor receptor (EGF-R) which play an important role in the growth process associated with many solid tumors. In this work, the whole antibody was digested with papain in order to generate a Fab fragment, derivatized with NHS-HYNIC-Tfa and radiolabel with technetium-99m (99mTc) as a potential agent of molecular imaging of cancer. Both, whole and fragment radiolabels were in-vivo and in-vitro characterized. Radiolabeling conditions with Tricine as coligand and quality controls were assessed to confirm the integrity of the labeled fragment. Biodistribution and imaging studies in normal and spontaneous adenocarcinoma mice were performed at different times to determine the in-vivo characteristics of the radiolabel fragment. Tumor localization was visualized by conventional gamma camera imaging studies, and the results were compared with the whole antibody. Also, an immunoreactivity assay was carried out for both. The results showed clearly the integrity of the nimotuzumab fragment and the affinity by the receptor was verified. Fab(nimotuzumab)-HYNIC was obtained with high purity and a simple strategy of radiolabeling was performed. Finally, a fast blood clearance was observed in the biodistribution studies increasing the tumor uptake of Fab(nimotuzumab)- HYNIC-99mTc over time, with tumor/muscle ratios of 3.81 ± 0.50, 5.16 ± 1.97 and 6.32 ± 1.98 at 1 h, 4 h and 24 h post injection. Urinary excretion resulted in 32.89 ± 3.91 %ID eliminated at 24 h. Scintigraphy images showed uptake in the tumor and the activity in non-target organs was consistent with the biodistribution data at the same time points. Hence, these preliminary results showed important further characteristic of Fab(nimotuzumab)-HYNIC-99mTc as a molecular imaging agent of cancer.

Pattern of myocardial 99mTc-HMDP uptake and impact on myocardial function in patients with transthyretin cardiac amyloidosis.

PubMed

Pradel, Sarah; Brun, Stéphanie; Victor, Gérard; Pascal, Pierre; Fournier, Pauline; Ribes, David; Lavie-Badie, Yoan; Galinier, Michel; Carrié, Didier; Berry, Isabelle; Lairez, Olivier

2018-06-07

The purpose of the study was to describe the pattern of 99m Tc-labeled phosphate agents myocardial uptake by scintigraphy and explore its impact on left ventricular (LV) functions in transthyretin cardiac amyloidosis (TTR-CA). Fifty patients with TTR-CA underwent 99m Tc- hydroxymethylene-diphosphonate ( 99m Tc-HMDP) scintigraphy and echocardiography with measure of LV thickness, longitudinal strain (LS), systolic and diastolic functions. Cardiac retention by scintigraphy was assessed by visual scoring and the heart/whole body (H/WB) ratio was calculated by dividing counts in the heart by counts in late whole-body images. The mean population age was 79 ± 10 years. Mean H/WB ratio was 12 ± 7. Myocardial 99m Tc-HMDP uptake on segments 5, 6, 7, 8, 11, 12, 13, 14, 16, and 17 was correlated with H/WB ratio. Mean LVEF and global LS were 51 ± 10% and - 10 ± 3%, respectively. H/WB ratio was correlated with global LS (R = 0.408, P = .003), Ea (R = - 0.566, P < .001) and mean left ventricular wall thickness (R = 0.476, P < .001) but not with LVEF (R = - 0.109, P = .453). Segmental myocardial uptake was slightly correlated with segmental LS (R = 0.152, P < .001). H/WB ratio was not correlated with NT-proBNP levels (R = 0.219, P = .148) neither E/Ea ratio (R = 0.204, P = .184). These findings show the relationship between bone tracer myocardial uptake and LV functions in patients with TTR cardiac amyloidosis.

Comparison of F(ab')2 versus Fab antivenom for pit viper envenomation: A prospective, blinded, multicenter, randomized clinical trial

PubMed Central

Ruha, Anne-Michelle; Seifert, Steven A.; Morgan, David L.; Lewis, Brandon J.; Arnold, Thomas C.; Clark, Richard F.; Meggs, William J.; Toschlog, Eric A.; Borron, Stephen W.; Figge, Gary R.; Sollee, Dawn R.; Shirazi, Farshad M.; Wolk, Robert; de Chazal, Ives; Quan, Dan; García-Ubbelohde, Walter; Alagón, Alejandro; Gerkin, Richard D.; Boyer, Leslie V.

2015-01-01

Background. Crotalidae Polyvalent Immune Fab (Ovine) has been the only antivenom commercially available in the US since 2007 for treatment of Crotalinae envenomation. Late coagulopathy can occur or recur after clearance of Fab antivenom, often after hospital discharge, lasting in some cases more than 2 weeks. There have been serious, even fatal, bleeding complications associated with recurrence phenomena. Frequent follow-up is required, and additional intervention or hospitalization is often necessary. F(ab')2 immunoglobulin derivatives have longer plasma half life than do Fab. We hypothesized that F(ab')2 antivenom would be superior to Fab in the prevention of late coagulopathy following treatment of patients with Crotalinae envenomation. Methods. We conducted a prospective, double-blind, randomized clinical trial, comparing late coagulopathy in snakebitten patients treated with F(ab')2 with maintenance doses [F(ab')2/F(ab')2], or F(ab')2 with placebo maintenance doses [F(ab')2/placebo], versus Fab with maintenance doses [Fab/Fab]. The primary efficacy endpoint was coagulopathy (platelet count < 150 K/mm3, fibrinogen level < 150 mg/dL) between end of maintenance dosing and day 8. Results. 121 patients were randomized at 18 clinical sites and received at least one dose of study drug. 114 completed the study. Of these, 11/37 (29.7%) in the Fab/Fab cohort experienced late coagulopathy versus 4/39 (10.3%, p < 0.05) in the F(ab')2/F(ab')2 cohort and 2/38 (5.3%, p < 0.05) in the F(ab')2/placebo cohort. The lowest heterologous protein exposure was with F(ab')2/placebo. No serious adverse events were related to study drug. In each study arm, one patient experienced an acute serum reaction and one experienced serum sickness. Conclusions. In this study, management of coagulopathic Crotalinae envenomation with longer-half-life F(ab')2 antivenom, with or without maintenance dosing, reduced the risk of subacute coagulopathy and bleeding following treatment of envenomation

Comparison of F(ab')2 versus Fab antivenom for pit viper envenomation: a prospective, blinded, multicenter, randomized clinical trial.

PubMed

Bush, Sean P; Ruha, Anne-Michelle; Seifert, Steven A; Morgan, David L; Lewis, Brandon J; Arnold, Thomas C; Clark, Richard F; Meggs, William J; Toschlog, Eric A; Borron, Stephen W; Figge, Gary R; Sollee, Dawn R; Shirazi, Farshad M; Wolk, Robert; de Chazal, Ives; Quan, Dan; García-Ubbelohde, Walter; Alagón, Alejandro; Gerkin, Richard D; Boyer, Leslie V

2015-01-01

Crotalidae Polyvalent Immune Fab (Ovine) has been the only antivenom commercially available in the US since 2007 for treatment of Crotalinae envenomation. Late coagulopathy can occur or recur after clearance of Fab antivenom, often after hospital discharge, lasting in some cases more than 2 weeks. There have been serious, even fatal, bleeding complications associated with recurrence phenomena. Frequent follow-up is required, and additional intervention or hospitalization is often necessary. F(ab')2 immunoglobulin derivatives have longer plasma half life than do Fab. We hypothesized that F(ab')2 antivenom would be superior to Fab in the prevention of late coagulopathy following treatment of patients with Crotalinae envenomation. We conducted a prospective, double-blind, randomized clinical trial, comparing late coagulopathy in snakebitten patients treated with F(ab')2 with maintenance doses [F(ab')2/F(ab')2], or F(ab')2 with placebo maintenance doses [F(ab')2/placebo], versus Fab with maintenance doses [Fab/Fab]. The primary efficacy endpoint was coagulopathy (platelet count < 150 K/mm(3), fibrinogen level < 150 mg/dL) between end of maintenance dosing and day 8. 121 patients were randomized at 18 clinical sites and received at least one dose of study drug. 114 completed the study. Of these, 11/37 (29.7%) in the Fab/Fab cohort experienced late coagulopathy versus 4/39 (10.3%, p < 0.05) in the F(ab')2/F(ab')2 cohort and 2/38 (5.3%, p < 0.05) in the F(ab')2/placebo cohort. The lowest heterologous protein exposure was with F(ab')2/placebo. No serious adverse events were related to study drug. In each study arm, one patient experienced an acute serum reaction and one experienced serum sickness. In this study, management of coagulopathic Crotalinae envenomation with longer-half-life F(ab')2 antivenom, with or without maintenance dosing, reduced the risk of subacute coagulopathy and bleeding following treatment of envenomation.

Diagnostic accuracy and functional parameters of myocardial perfusion scintigraphy using accelerated cardiac acquisition with IQ SPECT technique in comparison to conventional imaging.

PubMed

Pirich, Christian; Keinrath, Peter; Barth, Gabriele; Rendl, Gundula; Rettenbacher, Lukas; Rodrigues, Margarida

2017-03-01

IQ SPECT consists of a new pinhole-like collimator, cardio-centric acquisition, and advanced 3D iterative SPECT reconstruction. The aim of this paper was to compare diagnostic accuracy and functional parameters obtained with IQ SPECT versus conventional SPECT in patients undergoing myocardial perfusion scintigraphy with adenosine stress and at rest. Eight patients with known or suspected coronary artery disease underwent [99mTc] tetrofosmin gated SPECT. Acquisition was performed on a Symbia T6 equipped with IQ SPECT and on a conventional gamma camera system. Gated SPECT data were used to calculate functional parameters. Scores analysis was performed on a 17-segment model. Coronary angiography and clinical follow-up were considered as diagnostic reference standard. Mean acquisition time was 4 minutes with IQ SPECT and 21 minutes with conventional SPECT. Agreement degree on the diagnostic accuracy between both systems was 0.97 for stress studies, 0.91 for rest studies and 0.96 for both studies. Perfusion abnormalities scores obtained by using IQ SPECT and conventional SPECT were not significant different: SSS, 9.7±8.8 and 10.1±6.4; SRS, 7.1±6.1 and 7.5±7.3; SDS, 4.0±6.1 and 3.9±4.3, respectively. However, a significant difference was found in functional parameters derived from IQ SPECT and conventional SPECT both after stress and at rest. Mean LVEF was 8% lower using IQ SPECT. Differences in LVEF were found in patients with normal LVEF and patients with reduced LVEF. Functional parameters using accelerated cardiac acquisition with IQ SPECT are significantly different to those obtained with conventional SPECT, while agreement for clinical interpretation of myocardial perfusion scintigraphy with both techniques is high.

Prognostic impact of location and extent of vessel-related ischemia at myocardial perfusion scintigraphy in patients with or at risk for coronary artery disease.

PubMed

Nudi, Francesco; Schillaci, Orazio; Neri, Giandomenico; Pinto, Annamaria; Procaccini, Enrica; Vetere, Maurizio; Frati, Giacomo; Tomai, Fabrizio; Biondi-Zoccai, Giuseppe

2016-04-01

Myocardial perfusion scintigraphy (MPS) has an established diagnostic and prognostic role in patients with or at risk for coronary artery disease, with ischemia severity and extent having already been identified as key predictors. Whether this is affected by the location of myocardial ischemia is uncertain. We aimed at comparing the prognostic outlook of patients undergoing MPS according to the site of ischemia. Our institutional database was queried for subjects undergoing MPS, without myocardial necrosis or recent revascularization. We focused on the prognostic impact of location of vessel-related ischemia (VRI) at MPS, distinguishing four mutually exclusive groups: single-VRI involving left anterior descending (LAD), single-VRI not involving LAD, multi-VRI involving LAD, and multi-VRI not involving LAD. The primary outcome was the long-term (>1 year) rate of death or myocardial infarction (D/MI). A total of 13,254 patients were included. Moderate or severe VRI occurred in 2,627 (20%) patients. Clinical outcomes were significantly different among the groups of patients with moderate or severe VRI, including death, cardiac death, non-fatal myocardial infarction or their composites (overall P < .001). Specifically, and excluding subjects undergoing revascularization as first follow-up event, D/MI occurred in 8.4% of patients with single-VRI involving LAD, 5.5% of subjects with single-VRI not involving LAD, 16.5% of those with multi-VRI involving LAD, and 7.3% of patients with multi-VRI not involving LAD (overall P < .001). Even at incremental multivariable Cox proportional analysis, hierarchical VRI was independently associated with an increased risk of D/MI [hazard ratio = 1.17 (1.04-1.08) for each class increment, P = .010]. Location and extent of myocardial ischemia at MPS according to the VRI concept have a hierarchical predictive impact, with multi-VRI involving LAD being significantly and independently more prognostically ominous than other types of VRI.

Continuous IV Crotalidae Polyvalent Immune Fab (Ovine) (FabAV) for selected North American rattlesnake bite patients.

PubMed

Bush, Sean P; Seifert, Steven A; Oakes, Jennifer; Smith, Susan D; Phan, Tammy H; Pearl, Sarah R; Reibling, Ellen T

2013-07-01

In patients bitten by North American rattlesnakes and treated with Crotalidae Polyvalent Immune Fab (Ovine) (FabAV), late hematologic abnormalities-persistent, recurrent, or late, new onset of hypofibrinogenemia, prolonged PT/INR, prolonged PTT, and/or thrombocytopenia beyond 48 h post-envenomation-are common, difficult to manage, and may result in morbidity and mortality are common, difficult to manage, and may result in morbidity and mortality. The optimal management of late hematologic abnormalities, particularly the use of further treatment with antivenom, has not been well defined. The current FabAV treatment regimen is to give antivenom as a bolus dose over a one-hour period. We describe our experience using a continuous intravenous infusion of FabAV for late hematologic effects and/or associated bleeding complications in rattlesnake envenomation. This is a retrospective, observational case series of patients envenomated by North American rattlesnakes at three medical centers managed with a continuous intravenous infusion of FabAV for late hematologic abnormalities and/or associated bleeding complications. Indications, dilution and infusion protocols, and duration of therapy were individualized. Five cases were identified between July 2010 and September 2011. All patients had profound late hematologic abnormalities and/or were associated with bleeding complications. Several patients had received repeat bolus infusions of FabAV, with or without human blood products, with either inadequate or only transient beneficial response. All patients were then managed with a continuous intravenous infusion of FabAV and all appeared to respond to the continuous intravenous infusion of FabAV, titrated to effect, with cessation of progression and, in most cases, improvement in hematologic abnormalities. Rates of infusion varied from 2 to 4 vials per 24 h (mean = 3.1 ± 0.4 vials/day). The termination of FabAV infusion was between day 6 and day 14 from the time of

Tumour detection and localization using 99Tcm-labelled OV-TL 3 Fab' in patients suspected of ovarian cancer.

PubMed

Tibben, J G; Massuger, L F; Claessens, R A; Schijf, C P; Pak, K Y; Strijk, S P; Kenemans, P; Corstens, F H

1992-12-01

Fab' fragments of the monoclonal antibody OV-TL 3, that recognizes an ovarian carcinoma-associated antigen (OA3), were labelled with 99Tcm using D-glucarate as a ligand. Twenty patients suspected of having primary or recurrent ovarian cancer received intravenously 1 mg of the Fab' labelled with 740 MBq 99Tcm. Both planar and single photon emission computed tomographic (SPECT) scintigraphy were performed up to 30 h after intravenous infusion. In 19 out of 20 patients surgical and histopathological evaluation was performed between 2 and 6 days postinfusion. Imaging results were compared with X-ray computed tomography (CT), ultrasonography (US) and CA 125 serum level. Blood clearance was fast with median t1/2 beta of 9.5 h. Thirty-seven per cent of the injected dose (% ID) was excreted in the urine within the first 24 h, whereas 7% ID was excreted in the 24 h faeces. In one patient with an OA3 negative ovarian carcinoma, radioimmunoscintigraphy (RIS) did not visualize the tumour. In two other patients a benign ovarian cyst was found, also showing no elevated uptake. In 13 out of 17 patients ovarian cancer lesions were detected with RIS, whereas CT and US detected lesions in, respectively, 15 and 12 patients. Of 36 surgically defined tumour deposits larger than 1 cm in diameter, 53% were detected and localized with RIS, whereas CT and US detected 61 and 40%, respectively. Radioimmunoscintigraphy with 99Tcm-OV-TL 3 Fab' is less distressing for the patients but the overall imaging performance is not improved when compared with 111In-OV-TL 3 F(ab')2.

Sensitivity of technetium-99m-pyrophosphate scintigraphy in diagnosing cardiac amyloidosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Falk, R.H.; Lee, V.W.; Rubinow, A.

1983-03-01

To determine the value of technetium-99m-pyrophosphate myocardial scintigraphy in the diagnosis of amyloid heart disease this procedure was prospectively performed in 20 consecutive patients with biopsy-proven primary amyloidosis. Eleven patients had echocardiographic abnormalities compatible with amyloid cardiomyopathy, 9 of whom had congestive heart failure. Diffuse myocardial pyrophosphate uptake was of equal or greater intensity than that of the ribs in 9 of the 11 patients with echocardiograms suggestive of amyloidosis, but in only 2 of the 9 with normal echocardiograms, despite abnormal electrocardiograms (p less than 0.01). Increased wall thickness measured by M-mode echocardiography correlated with myocardial pyrophosphate uptake (rmore » . 0.68, p less than 0.01). None of 10 control patients with nonamyloid, nonischemic heart disease had a strongly positive myocardial pyrophosphate uptake. Thus, myocardial technetium-99m-pyrophosphate scanning is a sensitive and specific test for the diagnosis of cardiac amyloidosis in patients with congestive heart failure of obscure origin. It does not appear to be of value for the early detection of cardiac involvement in patients with known primary amyloidosis without echocardiographic abnormalities.« less

PSMA-targeted bispecific Fab conjugates that engage T cells.

PubMed

Patterson, James T; Isaacson, Jason; Kerwin, Lisa; Atassi, Ghazi; Duggal, Rohit; Bresson, Damien; Zhu, Tong; Zhou, Heyue; Fu, Yanwen; Kaufmann, Gunnar F

2017-12-15

Bioconjugate formats provide alternative strategies for antigen targeting with bispecific antibodies. Here, PSMA-targeted Fab conjugates were generated using different bispecific formats. Interchain disulfide bridging of an αCD3 Fab enabled installation of either the PSMA-targeting small molecule DUPA (SynFab) or the attachment of an αPSMA Fab (BisFab) by covalent linkage. Optimization of the reducing conditions was critical for selective interchain disulfide reduction and good bioconjugate yield. Activity of αPSMA/CD3 Fab conjugates was tested by in vitro cytotoxicity assays using prostate cancer cell lines. Both bispecific formats demonstrated excellent potency and antigen selectivity. Copyright © 2017. Published by Elsevier Ltd.

Normal results of post-race thallium-201 myocardial perfusion imaging in marathon runners with elevated serum MB creatine kinase levels

DOE Office of Scientific and Technical Information (OSTI.GOV)

Siegel, A.J.; Silverman, L.M.; Holman, B.L.

1985-10-01

Elevated cardiac enzyme values in asymptomatic marathon runners after competition can arise from skeletal muscle through exertional rhabdomyolysis, silent injury to the myocardium, or a combined tissue source. Peak post-race levels of the MB isoenzyme of creatine kinase are similar to values in patients with acute myocardial infarction. Previously reported normal results of infarct-avid myocardial scintigraphy with technetium 99m pyrophosphate in runners after competition suggest a non-cardiac source but cannot exclude silent injury to the myocardium. Therefore, thallium 201 myocardial perfusion imaging was performed in runners immediately after competition together with determination of sequential cardiac enzyme levels. Among 15 runnersmore » tested, the average peak in serum MB creatine kinase 24 hours after the race was 128 IU/liter with a cumulative MB creatine kinase release of 117 IU/liter; these values are comparable to those in patients with acute transmural myocardial infarction. Thallium 201 myocardial scintigraphic results were normal in five runners randomly selected from those who volunteered for determination of sequential blood levels. It is concluded that elevations of serum MB creatine kinase in marathon runners arise from a skeletal muscle source and that thallium 201 myocardial scintigraphy is useful to assess runners for myocardial injury when clinical questions arise.« less

Baculovirus display of functional antibody Fab fragments.

PubMed

Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

2015-08-01

The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

Reversibility by dipyridamole of thallium-201 myocardial scan defects in patients with sarcoidosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tellier, P.; Paycha, F.; Antony, I.

1988-08-01

In order to clarify the significance of anginal pain and myocardial thallium-201 scan defects in cardiac sarcoidosis, the pharmacologic effect of dipyridamole on myocardial perfusion was assessed by planar thallium-201 myocardial scintigraphy in patients with sarcoidosis. Thallium-201 myocardial scintigraphy was performed at rest and after 0.56 mg/kg intravenous dipyridamole during four minutes in 16 patients with sarcoidosis. The myocardial scan (45-degree and 70-degree left anterior oblique, and anterior views) was divided into 15 segments. Results were evaluated by the number of segmental defects and with a global perfusion score (from 0 to 60) by a semi-quantitative index depending on themore » size and severity of myocardial thallium-201 defects. Thirteen of the 16 patients showed partial or total reversion of their thallium-201 defects on redistribution scanning either at rest or after dipyridamole. The mean (+/- SD) number of myocardial perfusion defects that were present in all the patients decreased from 5.31 +/- 1.78 at rest to 3.25 +/- 2.52 after redistribution (p less than 0.001) and to 2.19 +/- 2.10 after dipyridamole (p less than 0.001). The mean global perfusion score increased from 53.2 +/- 3.0 at rest to 56.2 +/- 2.9 after redistribution (p less than 0.001) and to 57.2 +/- 2.7 after dipyridamole (p less than 0.001). A significant correlation (r = 0.82, p less than 0.001) was found between the increase of global perfusion score on redistribution and after dipyridamole. The reversibility of myocardial scan defects is a common finding in sarcoidosis. It makes unlikely the role of scar fibrosis or extensive confluent granulomas as a mechanism for such defects. The effect of dipyridamole suggests the presence of reversible disorders lying at the coronary microvascular level.« less

Controlled Fab installation onto polymeric micelle nanoparticles for tuned bioactivity

NASA Astrophysics Data System (ADS)

Chen, Shaoyi; Florinas, Stelios; Teitgen, Abigail; Xu, Ze-Qi; Gao, Changshou; Wu, Herren; Kataoka, Kazunori; Cabral, Horacio; Christie, R. James

2017-12-01

Antibodies and antigen-binding fragments (Fabs) can be used to modify the surface of nanoparticles for enhanced target binding. In our previous work, site-specific conjugation of Fabs to polymeric micelles using conventional methods was limited to approximately 30% efficiency, possibly due to steric hindrance related to macromolecular reactants. Here, we report a new method that enables conjugation of Fabs onto a micelle surface in a controlled manner with up to quantitative conversion of nanoparticle reactive groups. Variation of (i) PEG spacer length in a heterofunctionalized cross-linker and (ii) Fab/polymer feed ratios resulted in production of nanoparticles with a range of Fab densities on the surface up to the theoretical maximum value. The biological impact of variable Fab density was evaluated in vitro with respect to cell uptake and cytotoxicity of a drug-loaded (SN38) targeted polymeric micelle bearing anti-EphA2 Fabs. Fab conjugation increased cell uptake and potency compared with non-targeted micelles, although a Fab density of 60% resulted in decreased uptake and potency of the targeted micelles. Altogether, our findings demonstrate that conjugation strategies can be optimized to allow control of Fab density on the surface of nanoparticles and also that Fab density may need to be optimized for a given cell-surface target to achieve the highest bioactivity.

A single-domain antibody-linked Fab bispecific antibody Her2-S-Fab has potent cytotoxicity against Her2-expressing tumor cells.

PubMed

Li, Aifen; Xing, Jieyu; Li, Li; Zhou, Changhua; Dong, Bin; He, Ping; Li, Qing; Wang, Zhong

2016-12-01

Her2, which is frequently overexpressed in breast cancer, is one of the most studied tumor-associated antigens for cancer therapy. Anti-HER2 monoclonal antibody, trastuzumab, has achieved significant clinical benefits in metastatic breast cancer. In this study, we describe a novel bispecific antibody Her2-S-Fab targeting Her2 by linking a single domain anti-CD16 VHH to the trastuzumab Fab. The Her2-S-Fab antibody can be efficiently expressed and purified from Escherichia coli, and drive potent cancer cell killing in HER2-overexpressing cancer cells. In xenograft model, the Her2-S-Fab suppresses tumor growth in the presence of human immune cells. Our results suggest that the bispecific Her2-S-Fab may provide a valid alternative to Her2 positive cancer therapy.

FAB (Functionally Alert Behavior Strategies) to Improve Self-Control

ERIC Educational Resources Information Center

Pagano, John

2015-01-01

This paper describes the FAB (Functionally Alert Behavior) Strategies approach to improve behavior in children and adolescents with complex behavioral challenges. FAB Strategies include evidence-based environmental adaptations, sensory modulation, positive behavioral support, and physical self-regulation strategies. FAB Strategies can be used by…

Short-term outcomes after Fab antivenom therapy for severe crotaline snakebite.

PubMed

Lavonas, Eric J; Kokko, Jamie; Schaeffer, Tammi H; Mlynarchek, Sara L; Bogdan, Gregory M; Dart, Richard C

2011-02-01

We seek to determine the short-term outcomes associated with the use of Crotalidae polyvalent immune Fab (ovine) (CroFab; FabAV) therapy for severe crotaline snake envenomation and to better define the incidence of hypersensitivity reactions associated with FabAV use. We conducted a multicenter observational case series study of patients who received FabAV at 17 US hospitals in 2002 to 2004. A 7-point score incorporating local, systemic, and hematologic venom effects was used to grade envenomation severity before and after FabAV therapy. The primary outcome for response to therapy was the change in overall envenomation severity after FabAV administration. The primary safety outcomes were the rates of immediate hypersensitivity reactions and serum sickness. The outcome-evaluable population included 209 patients, of whom 28 had severe envenomation. All severely envenomated patients improved after receiving FabAV. The median severity scores of severely envenomated patients were 5 (interquartile range [IQR] 5 to 5) before FabAV, 1 (IQR 1 to 2) at the last FabAV loading dose, and 1 (IQR 0 to 1) at the last clinical observation. The proportion of patients with progressive pain, progressive swelling, cardiovascular effects, respiratory effects, neurologic effects, gastrointestinal effects, coagulopathy, and thrombocytopenia all improved after FabAV therapy. The safety population included 247 patients. Immediate hypersensitivity reactions were reported in 6.1% (95% confidence interval 3.4% to 9.8%) of patients. Serum sickness was reported in 5% (95% confidence interval 0.6% to 17%) of patients with a minimum of 6 days of follow-up after the last dose of FabAV. FabAV therapy is associated with clinical improvement in severe crotaline snake envenomation. Immediate hypersensitivity and serum sickness rates may be less than described in the FabAV prescribing information. Copyright © 2010. Published by Mosby, Inc.

Prevalence of ECG changes during adenosine stress and its association with perfusion defect on myocardial perfusion scintigraphy.

PubMed

Taywade, Sameer K; Ramaiah, Vijayaraghavan L; Basavaraja, Harish; Venkatasubramaniam, Parameswaran R; Selvakumar, Job

2017-04-01

Myocardial perfusion scintigraphy (MPS) is a valuable, noninvasive imaging modality in the evaluation of patients with coronary artery disease. Adenosine stress may occasionally be associated with ECG changes. This study evaluated the strength of association between adenosine stress-related ECG changes and perfusion defects on Tc-MPS. 117 (mean age: 61.25±9.27 years; sex: men 87, women 30) patients with known/suspected coronary artery disease underwent adenosine stress MPS. ECG was monitored continuously during adenosine stress for ST-depression. On the basis of the summed difference score, reversible perfusion defects were categorized as follows: normal: less than 4, mild: 4-8, moderate: 9-13, and severe: more than 13. ST-depression was observed in 27/117 (23.1%) and reversible perfusion defects were observed in 18/27 (66.66%) patients. 2/27, 6/27, and 10/27 patients had mild, moderate, and severe ischemia, respectively. 9/27 patients had normal perfusion. ECG changes and perfusion defects showed a moderate strength of association (correlation coefficient r=0.35, P=0.006). The sensitivity, specificity, positive predictive value, and negative predictive value of ECG findings for prediction of ischemia were 35.29, 86.36, 67.67, and 63.33%, respectively. ECG changes during adenosine stress are not uncommon. It shows a moderate strength of association with reversible perfusion defects. ECG changes during adenosine merit critical evaluation of MPS findings.

Prognostic significance of normal quantitative planar thallium-201 stress scintigraphy in patients with chest pain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wackers, F.J.; Russo, D.J.; Russo, D.

The prognostic significance of normal quantitative planar thallium-201 stress scintigraphy was evaluated in patients with a chest pain syndrome. The prevalence of cardiac events during follow-up was related to the pretest (that is, before stress scintigraphy) likelihood of coronary artery disease determined on the basis of symptoms, age, sex and stress electrocardiography. In a consecutive series of 344 patients who had adequate thallium-201 stress scintigrams, 95 had unequivocally normal studies by quantitative analysis. The pretest likelihood of coronary artery disease in the 95 patients had a bimodal distribution. During a mean follow-up period of 22 +/- 3 months, no patientmore » died. Three patients (3%) had a cardiac event: two of these patients (pretest likelihood of coronary artery disease 54 and 94%) had a nonfatal myocardial infarction 8 and 22 months, respectively, after stress scintigraphy, and one patient (pretest likelihood 98%) underwent percutaneous transluminal coronary angioplasty 16 months after stress scintigraphy for persisting anginal complaints. Three patients were lost to follow-up; all three had a low pretest likelihood of coronary artery disease. It is concluded that patients with chest pain and normal findings on quantitative thallium-201 scintigraphy have an excellent prognosis. Cardiac events are rare (infarction rate 1% per year) and occur in patients with a moderate to high pretest likelihood of coronary artery disease.« less

Immobilization and functional reconstitution of antibody Fab fragment by solid-phase refolding.

PubMed

Kumada, Yoichi; Hamasaki, Kyoto; Nakagawa, Aya; Sasaki, Eiju; Shirai, Tatsunori; Okumura, Masahiro; Inoue, Manami; Kishimoto, Michimasa

2013-12-31

In this study, we demonstrated the successful preparation of a Fab antibody-immobilized hydrophilic polystyrene (phi-PS) plate via one- and two-step solid-phase refolding methods. Both polystyrene-binding peptide (PS-tag)-fused Fd fragment of heavy chain (Fab H-PS) and full-length of light-chain (Fab L-PS) were individually produced in insoluble fractions of Escherichia coli cells, and they were highly purified in the presence of 8M of urea. Antigen-binding activities of Fab antibody immobilized were correctly recovered by the one-step solid-phase refolding method that a mixture of Fab H-PS and Fab L-PS was immobilized in the presence of 0.5-2M urea, followed by surface washing of the phi-PS plate with PBST. These results indicate that by genetic fusion of a PS-tag, a complex between Fab H and Fab L was efficiently immobilized on the surface of a phi-PS plate even in the presence of a low concentration of urea, and was then correctly refolded to retain its high antigen-binding activity via removal of the urea. A two-step solid-phase refolding method whereby Fab H-PS and Fab L-PS were successively refolded on the surface of a phi-PS plate also resulted in Fab antibody formation on the plate. Furthermore, both the binding affinity and the specificity of the Fab antibody produced by the two-step method were highly maintained, according to the results of sandwich ELISA and competitive ELISA using Fab antibody-immobilized plate via two-step solid-phase refolding. Thus, the solid-phase refolding method demonstrated in this study should be quite useful for the preparation of a Fab antibody-immobilized PS surface with high efficiency from individually produced Fab H-PS and Fab L-PS. This method will be applicable to the preparation of a large Fab antibody library on the surface of a PS plate for use in antibody screening. © 2013. Published by Elsevier B.V. All rights reserved.

Acute hypersensitivity reaction to Crotalidae polyvalent immune Fab (CroFab) as initial presentation of galactose-α-1,3-galactose (α-gal) allergy.

PubMed

Rizer, Justin; Brill, Kaitlin; Charlton, Nathan; King, Joshua

2017-08-01

Crotalidae polyvalent immune Fab antivenom (CroFab), commonly used for the treatment of clinically significant North American crotalinae envenomation, is generally well-tolerated. A novel form of anaphylaxis due to an IgE antibody response to the mammalian oligosaccharide galactose-α-1,3-galactose (α-gal) has been established following red-meat consumption as well as IV administration of cetuximab, which contain the α-gal epitope. We present a case of α-gal allergy discovered after acute hypersensitivity reaction to FabAV. A 61-year-old healthy female was bitten on her left ankle by Agkistrodon contortrix. Given the patient's rapid progression of pain and swelling, she was given FabAV. During infusion of FabAV, she developed diffuse hives over her entire body and itching, but denied respiratory or gastrointestinal symptoms and her vital signs remained stable. The FabAV was immediately discontinued and she received intravenous diphenhydramine and famotidine with gradual resolution of symptoms. On further discussion, she denied a history of α-gal or papaya allergy but rarely ate red meat and endorsed sustaining frequent tick bites. Subsequent antibody testing was significant for an α-1,3-galactose IgE concentration of 45,000 U/L (normal <3500 U/L), confirming α-gal allergy. To our knowledge, this is the first report of FabAV hypersensitivity associated with an underlying α-gal allergy.

Humoral anti-OV-TL 3 response after the intravenous administration of radiolabelled Fab' or F(ab')2 fragments in ovarian cancer patients.

PubMed

Tibben, J G; Thomas, C M; Massuger, L F; Segers, M F; Schijf, C P; Corstens, F H; Boerman, O C

1995-10-01

The human anti-mouse antibody (HAMA) response was determined in the serum of patients suspected of having ovarian cancer who underwent radioimmunoscintigraphy with either 99Tcm-OV-TL 3 Fab' (n = 20) or 111In-DTPA-OV-TL 3 F(ab')2 (n = 73). Blood samples were collected prior to and at several time points post-intravenous injection. The detection of HAMA was performed with an in-house OV-TL 3 F(ab')2-based sandwich-type immunoradiometric assay (IRMA). The homologous IRMA demonstrated that 8 of 20 (40%) patients had developed HAMA responses after injection of Fab' fragments and that 14 of 73 (19%) patients had developed HAMA responses after F(ab')2 administration. The subclass of the measured HAMA was analysed in a limited number of samples, showing IgG or IgM as well as mixed responses. The kinetics of the HAMA responses varied greatly. Our study showed the relevance of the sampling time and frequency: HAMA responses can be easily underestimated with a low sampling frequency. The homologous IRMA described in this study was able to quantify the OV-TL 3-specific HAMA responses. With additional assays, the subclass of the HAMA could be further analysed. Remarkably, the fraction of HAMA responders after injection of OV-TL 3 Fab' fragments was in the same range as the proportion of HAMA responders after F(ab')2 administration.

Assessment of Digoxin-Specific Fab Fragment Dosages in Digoxin Poisoning.

PubMed

Nordt, Sean Patrick; Clark, Richard F; Machado, Carol; Cantrell, F Lee

2016-01-01

Digoxin poisoning still remains a common cause of morbidity and mortality. Fortunately, digoxin-specific Fab fragments are commercially available as an antidote. However, these Fab fragments are several thousand dollars per vial. There is a standardized formula to calculate appropriate Fab fragment dosage based on the serum digoxin concentration. This can greatly reduce the amount of Fab fragment administered. There is also an empiric dosing guideline recommending 6-10 vials be given; however, this may result in higher amounts of Fab fragments being administered than required. We performed this study to assess the amounts of digoxin-specific Fab fragments administered in the treatment of digoxin poisonings recorded in a poison control system database from January 1, 2000, to December 31, 2009, in which digoxin serum concentrations were available. This was a retrospective study of 278 patients, 107 with acute poisonings (group A) and 171 following chronic poisoning (group B). In group A, the calculated Fab dose was higher than the calculated dose based on available concentrations in 39 (36%) of group A and 15 (9%) of group B patients. The average wholesale price cost of the excessive dosages ranged from $4818 to as high as $50,589 per patient. Our data suggests that clinician education on digoxin poisoning and the use of the standardized formula to calculate the Fab dose may decrease over utilization and decrease costs associated with the administration of digoxin-specific Fab fragments in the treatment of digoxin poisonings.

Single-reagent one-step procedures for the purification of ovine IgG, F(ab')2 and Fab antivenoms by caprylic acid.

PubMed

Al-Abdulla, Ibrahim; Casewell, Nicholas R; Landon, John

2014-01-15

Antivenoms are typically produced in horses or sheep and often purified using salt precipitation of immunoglobulins or F(ab')2 fragments. Caprylic (octanoic) acid fractionation of antiserum has the advantage of not precipitating the desired antibodies, thereby avoiding potential degradation that can lead to the formation of aggregates, which may be the cause of some adverse reactions to antivenoms. Here we report that when optimising the purification of immunoglobulins from ovine antiserum raised against snake venom, caprylic acid was found to have no effect on the activity of the enzymes pepsin and papain, which are employed in antivenom manufacturing to digest immunoglobulins to obtain F(ab')2 and Fab fragments, respectively. A "single-reagent" method was developed for the production of F(ab')2 antivenom whereby whole ovine antiserum was mixed with both caprylic acid and pepsin and incubated for 4h at 37°C. For ovine Fab antivenom production from whole antiserum, the "single reagent" comprised of caprylic acid, papain and l-cysteine; after incubation at 37°C for 18-20h, iodoacetamide was added to stop the reaction. Caprylic acid facilitated the precipitation of albumin, resulting in a reduced protein load presented to the digestion enzymes, culminating in substantial reductions in processing time. The ovine IgG, F(ab')2 and Fab products obtained using these novel caprylic acid methods were comparable in terms of yield, purity and specific activity to those obtained by multi-step conventional salt fractionation with sodium sulphate. Copyright © 2013 Elsevier B.V. All rights reserved.

Efficient production of Trastuzumab Fab antibody fragments in Brevibacillus choshinensis expression system.

PubMed

Mizukami, Makoto; Onishi, Hiromasa; Hanagata, Hiroshi; Miyauchi, Akira; Ito, Yuji; Tokunaga, Hiroko; Ishibashi, Matsujiro; Arakawa, Tsutomu; Tokunaga, Masao

2018-10-01

The Brevibacillus expression system has been successfully employed for the efficient productions of a variety of recombinant proteins, including enzymes, cytokines, antigens and antibody fragments. Here, we succeeded in secretory expression of Trastuzumab Fab antibody fragments using B. choshinensis/BIC (Brevibacillus in vivocloning) expression system. In the fed-batch high-density cell culture, recombinant Trastuzumab Fab with amino-terminal His-tag (His-BcFab) was secreted at high level, 1.25 g/liter, and Fab without His-tag (BcFab) at ∼145 mg/L of culture supernatant. His-BcFab and BcFab were purified to homogeneity using combination of conventional column chromatographies with a yield of 10-13%. This BcFab preparation exhibited native structure and functions evaluated by enzyme-linked immunosorbent assay, surface plasmon resonance, circular dichroism measurements and size exclusion chromatography. To our knowledge, this is the highest production of Fab antibody fragments in gram-positive bacterial expression/secretion systems. Copyright © 2018 Elsevier Inc. All rights reserved.

Oligovalent Fab display on M13 phage improved by directed evolution.

PubMed

Huovinen, Tuomas; Sanmark, Hanna; Ylä-Pelto, Jani; Vehniäinen, Markus; Lamminmäki, Urpo

2010-03-01

Efficient display of antibody on filamentous phage M13 coat is crucial for successful biopanning selections. We applied a directed evolution strategy to improve the oligovalent display of a poorly behaving Fab fragment fused to phage gene-3 for minor coat protein (g3p). The Fab displaying clones were enriched from a randomly mutated Fab gene library with polyclonal anti-mouse IgG antibodies. Contribution of each mutation to the improved phenotype of one selected mutant was studied. It was found out that two point mutations had significant contribution to the display efficiency of Fab clones superinfected with hyperphage. The most dramatic effect was connected to a start codon mutation, from AUG to GUG, of the PelB signal sequence preceding the heavy chain. The clone carrying this mutation, FabM(GUG), displayed Fab 19-fold better and yielded twofold higher phage titers than the original Fab.

Fab MOR03268 triggers absorption shift of a diagnostic dye via packaging in a solvent-shielded Fab dimer interface.

PubMed

Hillig, Roman C; Urlinger, Stefanie; Fanghänel, Jörg; Brocks, Bodo; Haenel, Cornelia; Stark, Yvonne; Sülzle, Detlev; Svergun, Dmitri I; Baesler, Siegfried; Malawski, Guido; Moosmayer, Dieter; Menrad, Andreas; Schirner, Michael; Licha, Kai

2008-03-14

Molecular interactions between near-IR fluorescent probes and specific antibodies may be exploited to generate novel smart probes for diagnostic imaging. Using a new phage display technology, we developed such antibody Fab fragments with subnanomolar binding affinity for tetrasulfocyanine, a near-IR in vivo imaging agent. Unexpectedly, some Fabs induced redshifts of the dye absorption peak of up to 44 nm. This is the largest shift reported for a biological system so far. Crystal structure determination and absorption spectroscopy in the crystal in combination with microcalorimetry and small-angle X-ray scattering in solution revealed that the redshift is triggered by formation of a Fab dimer, with tetrasulfocyanine being buried in a fully closed protein cavity within the dimer interface. The derived principle of shifting the absorption peak of a symmetric dye via packaging within a Fab dimer interface may be transferred to other diagnostic fluorophores, opening the way towards smart imaging probes that change their wavelength upon interaction with an antibody.

Structure determination of an Fab fragment that neutralizes human rhinovirus 14 and analysis of the Fab-virus complex.

PubMed

Liu, H; Smith, T J; Lee, W M; Mosser, A G; Rueckert, R R; Olson, N H; Cheng, R H; Baker, T S

1994-07-08

The crystal structure of Fab17-IA, an antigen-binding fragment from a murine immunoglobulin that neutralizes human rhinovirus 14 (HRV14), has been solved to 2.7 A resolution. Fab17-IA crystallized into three different space groups depending upon the method used to purify the intact antibody. The structure was determined by use of molecular and isomorphous replacement methods. The current model has a crystallographic R-factor of approximately 19% for 10,192 independent reflections between 8 and 2.7 A. Correlation coefficient calculations showed that the Fab17-IA structure can be fit into the Fab17-IA/HRV14 image reconstruction density to within 5 A positional accuracy and to within a few degrees of rotation. The resulting interface of the docked antibody was examined and showed extensive charge and shape complementarity with the virus surface that was supported by site-directed mutagenesis experiments. The success of this approach validates the utility of combining X-ray crystallography with cryo-electron microscopy of complex macromolecular assemblies.

A novel bispecific antibody, S-Fab, induces potent cancer cell killing.

PubMed

Li, Li; He, Ping; Zhou, Changhua; Jing, Li; Dong, Bin; Chen, Siqi; Zhang, Ning; Liu, Yawei; Miao, Ji; Wang, Zhong; Li, Qing

2015-01-01

Bispecific antibodies that engage immune cells to kill cancer cells have been actively studied in cancer immunotherapy. In this study, we present a novel bispecific format, S-Fab, fabricated by linking a single-domain anti-carcinoembryonic antigen VHH to a conventional anti-CD3 Fab. In contrast to most bispecific antibodies, the S-Fab bispecific antibody can be efficiently expressed and purified from bacteria. The purified S-Fab is stable in serum and is able to recruit T cells to drive potent cancer cell killing. In xenograft models, the S-Fab antibody suppresses tumor growth in the presence of human immune cells. Our study suggested that the bispecific S-Fab format can be applied to a wide range of immunotherapies.

Construction of an agglutination tool: recombinant Fab fragments biotinylated in vitro.

PubMed

Czerwinski, Marcin; Krop-Watorek, Anna; Wasniowska, Kazimiera; Smolarek, Dorota; Spitalnik, Steven L

2009-11-30

The pComb3H vector system is used for constructing and panning recombinant antibody libraries. It allows for expression of monovalent Fab fragments, either on the surface of M13 phage, or in the form of soluble proteins secreted into the periplasmic space of bacteria. We constructed a modified pComb3H vector containing cDNA encoding for a 23-amino acid fragment of the Escherichia coli biotin carboxy carrier protein (BCCP), which is an acceptor sequence for biotinylation. The vector was used to express the Fab fragment recognizing human glycophorin A. The purified Fab fragment containing this biotin acceptor sequence was effectively biotinylated in vitro using biotin ligase (BirA). The specificity and avidity of the biotinylated Fab fragments were similar to the previously produced, unmodified Fab fragments. An avidin-alkaline phosphatase conjugate was used to detect the recombinant Fab fragments, instead of secondary antibody. In addition, when biotinylated Fab fragments were mixed with avidin, red blood cells were directly agglutinated.

Evaluation of strategies to control Fab light chain dimer during mammalian expression and purification: A universal one-step process for purification of correctly assembled Fab.

PubMed

Spooner, Jennifer; Keen, Jenny; Nayyar, Kalpana; Birkett, Neil; Bond, Nicholas; Bannister, David; Tigue, Natalie; Higazi, Daniel; Kemp, Benjamin; Vaughan, Tristan; Kippen, Alistair; Buchanan, Andrew

2015-07-01

Fabs are an important class of antibody fragment as both research reagents and therapeutic agents. There are a plethora of methods described for their recombinant expression and purification. However, these do not address the issue of excessive light chain production that forms light chain dimers nor do they describe a universal purification strategy. Light chain dimer impurities and the absence of a universal Fab purification strategy present persistent challenges for biotechnology applications using Fabs, particularly around the need for bespoke purification strategies. This study describes methods to address light chain dimer formation during Fab expression and identifies a novel CH 1 affinity resin as a simple and efficient one-step purification for correctly assembled Fab. © 2015 Wiley Periodicals, Inc.

A Case Study of a High School Fab Lab

NASA Astrophysics Data System (ADS)

Lacy, Jennifer E.

This dissertation examines making and design-based STEM education in a formal makerspace. It focuses on how the design and implementation of a Fab Lab learning environment and curriculum affect how instructors and students see themselves engaging in science, and how the Fab Lab relates to the social sorting practices that already take place at North High School. While there is research examining design-based STEM education in informal and formal learning environments, we know little about how K-12 teachers define STEM in making activities when no university or museum partnership exists. This study sought to help fill this gap in the research literature. This case study of a formal makerspace followed instructors and students in one introductory Fab Lab course for one semester. Additional observations of an introductory woodworking course helped build the case and set it into the school context, and provided supplementary material to better understand the similarities and differences between the Fab Lab course and a more traditional design-based learning course. Using evidence from observational field notes, participant interviews, course materials, and student work, I found that the North Fab Lab relies on artifacts and rhetoric symbolic of science and STEM to set itself apart from other design-based courses at North High School. Secondly, the North Fab Lab instructors and students were unable to explain how what they were doing in the Fab Lab was science, and instead relied on vague and unsupported claims related to interdisciplinary STEM practices and dated descriptions of science. Lastly, the design and implementation of the Fab Lab learning environment and curriculum and its separation from North High School's low tech, design-based courses effectively reinforced social sorting practices and cultural assumptions about student work and intelligence.

Computational study of bindings of HK20 Fab and D5 Fab to HIV-1 gp41.

PubMed

Hartono, Yossa Dwi; Lazim, Raudah; Yip, Yew Mun; Zhang, Dawei

2012-02-15

Antibodies HK20 and D5 have been shown to target HIV-1 gp41, thereby inhibiting membrane fusion that facilitates viral entry. The binding picture is static, based on the X-ray crystal structures of the Fab regions and gp41 mimetic five-helix bundle. In this study, we carried out molecular dynamics simulation to provide the dynamic binding picture. Calculated binding free energies are within reasonable range of and follow the trend of the experimental values: -15.28 kcal/mol for HK20 Fab (expt. -11.60 kcal/mol) and -17.90 kcal/mol for D5 Fab (expt. -11.70 kcal/mol). Alanine scanning at protein-protein interface reveals that the highest contributors to binding for HK20 Fab are F54 and I56, both of V(H) region, as well as R30' of V(L) region; whereas for D5 Fab, F54 of V(H) region, as well as W32' and Y94' of V(L) region. HK20 F54 and I56, as well as D5 I52, F54, and T56, bind to the gp41 hydrophobic binding pocket, an important region targeted by many other fusion inhibitors. Hydrogen bonding analysis also identifies high-occupancy hydrogen bonds at the periphery of gp41 hydrophobic pocket. Considering that almost all interface residues are turn residues, further work may be directed to turn mimics. Pre-orientation by the hydrogen bonds to poise this particular turn towards the binding pocket may also be a point worth pursuing. Copyright © 2011 Elsevier Ltd. All rights reserved.

Iodine-123 metaiodobenzylguanidine scintigraphy and iodine-123 ioflupane single photon emission computed tomography in Lewy body diseases: complementary or alternative techniques?

PubMed

Treglia, Giorgio; Cason, Ernesto; Cortelli, Pietro; Gabellini, Anna; Liguori, Rocco; Bagnato, Antonio; Giordano, Alessandro; Fagioli, Giorgio

2014-01-01

To compare myocardial sympathetic imaging using (123)I-Metaiodobenzylguanidine (MIBG) scintigraphy and striatal dopaminergic imaging using (123)I-Ioflupane (FP-CIT) single photon emission computed tomography (SPECT) in patients with suspected Lewy body diseases (LBD). Ninety-nine patients who performed both methods within 2 months for differential diagnosis between Parkinson's disease (PD) and other parkinsonism (n = 68) or between dementia with Lewy bodies (DLB) and other dementia (n = 31) were enrolled. Sensitivity, specificity, accuracy, positive and negative predictive values of both methods were calculated. For (123) I-MIBG scintigraphy, the overall sensitivity, specificity, accuracy, positive and negative predictive values in LBD were 83%, 79%, 82%, 86%, and 76%, respectively. For (123)I-FP-CIT SPECT, the overall sensitivity, specificity, accuracy, positive and negative predictive values in LBD were 93%, 41%, 73%, 71%, and 80%, respectively. There was a statistically significant difference between these two methods in patients without LBD, but not in patients with LBD. LBD usually present both myocardial sympathetic and striatal dopaminergic impairments. (123)I-FP-CIT SPECT presents high sensitivity in the diagnosis of LBD; (123)I-MIBG scintigraphy may have a complementary role in differential diagnosis between PD and other parkinsonism. These scintigraphic methods showed similar diagnostic accuracy in differential diagnosis between DLB and other dementia. Copyright © 2012 by the American Society of Neuroimaging.

The Emerging Importance of IgG Fab Glycosylation in Immunity.

PubMed

van de Bovenkamp, Fleur S; Hafkenscheid, Lise; Rispens, Theo; Rombouts, Yoann

2016-02-15

Human IgG is the most abundant glycoprotein in serum and is crucial for protective immunity. In addition to conserved IgG Fc glycans, ∼15-25% of serum IgG contains glycans within the variable domains. These so-called "Fab glycans" are primarily highly processed complex-type biantennary N-glycans linked to N-glycosylation sites that emerge during somatic hypermutation. Specific patterns of Fab glycosylation are concurrent with physiological and pathological conditions, such as pregnancy and rheumatoid arthritis. With respect to function, Fab glycosylation can significantly affect stability, half-life, and binding characteristics of Abs and BCRs. Moreover, Fab glycans are associated with the anti-inflammatory activity of IVIgs. Consequently, IgG Fab glycosylation appears to be an important, yet poorly understood, process that modulates immunity. Copyright © 2016 by The American Association of Immunologists, Inc.

Crotalidae polyvalent immune Fab: in patients with North American crotaline envenomation.

PubMed

Keating, Gillian M

2011-04-01

Crotalidae polyvalent immune Fab is an antivenom comprising purified, sheep-derived, Fab IgG fragments and is indicated for use in patients with North American crotaline envenomation. Crotalidae polyvalent immune Fab is produced using four North American snake venoms: Crotalus atrox, Crotalus adamanteus, Crotalus scutulatus, and Agkistrodon piscivorus. Intravenous crotalidae polyvalent immune Fab was effective in patients aged ≥10 years who had minimal or moderate envenomation by a North American crotaline, who presented within 6 hours of the snakebite, and who had progression of the envenomation syndrome, according to the results of two prospective trials. One trial was a noncomparative, multicenter pilot study and the other trial was a randomized, open-label, multicenter trial in which patients received scheduled or 'as needed' administration of crotalidae polyvalent immune Fab after initial control had been achieved. A prospective, postmarketing trial demonstrated the efficacy of crotalidae polyvalent immune Fab in confirmed Crotalus viridis helleri envenomation (indicating cross-protection against a venom not used in its production). Results of these prospective trials are supported by the findings of additional (mainly retrospective) studies demonstrating the efficacy of crotalidae polyvalent immune Fab in patients with crotaline envenomation, including patients with severe envenomation, pediatric patients, and patients with symptoms of neurotoxicity. Despite treatment with crotalidae polyvalent immune Fab, patients may experience delayed-onset or recurrent venom effects (e.g. coagulopathy). Intravenous crotalidae polyvalent immune Fab was generally well tolerated; acute hypersensitivity reactions (e.g. urticaria, rash, pruritus) were the most commonly occurring adverse event. © 2011 Adis Data Information BV. All rights reserved.

Myocardial perfusion imaging study of CO(2)-induced panic attack.

PubMed

Soares-Filho, Gastão L F; Machado, Sergio; Arias-Carrión, Oscar; Santulli, Gaetano; Mesquita, Claudio T; Cosci, Fiammetta; Silva, Adriana C; Nardi, Antonio E

2014-01-15

Chest pain is often seen alongside with panic attacks. Moreover, panic disorder has been suggested as a risk factor for cardiovascular disease and even a trigger for acute coronary syndrome. Patients with coronary artery disease may have myocardial ischemia in response to mental stress, in which panic attack is a strong component, by an increase in coronary vasomotor tone or sympathetic hyperactivity setting off an increase in myocardial oxygen consumption. Indeed, coronary artery spasm was presumed to be present in cases of cardiac ischemia linked to panic disorder. These findings correlating panic disorder with coronary artery disease lead us to raise questions about the favorable prognosis of chest pain in panic attack. To investigate whether myocardial ischemia is the genesis of chest pain in panic attacks, we developed a myocardial perfusion study through research by myocardial scintigraphy in patients with panic attacks induced in the laboratory by inhalation of 35% carbon dioxide. In conclusion, from the data obtained, some hypotheses are discussed from the viewpoint of endothelial dysfunction and microvascular disease present in mental stress response. Copyright © 2014 Elsevier Inc. All rights reserved.

Crotalidae polyvalent immune Fab for the treatment of pediatric crotaline envenomation.

PubMed

Goto, Collin S; Feng, Sing-Yi

2009-04-01

Crotaline snakebites occur frequently in children, often resulting in significant morbidity. Crotalidae Polyvalent Immune Fab antivenom (FabAV) became available for clinical use in the US in 2000 and is currently the standard of care for the treatment of crotaline envenomation. The pediatric emergency care provider should be familiar with FabAV because its judicious use in affected children can greatly decrease morbidity caused by crotaline snakebites. This article will review the use of FabAV for the treatment of pediatric crotaline envenomation.

[Evaluation of myocardial uptake of beta-methyl-(123I)-iodophenylpentadecanoic acid (123I-BMIPP)].

PubMed

Momose, M; Kobayashi, H; Saito, K; Matsumoto, N; Maki, M; Hosoda, S; Kusakabe, K

1994-12-01

To evaluate the myocardial uptake of beta-methyl-(123I)-iodophenylpentadecanoic acid (123I-BMIPP), nineteen patients with ischemic heart disease including left ventricular hypertrophy (mean age 63 +/- 7.8, 14 males and 5 females) underwent BMIPP myocardial scintigraphy. Myocardial uptake (MU) of BMIPP to the total injected dose was calculated from anterior view of the planar image in all subjects, and was compared with plasma glucose (BS), triglyceride (TG), and free fatty acid (FFA). It was also compared with left ventricular mass (LVM) calculated with echocardiography. MU was not related to BS, TG, and FFA, however had the positive correlation with LVM (r = 0.676, p < 0.01). Myocardial uptake per left ventricular mass (MU/LVM) had the negative correlation with LVM (r = -0.671, p < 0.01). Further studies for the significance of MU/LVM will be required.

[Utility of early imaging of myocardial innervation scintigraphy in the diagnosis of Lewy Body Dementia].

PubMed

Camacho, V; Estorch, M; Marquié, M; Domènech, A; Flotats, A; Fernández, A; Duch, J; Geraldo, L L; Deportos, J; Artigas, C; Lleó, A; Carrió, I

2013-03-01

The importance of accurate and early diagnosis of dementia with Lewy bodies (DLB) lies in its pharmacological management. Delayed imaging of cardiac (123)I-MIBG scintigraphy allows differentiation between DLB and other neurodegenerative diseases with cognitive impairment. The aim of this study was to assess the utility of early imaging of cardiac (123)I-MIBG scintigraphy for differentiating DLB from others neurodegenerative disease with cognitive impairment. We assess retrospectively 106 patients (51 men, mean age 78 years) with cognitive impairment that underwent a cardiac (123)I-MIBG study. Planar images were acquired in anterior view of the thorax 15min (early) and 4h (delayed) after tracer administration. The heart-to-mediastinum ratios (HMR) at 15m (HMR15m) and at 4h (HMR4h) were obtained. After four years, 52 patients were diagnosed of DLB.HMR15m and HMR4h were significantly inferior in DLB respect to the others neurodegenerative diseases (1,27±0,15 vs 1,76±0,15,p<0,05) and (1,14±0,13 vs 1,68±0,19,p<0.01), respectively. The ROC analysis showed a HMR15m cut off point of 1.56 to differentiated DLB from the other dementias with a sensitivity and a specificity of 98%. Early imaging of cardiac (123)I-MIBG scintigraphy can help to differentiate DLB from other neurodegenerative diseases with cognitive impairment. Copyright © 2012 Elsevier España, S.L. y SEMNIM. All rights reserved.

Fab-based bispecific antibody formats with robust biophysical properties and biological activity.

PubMed

Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Lewis, Steven M; Lieu, Ricky L; Weldon, Caroline; Torres, Carina; Fine, Cody; Batt, Micheal A; Fitchett, Jonathan R; Glasebrook, Andrew L; Kuhlman, Brian; Demarest, Stephen J

2015-01-01

A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity.

Fab-based bispecific antibody formats with robust biophysical properties and biological activity

PubMed Central

Wu, Xiufeng; Sereno, Arlene J; Huang, Flora; Lewis, Steven M; Lieu, Ricky L; Weldon, Caroline; Torres, Carina; Fine, Cody; Batt, Micheal A; Fitchett, Jonathan R; Glasebrook, Andrew L; Kuhlman, Brian; Demarest, Stephen J

2015-01-01

A myriad of innovative bispecific antibody (BsAb) platforms have been reported. Most require significant protein engineering to be viable from a development and manufacturing perspective. Single-chain variable fragments (scFvs) and diabodies that consist only of antibody variable domains have been used as building blocks for making BsAbs for decades. The drawback with Fv-only moieties is that they lack the native-like interactions with CH1/CL domains that make antibody Fab regions stable and soluble. Here, we utilize a redesigned Fab interface to explore 2 novel Fab-based BsAbs platforms. The redesigned Fab interface designs limit heavy and light chain mixing when 2 Fabs are co-expressed simultaneously, thus allowing the use of 2 different Fabs within a BsAb construct without the requirement of one or more scFvs. We describe the stability and activity of a HER2×HER2 IgG-Fab BsAb, and compare its biophysical and activity properties with those of an IgG-scFv that utilizes the variable domains of the same parental antibodies. We also generated an EGFR × CD3 tandem Fab protein with a similar format to a tandem scFv (otherwise known as a bispecific T cell engager or BiTE). We show that the Fab-based BsAbs have superior biophysical properties compared to the scFv-based BsAbs. Additionally, the Fab-based BsAbs do not simply recapitulate the activity of their scFv counterparts, but are shown to possess unique biological activity. PMID:25774965

The use and tolerability of Crotalidae Polyvalent Immune FAB (Ovine) in pediatric envenomations.

PubMed

Farrar, Henry C; Grayham, Taylor; Bolden, Branson; Vyas, Dileep; Graham, James; James, Laura P

2012-10-01

There are limited data on the use of Crotalidae Polyvalent Immune FAB-Ovine (CroFab) in the management of crotalid envenomations in children. Thus, the primary objective of this retrospective chart review was to evaluate the safety and tolerability of CroFab in a pediatric population. Over an 8-year time period at this institution, there were 204 admissions for snakebite of which 82 received CroFab. Children who received CroFab were more often associated with bites to the hands and fingers and tended to have more significant envenomations as indicated by longer hospital stays, greater tissue injury, and a tendency to require surgery more often. Six (7.3%) of the 82 patients who received CroFab experienced an adverse drug reaction. Reactions consisted of allergic symptoms that were mild, responded to minimal interventions, and did not limit the subsequent use of CroFab. It is concluded that CroFab use is typically well tolerated in pediatric patients.

Meditope-Fab interaction: threading the hole.

PubMed

Bzymek, Krzysztof P; Ma, Yuelong; Avery, Kendra N; Horne, David A; Williams, John C

2017-12-01

Meditope, a cyclic 12-residue peptide, binds to a unique binding side between the light and heavy chains of the cetuximab Fab. In an effort to improve the affinity of the interaction, it was sought to extend the side chain of Arg8 in the meditope, a residue that is accessible from the other side of the meditope binding site, in order to increase the number of interactions. These modifications included an n-butyl and n-octyl extension as well as hydroxyl, amine and carboxyl substitutions. The atomic structures of the complexes and the binding kinetics for each modified meditope indicated that each extension threaded through the Fab `hole' and that the carboxyethylarginine substitution makes a favorable interaction with the Fab, increasing the half-life of the complex by threefold compared with the unmodified meditope. Taken together, these studies provide a basis for the design of additional modifications to enhance the overall affinity of this unique interaction.

Tracheal intubation prevented with administration of Fab antivenom after severe crotaline envenomation.

PubMed

Bebarta, Vikhyat S; Ferre, Robinson M; Peck, Michael

2010-07-01

Crotaline snake envenomations are common, but severe crotaline envenomations are infrequent. Death from severe envenomation is usually from upper airway edema and respiratory failure. Published reports of severe respiratory compromise and anaphylactoid reactions are rare. Currently, FabAV (Crotalidae polyvalent immune Fab [Ovine] [CroFab]) is the mainstay of crotaline envenomation treatment; however, FabAV has been approved for only mild and moderate envenomations. We describe a case of a male with severe systemic effects and airway compromise after crotaline envenomation. The patient's systemic effects and upper airway edema substantially improved after antivenom infusion and before epinephrine administration. Endotracheal intubation was averted, clinical deterioration was avoided, and improvement occurred after prompt FabAV use. Fab antivenom likely prevented endotracheal intubation in our case of severe crotaline envenomation. Published by Elsevier Inc.

Pygmy rattlesnake envenomation treated with Crotalidae Polyvalent Immune Fab Antivenom.

PubMed

King, Andrew M; Crim, William S; Menke, Nathan B; Pizon, Anthony F

2012-12-01

Documented envenomations by the pygmy rattlesnake (Sistrurus miliarius barbouri) are rare. While there have been no documented fatalities, several older case reports describe significant morbidity. We describe the first known case of pygmy rattlesnake envenomation that was treated with Crotalidae Polyvalent Immune Fab Antivenom (CroFab®). A 28-year-old man with no significant past medical history presented after being envenomated on the right hand by his friend's pet pygmy rattlesnake. He developed swelling and pain in his hand and forearm. He responded well to a ten vial loading dose and a 18 h maintenance protocol of CroFab and was discharged the following day without developing any hematological or electrolyte derangements. This is the first documented use of CroFab for S. m. barbouri envenomation. The outcome of this case suggests that CroFab is a safe treatment modality in this setting. Copyright © 2012 Elsevier Ltd. All rights reserved.

Preparation of crotaline F-ab antivenom (CroFab) with automated mixing methods: in vitro observations.

PubMed

Vohra, Rais; Kelner, Michael; Clark, Richard F

2009-01-01

Crotaline Polyvalent Ovine Fab antivenom (CroFab, Savage Laboratories and Protherics Inc., Brentwood, TN, USA) preparation requires that the lyophilized powder be manually reconstituted before use. We compared automated methods for driving the product into solution with the standard manual method of reconstitution, and the effect of repeated rinsing of the product vial, on the per-vial availability of antivenom. Normal saline (NS, 10 mL) was added to 12 vials of expired CroFab. Vials were assigned in pairs to each of six mixing methods, including one pair mixed manually as recommended by the product package insert. Each vial's contents were diluted to a final volume of 75 mL of normal saline. Protein concentration was measured with a colorimetric assay. The fluid left in each vial was removed and the vial was washed with 10 mL NS. Total protein yield from each step was calculated. There was no significant change in protein yield among three of five automated mixing methods when compared to manual reconstitution. Repeat rinsing of the product vial with an additional 10 mLs of fluid added to the protein yield regardless of the mixing method used. We found slightly higher protein yields with all automated methods compared to manual mixing, but only two of five comparisons with the standard mixing method demonstrated statistical significance. However, for all methods tested, the addition of a second rinsing and recovery step increased the amount of protein recovered considerably, presumably by allowing solution of protein trapped in the foamy residues. Automated mixing methods and repeat rinsing of the product vial may allow higher protein yields in the preparation of CroFab antivenom.

Comparing domain interactions within antibody Fabs with kappa and lambda light chains.

PubMed

Toughiri, Raheleh; Wu, Xiufeng; Ruiz, Diana; Huang, Flora; Crissman, John W; Dickey, Mark; Froning, Karen; Conner, Elaine M; Cujec, Thomas P; Demarest, Stephen J

2016-10-01

IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cβ), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.

Optimal expression of a Fab-effector fusion protein in Escherichia coli by removing the cysteine residues responsible for an interchain disulfide bond of a Fab molecule.

PubMed

Kang, Hyeon-Ju; Kim, Hye-Jin; Jung, Mun-Sik; Han, Jae-Kyu; Cha, Sang-Hoon

2017-04-01

Development of novel bi-functional or even tri-functional Fab-effector fusion proteins would have a great potential in the biomedical sciences. However, the expression of Fab-effector fusion proteins in Escherichia coli is problematic especially when a eukaryotic effector moiety is genetically linked to a Fab due to the lack of proper chaperone proteins and an inappropriate physicochemical environment intrinsic to the microbial hosts. We previously reported that a human Fab molecule, referred to as SL335, reactive to human serum albumin has a prolonged in vivo serum half-life in rats. We, herein, tested six discrete SL335-human growth hormone (hGH) fusion constructs as a model system to define an optimal Fab-effector fusion format for E. coli expression. We found that one variant, referred to as HserG/Lser, outperformed the others in terms of a soluble expression yield and functionality in that HserG/Lser has a functional hGH bioactivity and possesses an serum albumin-binding affinity comparable to SL335. Our results clearly demonstrated that the genetic linkage of an effector domain to the C-terminus of Fd (V H +C H1 ) and the removal of cysteine (Cys) residues responsible for an interchain disulfide bond (IDB) ina Fab molecule optimize the periplasmic expression of a Fab-effector fusion protein in E. coli. We believe that our approach can contribute the development of diverse bi-functional Fab-effector fusion proteins by providing a simple strategy that enables the reliable expression of a functional fusion proteins in E. coli. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

Targeting human prostate cancer with 111In-labeled D2B IgG, F(ab')2 and Fab fragments in nude mice with PSMA-expressing xenografts.

PubMed

Lütje, Susanne; van Rij, Catharina M; Franssen, Gerben M; Fracasso, Giulio; Helfrich, Wijnand; Eek, Annemarie; Oyen, Wim J; Colombatti, Marco; Boerman, Otto C

2015-01-01

D2B is a new monoclonal antibody directed against an extracellular domain of prostate-specific membrane antigen (PSMA), which is overexpressed in prostate cancer. The potential of D2B IgG, and F(ab')2 and Fab fragments of this antibody for targeting prostate cancer was determined in mice bearing subcutaneous prostate cancer xenografts. The optimal time point for imaging was determined in biodistribution and microSPECT imaging studies with (111)In-D2B IgG, (111)In-capromab pendetide, (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments in mice with PSMA-expressing LNCaP and PSMA-negative PC3 tumors at several time points after injection. All (111)In-labeled antibody formats specifically accumulated in the LNCaP tumors, with highest uptake of (111)In-D2B IgG and (111)In-capromab pendetide at 168 h p.i. (94.8 ± 19.2% injected dose per gram (ID/g) and 16.7 ± 2.2% ID/g, respectively), whereas uptake of (111)In-D2B F(ab')2 and (111)In-D2B Fab fragments peaked at 24 h p.i. (12.1 ± 3.0% ID/g and 15.1 ± 2.9% ID/g, respectively). Maximum LNCaP tumor-to-blood ratios were 13.0 ± 2.3 (168 h p.i.), 6.2 ± 0.7 (24 h p.i.), 23.0 ± 4.0 (24 h p.i.) and 4.5 ± 0.6 (168 h p.i.) for (111)In-D2B IgG, (111)In-F(ab')2, (111)In-Fab and (111)In-capromab pendetide, respectively. LNCaP tumors were clearly visualized with microSPECT with all antibody formats. This study demonstrates the feasibility of D2B IgG, F(ab')2 and Fab fragments for targeting PSMA-expressing prostate cancer xenografts. Copyright © 2014 John Wiley & Sons, Ltd.

Complex binding of the FabR repressor of bacterial unsaturated fatty acid biosynthesis to its cognate promoters.

PubMed

Feng, Youjun; Cronan, John E

2011-04-01

Two transcriptional regulators, the FadR activator and the FabR repressor, control biosynthesis of unsaturated fatty acids in Escherichia coli. FabR represses expression of the two genes, fabA and fabB, required for unsaturated fatty acid synthesis and has been reported to require the presence of an unsaturated thioester (of either acyl carrier protein or CoA) in order to bind the fabA and fabB promoters in vitro. We report in vivo experiments in which unsaturated fatty acid synthesis was blocked in the absence of exogenous unsaturated fatty acids in a ΔfadR strain and found that the rates of transcription of fabA and fabB were unaffected by the lack of unsaturated thioesters. To examine the discrepancy between our in vivo results and the prior in vitro results we obtained active, natively folded forms of the E. coli and Vibrio cholerae FabRs by use of an in vitro transcription-translation system. We report that FabR bound the intact promoter regions of both fabA and fabB in the absence of unsaturated acyl thioesters, but bound the two promoters differently. Native FabR bound the fabA promoter region provided that the canonical FabR binding site is extended by inclusion of flanking sequences that overlap the neighbouring FadR binding site. In contrast, although binding to the fabB operator also required a flanking sequence, a non-specific sequence could suffice. However, unsaturated thioesters did allow FabR binding to the minimal FabR operator sites of both promoters which otherwise were not bound. Thus unsaturated thioester ligands were not essential for FabR/target DNA interaction, but acted to enhance binding. The gel mobility shift data plus in vivo expression data indicate that despite the remarkably similar arrangements of promoter elements, FadR predominately regulates fabA expression whereas FabR is the dominant regulator of fabB expression. We also report that E. coli fabR expression is not autoregulated. Complementation, qRT-PCR and fatty acid

21 CFR 866.5520 - Immunoglobulin G (Fab fragment specific) immunological test system.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Immunoglobulin G (Fab fragment specific... Test Systems § 866.5520 Immunoglobulin G (Fab fragment specific) immunological test system. (a) Identification. An immunoglobulin G (Fab fragment specific) immunological test system is a device that consists...

Landiolol suppression of electrical storm of torsades de pointes in patients with congenital long-QT syndrome type 2 and myocardial ischemia.

PubMed

Kitajima, Ryota; Aiba, Takeshi; Kamakura, Tsukasa; Ishibashi, Kohei; Wada, Mitsuru; Inoue, Yuko; Miyamoto, Koji; Okamura, Hideo; Noda, Takashi; Nagase, Satoshi; Kataoka, Yu; Asaumi, Yasuhide; Noguchi, Teruo; Yasuda, Satoshi; Kusano, Kengo

2017-10-01

A 76-year-old man who had been diagnosed with long-QT syndrome type 2 had frequent syncopal attacks. The electrocardiogram was monitored, and frequent torsades de pointes (TdP) was detected despite administration of conventional medications: oral propranolol, verapamil, intravenous magnesium sulfate, verapamil, and lidocaine. In contrast, 2 μg/kg/min landiolol could completely suppress TdP. Subsequently, an implantable cardioverter defibrillator was placed, and he was diagnosed with silent myocardial ischemia using myocardial perfusion scintigraphy and coronary angiography. This is the first case report wherein landiolol effectively suppressed TdP due to long-QT syndrome with silent myocardial ischemia.

Introduction of a glycosylation site in the constant region decreases the aggregation of adalimumab Fab.

PubMed

Nakamura, Hitomi; Oda-Ueda, Naoko; Ueda, Tadashi; Ohkuri, Takatoshi

2018-06-18

The production of therapeutic monoclonal antibodies is costly; therefore, antigen-binding fragments (Fabs) can be used instead. However, their tendency toward aggregation can reduce the half-life in the plasma and the therapeutic effectiveness. To examine the effect of glycosylation on the properties of the Fab of a therapeutic antibody, an N-glycosylation site was introduced at position 178 of the H-chain constant region of adalimumab Fab through site-directed mutagenesis of L178 N (H:L178 N Fab), and then H:L178 N Fab was expressed in Pichia pastoris. SDS-PAGE analysis with treatment of N-glycosidase F or periodic acid-Schiff reagent showed that H:L178 N Fab contained a relatively low glycan level. Moreover, the H:L178 N mutation did not decrease the binding activity and thermal stability of Fab, and H:L178 N Fab was more resistant to protease digestion than wild-type Fab. The aggregation of Fab induced by pH-shift stress was measured by monitoring the optical density at 350 nm. Although the wild-type Fab showed a large increase in optical density with an increase of protein concentration, no such increase of turbidity during aggregation was found in H:L178 N Fab. These results demonstrated that glycosylation at position 178 of the H-chain constant region of adalimumab Fab can prevent protein aggregation, and therefore serve as a potentially effective platform for drug development. Copyright © 2018. Published by Elsevier Inc.

The community FabLab platform: applications and implications in biomedical engineering.

PubMed

Stephenson, Makeda K; Dow, Douglas E

2014-01-01

Skill development in science, technology, engineering and math (STEM) education present one of the most formidable challenges of modern society. The Community FabLab platform presents a viable solution. Each FabLab contains a suite of modern computer numerical control (CNC) equipment, electronics and computing hardware and design, programming, computer aided design (CAD) and computer aided machining (CAM) software. FabLabs are community and educational resources and open to the public. Development of STEM based workforce skills such as digital fabrication and advanced manufacturing can be enhanced using this platform. Particularly notable is the potential of the FabLab platform in STEM education. The active learning environment engages and supports a diversity of learners, while the iterative learning that is supported by the FabLab rapid prototyping platform facilitates depth of understanding, creativity, innovation and mastery. The product and project based learning that occurs in FabLabs develops in the student a personal sense of accomplishment, self-awareness, command of the material and technology. This helps build the interest and confidence necessary to excel in STEM and throughout life. Finally the introduction and use of relevant technologies at every stage of the education process ensures technical familiarity and a broad knowledge base needed for work in STEM based fields. Biomedical engineering education strives to cultivate broad technical adeptness, creativity, interdisciplinary thought, and an ability to form deep conceptual understanding of complex systems. The FabLab platform is well designed to enhance biomedical engineering education.

CroFab reconstitution in various media: an in vitro solubility study.

PubMed

Vohra, Rais; Clark, Rick; Kelner, Michael

2008-11-01

We investigated the solubility of Crotalidae Polyvalent Ovine Immune Fab antivenom (CroFab, Savage Labs and Protherics Inc., Brentwood, TN, USA) in solutions not listed in the Food and Drug Administration (FDA)-approved product package insert. We also assessed whether adsorption to plastic tubing occurs with CroFab preparations. Nine vials of expired CroFab were divided into three groups according to the solution used for reconstitution. Assignment to the solution groups of normal saline, lactated Ringer's solution, or half-normal saline (NS, LR, 1/2NS) was blinded. The antivenom was diluted to a final volume of 75 mL of test solution. Protein concentration was measured after reconstitution, after storage at 4-6 degrees C for 4 h, and after passage through plastic intravenous (IV) tubing. Higher measured protein yields were noted when half-normal saline was used in comparison with normal saline at each step of the study. Lactated Ringer's solution yielded higher protein concentrations than normal saline only at one out of the three measurement steps. There was no adsorption effect when CroFab was infused through plastic IV tubing. These data suggest that CroFab is slightly more soluble in the hypotonic solution we tested, and the amounts of measured antivenom did not diminish after 4 h of refrigeration or passage through plastic tubing. Our study may be of relevance when clinicians or pharmacists mix CroFab into non-standard solutions.

Structural features of Fab fragments of rheumatoid factor IgM-RF in solution

DOE Office of Scientific and Technical Information (OSTI.GOV)

Volkov, V. V., E-mail: vvo@ns.crys.ras.ru; Lapuk, V. A.; Shtykova, E. V.

The structural features of the Fab fragments of monoclonal (Waldenstroem's disease) immunoglobulin M (IgM) and rheumatoid immunoglobulin M (IgM-RF) were studied by a complex of methods, including small-angle X-ray scattering (SAXS), electron spin resonance (ESR), and mass spectrometry (MS). The Fab-RF fragment was demonstrated to be much more flexible in the region of interdomain contacts, the molecular weights and the shapes of the Fab and Fab-RF macromolecules in solution being only slightly different. According to the ESR data, the rotational correlation time for a spin label introduced into the peptide sequence for Fab is twice as large as that formore » Fab-RF (21{+-}2 and 11{+-}1 ns, respectively), whereas the molecular weights of these fragments differ by only 0.5% (mass-spectrometric data), which correlates with the results of molecular-shape modeling by small-angle X-ray scattering. The conclusion about the higher flexibility of the Fab-RF fragment contributes to an understanding of the specificity of interactions between the rheumatoid factor and the antigens of the own organism.« less

Improved Fab presentation on phage surface with the use of molecular chaperone coplasmid system.

PubMed

Loh, Qiuting; Leong, Siew Wen; Tye, Gee Jun; Choong, Yee Siew; Lim, Theam Soon

2015-05-15

The low presentation efficiency of Fab (fragment antigen binding) fragments during phage display is largely due to the complexity of disulphide bond formation. This can result in the presentation of Fab fragments devoid of a light chain during phage display. Here we propose the use of a coplasmid system encoding several molecular chaperones (DsbA, DsbC, FkpA, and SurA) to improve Fab packaging. A comparison was done using the Fab fragment from IgG and IgD. We found that the use of the coplasmid during phage packaging was able to improve the presentation efficiency of the Fab fragment on phage surfaces. A modified version of panning using the coplasmid system was evaluated and was successful at enriching Fab binders. Therefore, the coplasmid system would be an attractive alternative for improved Fab presentation for phage display. Copyright © 2015 Elsevier Inc. All rights reserved.

Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex.

PubMed

Wolle, Daniel; Cleard, Fabienne; Aoki, Tsutomu; Deshpande, Girish; Schedl, Paul; Karch, Francois

2015-11-01

Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Functional Requirements for Fab-7 Boundary Activity in the Bithorax Complex

PubMed Central

Wolle, Daniel; Cleard, Fabienne; Aoki, Tsutomu; Deshpande, Girish; Karch, Francois

2015-01-01

Chromatin boundaries are architectural elements that determine the three-dimensional folding of the chromatin fiber and organize the chromosome into independent units of genetic activity. The Fab-7 boundary from the Drosophila bithorax complex (BX-C) is required for the parasegment-specific expression of the Abd-B gene. We have used a replacement strategy to identify sequences that are necessary and sufficient for Fab-7 boundary function in the BX-C. Fab-7 boundary activity is known to depend on factors that are stage specific, and we describe a novel ∼700-kDa complex, the late boundary complex (LBC), that binds to Fab-7 sequences that have insulator functions in late embryos and adults. We show that the LBC is enriched in nuclear extracts from late, but not early, embryos and that it contains three insulator proteins, GAF, Mod(mdg4), and E(y)2. Its DNA binding properties are unusual in that it requires a minimal sequence of >65 bp; however, other than a GAGA motif, the three Fab-7 LBC recognition elements display few sequence similarities. Finally, we show that mutations which abrogate LBC binding in vitro inactivate the Fab-7 boundary in the BX-C. PMID:26303531

Neutron Reflection Study of Surface Adsorption of Fc, Fab, and the Whole mAb.

PubMed

Li, Zongyi; Li, Ruiheng; Smith, Charles; Pan, Fang; Campana, Mario; Webster, John R P; van der Walle, Christopher F; Uddin, Shahid; Bishop, Steve M; Narwal, Rojaramani; Warwicker, Jim; Lu, Jian Ren

2017-07-12

Characterizing the influence of fragment crystallization (Fc) and antigen-binding fragment (Fab) on monoclonal antibody (mAb) adsorption at the air/water interface is an important step to understanding liquid mAb drug product stability during manufacture, shipping, and storage. Here, neutron reflection is used to study the air/water adsorption of a mAb and its Fc and Fab fragments. By varying the isotopic contrast, the adsorbed amount, thickness, orientation, and immersion of the adsorbed layers could be determined unambiguously. While Fc adsorption reached saturation within the hour, its surface adsorbed amount showed little variation with bulk concentration. In contrast, Fab adsorption was slower and the adsorbed amount was concentration dependent. The much higher Fc adsorption, as compared to Fab, was linked to its lower surface charge. Time and concentration dependence of mAb adsorption was dominated by Fab behavior, although both Fab and Fc behaviors contributed to the amount of mAb adsorbed. Changing the pH from 5.5 to 8.8 did not much perturb the adsorbed amount of Fc, Fab, or mAb. However, a small decrease in adsorption was observed for the Fc over pH 8-8.8 and vice versa for the Fab and mAb, consistent with a dominant Fab behavior. As bulk concentration increased from 5 to 50 ppm, the thicknesses of the Fc layers were almost constant at 40 Å, while Fab and mAb layers increased from 45 to 50 Å. These results imply that the adsorbed mAb, Fc, and Fab all retained their globular structures and were oriented with their short axial lengths perpendicular to the interface.

Comparative study of thiophilic functionalised matrices for polyclonal F(ab')2 purification.

PubMed

Kumpalume, Peter; Slater, Nigel K H

2004-01-02

Thiophilic adsorbents have been developed using divinyl sulfone or epoxy activated Streamline quartz base matrix. Their capacity and selectivity for binding polyclonal F(ab')2 fragments generated by whole serum proteolysis was tested. Except for epoxy activated guanidine, all the adsorbents displayed high selectivity for F(ab')2 with dynamic binding capacities ranging from 3 to 10 mg/ml of adsorbent. Thiol immobilised ligands adsorbed more F(ab')2 and the recovery was equal to or more than that from amino immobilised ligands. All adsorbents showed good selectivity for IgG and the dynamic binding capacities were better than for F(ab')2.

CIM for 300-mm semiconductor fab

NASA Astrophysics Data System (ADS)

Luk, Arthur

1997-08-01

Five years ago, factory automation (F/A) was not prevalent in the fab. Today facing the drastically changed market and the intense competition, management request the plant floor data be forward to their desktop computer. This increased demand rapidly pushed F/A to the computer integrated manufacturing (CIM). Through personalization, we successfully reduced a computer size, let them can be stored on our desktop. PC initiates a computer new era. With the advent of the network, the network computer (NC) creates fresh problems for us. When we plan to invest more than $3 billion to build new 300 mm fab, the next generation technology raises a challenging bar.

Immobilization of Fab' fragments onto substrate surfaces: A survey of methods and applications.

PubMed

Crivianu-Gaita, Victor; Thompson, Michael

2015-08-15

Antibody immobilization onto surfaces has widespread applications in many different fields. It is desirable to bind antibodies such that their fragment-antigen-binding (Fab) units are oriented away from the surface in order to maximize analyte binding. The immobilization of only Fab' fragments yields benefits over the more traditional whole antibody immobilization technique. Bound Fab' fragments display higher surface densities, yielding a higher binding capacity for the analyte. The nucleophilic sulfide of the Fab' fragments allows for specific orientations to be achieved. For biosensors, this indicates a higher sensitivity and lower detection limit for a target analyte. The last thirty years have shown tremendous progress in the immobilization of Fab' fragments onto gold, Si-based, polysaccharide-based, plastic-based, magnetic, and inorganic surfaces. This review will show the current scope of Fab' immobilization techniques available and illustrate methods employed to minimize non-specific adsorption of undesirables. Furthermore, a variety of examples will be given to show the versatility of immobilized Fab' fragments in different applications and future directions of the field will be addressed, especially regarding biosensors. Copyright © 2015 Elsevier B.V. All rights reserved.

[Myocardial imaging in acute myocardial infarction using beta-methyl-p-(123I)-iodophenylpentadecanoic acid: comparison with 201Tl imaging and wall motion].

PubMed

Naruse, H; Itano, M; Kondo, T; Kogame, T; Yamamoto, J; Morita, M; Kawamoto, H; Fukutake, N; Ohyanagi, M; Iwasaki, T

1992-01-01

Myocardial imaging using beta-methyl-p-(123I)-iodophenylpentadecanoic acid (BMIPP) was performed in 11 patients with acute myocardial infarction. The left ventricular images were divided into 12 segments, and myocardial imagings with BMIPP were compared with coronary angiography (CAG), thallium-201 myocardial scintigraphy (TL) and wall motion obtained by two-dimensional echocardiography (WM). When the culprit lesion was at the proximal point of the left anterior descending artery (LAD), all segments showed depressed uptake. In 3 cases with single vessel disease of the LAD, inferior wall of the basis showed reduced uptake of BMIPP despite the location of the culprit lesion. In cases with discordant uptake between the two tracers, BMIPP frequently showed more severely depressed uptake than TL in the subacute phase, although the uptake of BMIPP correlated with that of TL (tau = 0.82, p less than 0.001). In such cases, the discordance was related to the improvement in WM from the acute phase to the convalescent phase. BMIPP uptake correlated with WM in the subacute phase (tau = 0.50, p less than 0.001). BMIPP showed more severely depressed uptake while WM showed mild asynergy in most cases in which discordance was found between the BMIPP and WM findings. However, there was no correlation between the change in WM from the acute to subacute phases, or the uptakes of BMIPP and TL alone. We concluded that the myocardial condition can be evaluated in detail in acute myocardial infarction by comparing the findings of BMIPP with those of TL and WM.

Prevalence of silent myocardial ischaemia during exercise stress testing. Its relation to effort tolerance and myocardial perfusion abnormalities.

PubMed

Fragasso, G; Sciammarella, M G; Rossetti, E E; Xuereb, R G; Xuereb, M; Bonetti, F; Carandente, O M; Margonato, A; Chierchia, S L

1992-07-01

The number of underperfused myocardial segments, the extent of coronary artery disease and the severity of impairment of coronary flow reserve were compared in 147 consecutive patients exhibiting painful or painless ischaemic ST segment depression on exercise testing. Of 147 patients, only 61 (41%) experienced angina (group 1) whilst 86 (59%) did not (group 2). In the two groups coronary disease was comparable for both extent and distribution, and neither the location of transient perfusion defects nor their relation to areas of old myocardial necrosis appeared to influence the presence or absence of chest pain. However, exercise duration, exercise time and rate-pressure product at the beginning of ischaemia were lower in group 1. Furthermore, a greater proportion of asymptomatic patients had only one ischaemic segment on 99mTc-MIBI perfusion scintigraphy. We conclude that: (1) in patients with effort angina and coronary disease, the incidence of electrocardiographic silent ischaemic events induced by exercise is similar to that observed in studies based on continuous ECG monitoring. (2) Exertional angina is more frequently associated with greater ischaemic areas and with more severe degrees of impairment of residual coronary flow reserve. (3) The presence of an old myocardial infarction does not appear to influence the incidence of ischaemic cardiac pain.

IgG Fab Fragments Forming Bivalent Complexes by a Conformational Mechanism That Is Reversible by Osmolytes*

PubMed Central

Nelson, Alfreda D.; Hoffmann, Michele M.; Parks, Christopher A.; Dasari, Surendra; Schrum, Adam G.; Gil, Diana

2012-01-01

Generated by proteolytic cleavage of immunoglobulin, Fab fragments possess great promise as blocking reagents, able to bind receptors or other targets without inducing cross-linking. However, aggregation of Fab preparations is a common occurrence, which generates intrinsic stimulatory capacity and thwarts signal blockade strategies. Using a panel of biochemical approaches, including size exclusion chromatography, SDS-PAGE, mass spectrometry, and cell stimulation followed by flow cytometry, we have measured the oligomerization and acquisition of stimulatory capacity that occurs in four monoclonal IgG Fabs specific for TCR/CD3. Unexpectedly, we observed that all Fabs spontaneously formed complexes that were precisely bivalent, and these bivalent complexes possessed most of the stimulatory activity of each Fab preparation. Fabs composing bivalent complexes were more susceptible to proteolysis than monovalent Fabs, indicating a difference in conformation between the Fabs involved in these two different states of valency. Because osmolytes represent a class of compounds that stabilize protein folding and conformation, we sought to determine the extent to which the amino acid osmolyte l-proline might impact bivalent Fab complexation. We found that l-proline (i) inhibited the adoption of the conformation associated with bivalent complexation, (ii) preserved Fab monovalency, (iii) reversed the conformation of preformed bivalent Fabs to that of monovalent Fabs, and (iv) separated a significant percentage of preformed bivalent complexes into monovalent species. Thus, Fab fragments can adopt a conformation that is compatible with folding or packing of a bivalent complex in a process that can be inhibited by osmolytes. PMID:23109335

The Work Disability Functional Assessment Battery (WD-FAB): Feasibility and Psychometric Properties

PubMed Central

Meterko, Mark; Marfeo, Elizabeth E.; McDonough, Christine M.; Jette, Alan M.; Ni, Pengsheng; Bogusz, Kara; Rasch, Elizabeth K; Brandt, Diane E.; Chan, Leighton

2015-01-01

Objectives To assess the feasibility and psychometric properties of eight scales covering two domains of the newly developed Work Disability Functional Assessment Battery (WD-FAB): physical function (PF) and behavioral health (BH) function. Design Cross-sectional. Setting Community. Participants Adults unable to work due to a physical (n=497) or mental (n=476) disability. Interventions None. Main Outcome Measures Each disability group responded to a survey consisting of the relevant WD-FAB scales and existing measures of established validity. The WD-FAB scales were evaluated with regard to data quality (score distribution; percent “I don’t know” responses), efficiency of administration (number of items required to achieve reliability criterion; time required to complete the scale) by computerized adaptive testing (CAT), and measurement accuracy as tested by person fit. Construct validity was assessed by examining both convergent and discriminant correlations between the WD-FAB scales and scores on same-domain and cross-domain established measures. Results Data quality was good and CAT efficiency was high across both WD-FAB domains. Measurement accuracy was very good for the PF scales; BH scales demonstrated more variability. Construct validity correlations, both convergent and divergent, between all WD-FAB scales and established measures were in the expected direction and range of magnitude. Conclusions The data quality, CAT efficacy, person fit and construct validity of the WD-FAB scales were well supported and suggest that the WD-FAB could be used to assess physical and behavioral health function related to work disability. Variation in scale performance suggests the need for future work on item replenishment and refinement, particularly regarding the Self-Efficacy scale. PMID:25528263

Promoter engineering to optimize recombinant periplasmic Fab' fragment production in Escherichia coli.

PubMed

Schofield, Desmond M; Templar, Alex; Newton, Joseph; Nesbeth, Darren N

2016-07-08

Fab' fragments have become an established class of biotherapeutic over the last two decades. Likewise, developments in synthetic biology are providing ever more powerful techniques for designing bacterial genes, gene networks and entire genomes that can be used to improve industrial performance of cells used for production of biotherapeutics. We have previously observed significant leakage of an exogenous therapeutic Fab' fragment into the growth medium during high cell density cultivation of an Escherichia coli production strain. In this study we sought to apply a promoter engineering strategy to address the issue of Fab' fragment leakage and its consequent bioprocess challenges. We used site directed mutagenesis to convert the Ptac promoter, present in the plasmid, pTTOD-A33 Fab', to a Ptic promoter which has been shown by others to direct expression at a 35% reduced rate compared to Ptac . We characterized the resultant production trains in which either Ptic or Ptac promoters direct Fab' fragment expression. The Ptic promoter strain showed a 25-30% reduction in Fab' expression relative to the original Ptac strain. Reduced Fab' leakage and increased viability over the course of a fed-batch fermentation were also observed for the Ptic promoter strain. We conclude that cell design steps such as the Ptac to Ptic promoter conversion reported here, can yield significant process benefit and understanding with respect to periplasmic Fab' fragment production. It remains an open question as to whether the influence of transgene expression on periplasmic retention is mediated by global metabolic burden effects or periplasm overcapacity. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:840-847, 2016. © 2016 American Institute of Chemical Engineers.

Locking the Elbow: Improved Antibody Fab Fragments as Chaperones for Structure Determination.

PubMed

Bailey, Lucas J; Sheehy, Kimberly M; Dominik, Pawel K; Liang, Wenguang G; Rui, Huan; Clark, Michael; Jaskolowski, Mateusz; Kim, Yejoon; Deneka, Dawid; Tang, Wei-Jen; Kossiakoff, Anthony A

2018-02-02

Antibody Fab fragments have been exploited with significant success to facilitate the structure determination of challenging macromolecules as crystallization chaperones and as molecular fiducial marks for single particle cryo-electron microscopy approaches. However, the inherent flexibility of the "elbow" regions, which link the constant and variable domains of the Fab, can introduce disorder and thus diminish their effectiveness. We have developed a phage display engineering strategy to generate synthetic Fab variants that significantly reduces elbow flexibility, while maintaining their high affinity and stability. This strategy was validated using previously recalcitrant Fab-antigen complexes where introduction of an engineered elbow region enhanced crystallization and diffraction resolution. Furthermore, incorporation of the mutations appears to be generally portable to other synthetic antibodies and may serve as a universal strategy to enhance the success rates of Fabs as structure determination chaperones. Copyright © 2017 Elsevier Ltd. All rights reserved.

High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation.

PubMed

Mendler, Claudia T; Friedrich, Lars; Laitinen, Iina; Schlapschy, Martin; Schwaiger, Markus; Wester, Hans-Jürgen; Skerra, Arne

2015-01-01

Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions.

A novel engineered interchain disulfide bond in the constant region enhances the thermostability of adalimumab Fab.

PubMed

Nakamura, Hitomi; Oda-Ueda, Naoko; Ueda, Tadashi; Ohkuri, Takatoshi

2018-01-01

We constructed a system for expressing the Fab of the therapeutic human monoclonal antibody adalimumab at a yield of 20 mg/L in the methylotrophic yeast Pichia pastoris. To examine the contribution of interchain disulfide bonds to conformational stability, we prepared adalimumab Fab from which the interchain disulfide bond at the C-terminal region at both the CH 1 and CL domains was deleted by substitution of Cys with Ala (Fab ΔSS ). DSC measurements showed that the Tm values of Fab ΔSS were approximately 5 °C lower than those of wild-type Fab, suggesting that the interchain disulfide bond contributes to conformational thermostability. Using computer simulations, we designed a novel interchain disulfide bond outside the C-terminal region to increase the stability of Fab ΔSS . The resulting Fab (mutSS Fab ΔSS ) had the mutations H:V177C and L:Q160C in Fab ΔSS , confirming the formation of the disulfide bond between CH 1 and CL. The thermostability of mutSS Fab ΔSS was approximately 5 °C higher than that of Fab ΔSS . Therefore, the introduction of the designed interchain disulfide bond enhanced the thermostability of Fab ΔSS and mitigated the destabilization caused by partial reduction of the interchain disulfide bond at the C-terminal region, which occurs in site-specific modification such as PEGylation. Copyright © 2017 Elsevier Inc. All rights reserved.

Uptake Index of 123I-metaiodobenzylguanidine Myocardial Scintigraphy for Diagnosing Lewy Body Disease

PubMed Central

Kamiya, Yoshito; Ota, Satoru; Okumiya, Shintaro; Yamashita, Kosuke; Takaki, Akihiro; Ito, Shigeki

2017-01-01

Objective(s): Iodine-123 metaiodobenzylguanidine (123I-MIBG) myocardial scintigraphy has been used to evaluate cardiac sympathetic denervation in Lewy body disease (LBD), including Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). The heart-to-mediastinum ratio (H/M) in PD and DLB is significantly lower than that in Parkinson’s plus syndromes and Alzheimer’s disease. Although this ratio is useful for distinguishing LBD from non-LBD, it fluctuates depending on the system performance of the gamma cameras. Therefore, a new, simple quantification method using 123I-MIBG uptake analysis is required for clinical study. The purpose of this study was to develop a new uptake index with a simple protocol to determine 123I-MIBG uptake on planar images. Methods: The 123I-MIBG input function was obtained from the input counts of the pulmonary artery (PA), which were assessed by analyzing the PA time-activity curves. The heart region of interest used for determining the H/M was used for calculating the uptake index, which was obtained by dividing the heart count by the input count. Results: Forty-eight patients underwent 123I-MIBG chest angiography and planar imaging, after clinical feature assessment and tracer injection. The H/M and 123I-MIBG uptake index were calculated and correlated with clinical features. Values for LBD were significantly lower than those for non-LBD in all analyses (P<0.001). The overlapping ranges between non-LBD and LBD were 2.15 to 2.49 in the H/M method, and 1.04 to 1.22% in the uptake index method. The diagnostic accuracy of the uptake index (area under the curve (AUC), 0.98; sensitivity, 96%; specificity, 91%; positive predictive value (PPV), 90%; negative predictive value (NPV), 93%; and accuracy, 92%) was approximately equal to that of the H/M (AUC, 0.95; sensitivity, 93%; specificity, 91%; PPV, 90%; NPV, 93%; and accuracy, 92%) for discriminating patients with LBD and non-LBD. Conclusion: A simple uptake index method was

Initial postmarketing experience with crotalidae polyvalent immune Fab for treatment of rattlesnake envenomation.

PubMed

Ruha, Anne-Michelle; Curry, Steven C; Beuhler, Michael; Katz, Ken; Brooks, Daniel E; Graeme, Kimberlie A; Wallace, Kevin; Gerkin, Richard; Lovecchio, Frank; Wax, Paul; Selden, Brad

2002-06-01

We describe our postmarketing experience with patients receiving Crotalidae polyvalent immune Fab (CroFab; FabAV) antivenom for treatment of rattlesnake envenomation. The charts of 28 patients admitted between March 1 and September 9, 2001, with rattlesnake envenomation and treated with FabAV were reviewed for demographic information, time until antivenom treatment, laboratory findings, evidence of hypersensitivity reaction, length of hospital stay, and readmission to the hospital. All patients had swelling, 20 patients had elevated prothrombin times (>14 seconds), 12 patients had low fibrinogen levels (<170 mg/dL), and 6 patients had thrombocytopenia (platelet count <120,000/mm(3)) on presentation. The total dose of FabAV ranged from 10 to 47 vials per patient. Hypofibrinogenemia was resistant to FabAV in some patients. On follow-up, recurrence of coagulopathy was detected in 3 patients, and recurrence of thrombocytopenia was detected in 1 patient. Two patients demonstrated delayed-onset severe thrombocytopenia. Recurrence or delayed-onset toxicity might have been underestimated because of incomplete follow-up in some patients. No acute hypersensitivity reactions occurred. Two patients reported mild symptoms of possible serum sickness on follow-up. FabAV effectively controlled the effects of envenomation; however, initial control of coagulopathy was difficult to achieve in some cases, and recurrence or delayed-onset hematotoxicity was common. When initially managing hematotoxicity, a trend toward normalization of laboratory values might be a more reasonable end point for FabAV treatment than attainment of normal reference values in nonbleeding patients.

The reliability and validity of the Turkish version of Fullerton Advanced Balance (FAB-T) scale.

PubMed

Iyigun, Gozde; Kirmizigil, Berkiye; Angin, Ender; Oksuz, Sevim; Can, Filiz; Eker, Levent; Rose, Debra J

2018-06-04

The aim of this study was to evaluate the reliability and validity of the Turkish version of the FAB(FAB-T) scale in the older Turkish adults. The reliability and validity of the scale was tested on 200 community-dwelling older adults. FAB-T scale was scored by different physiotherapists on different days to evaluate inter-rater and intrarater reliability. The Berg Balance Scale (BBS) was used for the evaluation of convergent validity, and the content validity of the FAB-T scale was investigated. The FAB-T scale showed very high inter- and intra-rater reliability. For inter-rater agreement, on the individual test items and total score ICC values were 0.92 (95 %CI; 0.90-0.94) and 0.96 (95% CI; 0.95-0.97) respectively. The intra-rater agreement, on the individual test items and total score ICC values were 0.93 (95 %CI; 0.91- 0.95) and 0.96 (95% CI; 0.95- 0.97) respectively. There was a good agreement between the FAB-T and BBS scales. A high correlation was found between the BBS and FAB-T scales [rho = 0.70 (%95 CI; 0.62-0.76)] indicating good convergent validity. Considering the content validity of the FAB-T scale, no floor (floor score: 0%) or ceiling (ceiling score: 6.5%) effect was detected. The FAB-T scale was successfully translated from the original English version (FAB) and demonstrated strong psychometric features. It was found that the FAB-T scale has very high inter-rater and intra-rater reliability. Considering the convergent validity, the scale has high correlation with the BBS. The FAB-T has no floor and ceiling effect. Copyright © 2018 Elsevier B.V. All rights reserved.

Initial experience with Crotalidae polyvalent immune Fab (ovine) antivenom in the treatment of copperhead snakebite.

PubMed

Lavonas, Eric J; Gerardo, Charles J; O'Malley, Gerald; Arnold, Thomas C; Bush, Sean P; Banner, William; Steffens, Mark; Kerns, William P

2004-02-01

Crotalidae polyvalent immune Fab (ovine) (CroFab; FabAV) effectively treats patients bitten by rattlesnakes. The copperhead snake (Agkistrodon contortrix) caused 37% of venomous snakebites reported to US poison centers in 2001 and is the major envenomating reptile in the southeastern United States. FabAV has not been tested in human beings envenomated by copperhead snakes. In this preliminary study, we performed a retrospective chart review of all copperhead snake envenomations reported to the Carolinas Poison Center that were treated with FabAV. Progression of limb swelling, coagulopathy, and hemodynamic status before and after FabAV administration, adverse effects of FabAV therapy, and recurrence phenomena were recorded. Of approximately 400 copperhead envenomation cases reported to the poison center during the study period, 32 received FabAV and were included. Most patients had moderate envenomation. The median time to FabAV administration was 4.0 hours. The median time to achieve initial control was 1.0 hour, with a median dose of 4 vials of FabAV. A rapid initial response, defined as cessation of the progression of local tissue injury within 4 hours of FabAV administration, occurred in 28 cases (88%; 95% confidence interval [CI] 76% to 99%). Four cases (13%; 95% CI 1% to 24%) were considered treatment failures. Recurrent swelling occurred in 6 cases (19%; 95% CI 5% to 32%). The incidence of recurrent swelling was not reduced by administration of repeated doses of antivenom on a planned schedule. One patient developed late-onset coagulopathy. One minor allergic reaction was observed. In this select group of patients bitten by copperhead snakes, local tissue effects of envenomation halted promptly after FabAV treatment in most cases. Treatment failures occurred, and recurrence of swelling and defibrination syndrome was sometimes problematic. Time to return to work and long-term limb function were not assessed. A controlled trial with long-term follow-up is

Structural and dynamical aspects of Streptococcus gordonii FabH through molecular docking and MD simulations.

PubMed

Shamim, Amen; Abbasi, Sumra Wajid; Azam, Syed Sikander

2015-07-01

β-Ketoacyl-ACP-synthase III (FabH or KAS III) has become an attractive target for the development of new antibacterial agents which can overcome the multidrug resistance. Unraveling the fatty acid biosynthesis (FAB) metabolic pathway and understanding structural coordinates of FabH will provide valuable insights to target Streptococcus gordonii for curing oral infection. In this study, we designed inhibitors against therapeutic target FabH, in order to block the FAB pathway. As compared to other targets, FabH has more interactions with other proteins, located on the leading strand with higher codon adaptation index value and associated with lipid metabolism category of COG. Current study aims to gain in silico insights into the structural and dynamical aspect of S. gordonii FabH via molecular docking and molecular dynamics (MD) simulations. The FabH protein is catalytically active in dimerization while it can lock in monomeric state. Current study highlights two residues Pro88 and Leu315 that are close to each other by dimerization. The active site of FabH is composed of the catalytic triad formed by residues Cys112, His249, and Asn279 in which Cys112 is involved in acetyl transfer, while His249 and Asn279 play an active role in decarboxylation. Docking analysis revealed that among the studied compounds, methyl-CoA disulfide has highest GOLD score (82.75), binding affinity (-11 kcal/mol) and exhibited consistently better interactions. During MD simulations, the FabH structure remained stable with the average RMSD value of 1.7 Å and 1.6 Å for undocked protein and docked complex, respectively. Further, crucial hydrogen bonding of the conserved catalytic triad for exhibiting high affinity between the FabH protein and ligand is observed by RDF analysis. The MD simulation results clearly demonstrated that binding of the inhibitor with S. gordonii FabH enhanced the structure and stabilized the dimeric FabH protein. Therefore, the inhibitor has the potential to become

Early diagnosis of interferon-induced myocardial disorder in patients with chronic hepatitis C: evaluation by myocardial imaging with 123I-BMIPP.

PubMed

Kondo, Y; Yukinaka, M; Nomura, M; Nakaya, Y; Ito, S

2000-01-01

Interferon (IFN) therapy for chronic hepatitis C is sometimes associated with cardiac complications. In the present study, we performed myocardial imaging with 123I-labeled beta-methyl-p-iodophenylpentadecanoic acid (123I-BMIPP) in order to evaluate myocardial disorders caused by IFN. We studied 40 healthy subjects (H group) and 25 patients with chronic hepatitis C who had been treated with IFN (IFN group). A Holter electrocardiogram (ECG) was performed and the autonomic nervous function was assessed by analyzing the spectral variability and 1/f fluctuation of heart rate. Myocardial planner imaging with 123I-BMIPP was performed to obtain the time activity curve for 20min immediately after administration of 123I-BMIPP (dynamic study). Early and delayed myocardial single photon emission computed tomography (SPECT) images were expressed as Bull's eyes and the myocardium was divided into four segments to calculate the washout rate for each segment on early and late SPECT images (early and late SPECT study). No significant differences in autonomic nervous function were observed between the two groups in heart rate variability. In a dynamic study, the reduction rate from the time activity curve was significantly higher in the IFN group compared with the H group (reduction rate, IFN group, 5.3 +/- 3.7% vs H group, 1.2 +/- 3.3%; P < 0.05). In the early and delayed myocardial SPECT study, the washout rate for the IFN group was significantly increased in all myocardial areas compared to that in the H group. However, the metabolic disorder of fatty acids caused by IFN was reversed on the second 123I-BMIPP myocardial scintigraphy examination several months after IFN therapy. These results indicate that metabolic disorders of fatty acids caused by IFN therapy can be detected before abnormalities are observed by Holter-ECG or echocardiography.

A human Fab exclusively binding to the extracellular domain of LMP2A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cao, Qing; Zhang, Dawei; Mao, Yuan

In the areas of North Africa, Southeast Asia as well as South China, Nasopharyngeal carcinoma (NPC) is among the most widespread cancers. Plenty of research findings confirmed that Epstein-Barr virus (EBV) played a crucial role in NPC. EBV-encoded Latent membrane protein 2A (LMP2A) which continuously expressed in cell membrane protein induced an epithelial-mesenchymal transition and increased the number of side population stem-like cancer cells in NPC. This reveals that LMP2A could contribute to the development and recurrence in NPC. Above evidences suggest that LMP2A could be the potential target molecule in the treatment of NPC. In the current study, amore » novel human antibody Fab (Fab29) against the extracellular domain of LMP2A was produced with success. Through immunofluorescence experiment it was proved that human antibody Fab29 exclusively combined the surface of SUNE cells (LMP2A-positive). Then flow cytometry result exhibited that the fluorescent intensities of SUNE cells and CNE cells were distinct (96.89% and 0.02% respectively). After that, it was shown by affinity test that the Fab29 fragment had high affinity (KD (M) 1.79E-09) with LMP2A. It was also revealed by immunohistochemical analysis that the Fab29 fragment could combine with LMP2A-positive human NPC tissues in comparison with the control group. Finally, the MTT result indicated that the Fab29 fragment could inhibit the proliferation of LMP2A-positive NPC cells. The inhibiting rate to SUNE cell proliferation reached a peak by Fab29 (19.67%) compared with unrelated Fab and CNE with Fab29 at a concentration of 500 μg/L in first 24 h and in the next 24 h the inhibition rate grew to 22.54%. In brief, it was shown that Fab29, a characteristic human antibody, could recognize LMP2A protein and inhibit the proliferation of LMP2A-expressing NPC cells in vitro.« less

FabQ, a Dual-Function Dehydratase/Isomerase, Circumvents the Last Step of the Classical Fatty Acid Synthesis Cycle

PubMed Central

Bi, Hongkai; Wang, Haihong; Cronan, John E.

2015-01-01

SUMMARY In the classical anaerobic pathway of unsaturated fatty acid biosynthesis, that of Escherichia coli, the double bond is introduced into the growing acyl chain by the FabA dehydratase/isomerase. Another dehydratase, FabZ, functions in the chain elongation cycle. In contrast, Aerococcus viridans has only a single FabA/FabZ homolog we designate FabQ. FabQ can not only replace the function of E. coli FabZ in vivo, but it also catalyzes the isomerization required for unsaturated fatty acid biosynthesis. Most strikingly, FabQ in combination with E. coli FabB imparts the surprising ability to bypass reduction of the trans-2-acyl-ACP intermediates of classical fatty acid synthesis. FabQ allows elongation by progressive isomerization reactions to form the polyunsaturated fatty acid, 3-hydroxy-cis-5, 7-hexadecadienoic acid, both in vitro and in vivo. FabQ therefore provides a potential pathway for bacterial synthesis of polyunsaturated fatty acids. PMID:23972938

The structure of (3R)-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from Pseudomonas aeruginosa.

PubMed

Kimber, Matthew S; Martin, Fernando; Lu, Yingjie; Houston, Simon; Vedadi, Masoud; Dharamsi, Akil; Fiebig, Klaus M; Schmid, Molly; Rock, Charles O

2004-12-10

Type II fatty acid biosynthesis systems are essential for membrane formation in bacteria, making the constituent proteins of this pathway attractive targets for antibacterial drug discovery. The third step in the elongation cycle of the type II fatty acid biosynthesis is catalyzed by beta-hydroxyacyl-(acyl carrier protein) (ACP) dehydratase. There are two isoforms. FabZ, which catalyzes the dehydration of (3R)-hydroxyacyl-ACP to trans-2-acyl-ACP, is a universally expressed component of the bacterial type II system. FabA, the second isoform, as has more limited distribution in nature and, in addition to dehydration, also carries out the isomerization of trans-2- to cis-3-decenoyl-ACP as an essential step in unsaturated fatty acid biosynthesis. We report the structure of FabZ from the important human pathogen Pseudomonas aeruginosa at 2.5 A of resolution. PaFabZ is a hexamer (trimer of dimers) with the His/Glu catalytic dyad located within a deep, narrow tunnel formed at the dimer interface. Site-directed mutagenesis experiments showed that the obvious differences in the active site residues that distinguish the FabA and FabZ subfamilies of dehydratases do not account for the unique ability of FabA to catalyze isomerization. Because the catalytic machinery of the two enzymes is practically indistinguishable, the structural differences observed in the shape of the substrate binding channels of FabA and FabZ lead us to hypothesize that the different shapes of the tunnels control the conformation and positioning of the bound substrate, allowing FabA, but not FabZ, to catalyze the isomerization reaction.

Fab fragments of ovine antibody to colchicine enhance its clearance in the rat.

PubMed

Peake, Philip W; Pianta, Timothy J; Succar, Lena; Fernando, Mangalee; Buckley, Nicholas A; Endre, Zoltan H

2015-06-01

Colchicine is an anti-inflammatory alkaloid used for the treatment of acute gout, but has a narrow therapeutic index. Colchicine overdoses are relatively rare, but have high mortality requiring rapid treatment. To evaluate the ability of a newly available ovine fragment antigen-binding (Fab) antibody to colchicine (ColchiFab(™)) to protect rats against renal and other injury 24 h after colchicine ingestion. Rats were gavaged with colchicine (5 mg/kg), then 2 h later injected intraperitoneally with 5 ml of sterile saline, or Fab anti-colchicine, a newly available ovine antibody to colchicine. Samples of blood were taken at 1, 2, 5 and 24 h after gavage, and urine was collected from 5 to 24 h after gavage. Concentrations of colchicine in tissue, blood and urine were measured by liquid chromatography/mass spectrometry, concentrations of Fab anti-colchicine, urinary neutrophil gelatinase-associated lipocalin (NGAL) and kidney injury molecule-1 or KIM-1 by enzyme-linked immunosorbent assay or ELISA, while concentrations of creatine kinase and creatinine (Cr) were measured enzymatically. Colchicine equilibrated rapidly throughout the body and increased serum creatine kinase. Fab anti-colchicine also rapidly redistributed to the blood and remained at high concentrations over 24 h. Fab anti-colchicine caused a rapid 7.1-fold increase in serum colchicine level, followed by excretion of both colchicine and Fab anti-colchicine through the urine. This was associated with the accumulation of colchicine in the kidney, a reversal of colchicine-induced diarrhoea, and increasing urinary NGAL level; from 168 ± 48 to 477 ± 255 ng/mmol Cr [mean ± standard deviation or SD]. Fab anti-colchicine greatly increased the clearance of colchicine, although increasing NGAL level suggested the presence of mild kidney damage. These data suggest clinical utility for Fab anti-colchicine in the treatment of colchicine overdose.

Characterization of deamidation at Asn138 in L-chain of recombinant humanized Fab expressed from Pichia pastoris.

PubMed

Ohkuri, Takatoshi; Murase, Eri; Sun, Shu-Lan; Sugitani, Jun; Ueda, Tadashi

2013-10-01

A method was previously established for evaluating Asn deamidation by matrix-assisted laser desorption/ionization time of flight-mass spectrometry using endoproteinase Asp-N. In this study, we demonstrated that this method could be applied to the identification of the deamidation site of the humanized fragment antigen-binding (Fab). First, a system for expressing humanized Fab from methylotrophic yeast Pichia pastoris was constructed, resulting in the preparation of ∼30 mg of the purified humanized Fab from 1 l culture. Analysis of the L-chain derived from recombinant humanized Fab that was heated at pH 7 and 100°C for 1 h showed the deamidation at Asn138 in the constant region. Then, we prepared L-N138D Fab and L-N138A Fab and examined their properties. The circular dichroism (CD) spectrum of the L-N138D Fab was partially different from that of the wild-type Fab. The measurement of the thermostability showed that L-N138D caused a significant decrease in the thermostability of Fab. On the other hand, the CD spectrum and thermostability of L-N138A Fab showed the same behaviour as the wild-type Fab. Thus, it was suggested that the introduction of a negative charge at position 138 in the L-chain by the deamidation significantly affected the stability of humanized Fab.

Fab fragment labeled with ICG-derivative for detecting digestive tract cancer.

PubMed

Yano, Hiromi; Muguruma, Naoki; Ito, Susumu; Aoyagi, Eriko; Kimura, Tetsuo; Imoto, Yoshitaka; Cao, Jianxin; Inoue, Shohei; Sano, Shigeki; Nagao, Yoshimitsu; Kido, Hiroshi

2006-09-01

In previous studies, we generated infrared ray fluorescence-labeled monoclonal antibodies and developed an infrared ray fluorescence endoscope capable of detecting the monoclonal antibodies to establish a novel diagnostic technique for gastrointestinal cancer. Although the whole IgG molecule has commonly been used for preparation of labeled antibodies, labeled IgG displays insufficient sensitivity and specificity, probably resulting from non-specific binding of the Fc fragment to target cells or interference between fluorochromes on the identical labeled antibody, which might be caused by molecular structure. In this in vitro study, we characterized an Fc-free fluorescence-labeled Fab fragment, which was expected to yield more specific binding to target cells than the whole IgG molecule. An anti-mucin antibody and ICG-ATT, an ICG derivative, were used as the labeled antibody and labeling compound, respectively. Paraffin sections of excised gastric cancer tissues were subjected to staining. The labeled whole IgG molecule (ICG-ATT-labeled IgG) and the labeled Fab fragment (ICG-ATT-labeled Fab) were prepared according to a previous report, and the fluorescence properties, antibody activities, and features of fluorescence microscope images obtained from paraffin sections were compared. Both ICG-ATT-labeled Fab and ICG-ATT-labeled IgG were excited by a near infrared ray of 766nm, and maximum emission occurred at 804nm. Antibody activities of ICG-ATT-labeled Fab were shown to be similar to those of unlabeled anti-MUC1 antibody. The fluorescence intensity obtained from paraffin sections of excised gastric cancer tissues revealed a tendency to be greater with ICG-ATT-labeled Fab than with ICG-ATT-labeled IgG. The infrared ray fluorescence-labeled Fab fragment was likely to be more specific than the conventionally labeled antibodies. Fragmentation of antibodies is considered to contribute to improved sensitivity and specificity of labeled antibodies for detection of micro

Resistance to AFN-1252 Arises from Missense Mutations in Staphylococcus aureus Enoyl-acyl Carrier Protein Reductase (FabI)*

PubMed Central

Yao, Jiangwei; Maxwell, John B.; Rock, Charles O.

2013-01-01

AFN-1252 is a potent antibiotic against Staphylococcus aureus that targets the enoyl-acyl carrier protein reductase (FabI). A thorough screen for AFN-1252-resistant strains was undertaken to identify the spectrum of mechanisms for acquired resistance. A missense mutation in fabI predicted to encode FabI(M99T) was isolated 49 times, and a single isolate was predicted to encode FabI(Y147H). AFN-1252 only bound to the NADPH form of FabI, and the close interactions between the drug and Met-99 and Tyr-147 explained how the mutations would result in resistant enzymes. The clone expressing FabI(Y147H) had a pronounced growth defect that was rescued by exogenous fatty acid supplementation, and the purified protein had less than 5% of the enzymatic activity of FabI. FabI(Y147F) was also catalytically defective but retained its sensitivity to AFN-1252, illustrating the importance of the conserved Tyr-147 hydroxyl group in FabI function. The strains expressing FabI(M99T) exhibited normal growth, and the biochemical properties of the purified protein were indistinguishable from those of FabI. The AFN-1252 Kiapp increased from 4 nm in FabI to 69 nm in FabI(M99T), accounting for the increased resistance of the corresponding mutant strain. The low activity of FabI(Y147H) precluded an accurate Ki measurement. The strain expressing FabI(Y147H) was also resistant to triclosan; however, the strain expressing FabI(M99T) was more susceptible. Strains with higher levels of AFN-1252 resistance were not obtained. The AFN-1252-resistant strains remained sensitive to submicromolar concentrations of AFN-1252, which blocked growth through inhibition of fatty acid biosynthesis at the FabI step. PMID:24189061

Recurrent hemorrhage after western diamondback rattlesnake envenomation treated with crotalidae polyvalent immune fab (ovine).

PubMed

Fazelat, Joyia; Teperman, Sheldon H; Touger, Michael

2008-11-01

Recurrent coagulopathy has been observed in patients after rattlesnake envenomation treated with Crotalidae Polyvalent Immune Fab (ovine) [FabAV]. While recurrent coagulopathy is well documented in the literature, clinically significant sequelae have not been reported. We present a case of recurrent thrombocytopenia after western diamondback envenomation treated with FabAV, resulting in an extensive recurrent local hemorrhage. A 24-year-old male presented to our emergency department several hours after western diamondback envenomation. He sustained bites to both hands and the right flank by leaning over his pet "snake enclosure." On presentation, the patient was hypotensive, tachycardic, and thrombocytopenic with a platelet count of 17/nl. Antivenom therapy was initiated according to the standard FabAV protocol. However, sixteen hours after completion of the recommended FabAV infusion, the patient experienced a recurrent thrombocytopenia with a dramatic seventeen point drop in hematocrit. The source of bleeding was clinically attributed to an expanding hematoma at the site of envenomation. FabAV has become the standard treatment for symptomatic crotalid envenomation. However, the pharmacokinetics of this drug predispose it to recurrent coagulopathies. While studies have shown persistent and recurrent coagulopathic derangements after FabAV therapy, no clinically significant sequelae have been reported. This report highlights the potential for recurrent local hemorrhagic complications following rattlesnake envenomation, even after treatment guided by the current FabAV protocol. Recurrent coagulopathy following FabAV therapy can result in clinically significant hemorrhage, supporting the observation that extended repeat dosing may be necessary to adequately treat subjects of rattlesnake envenomation.

High contrast tumor imaging with radio-labeled antibody Fab fragments tailored for optimized pharmacokinetics via PASylation

PubMed Central

Mendler, Claudia T; Friedrich, Lars; Laitinen, Iina; Schlapschy, Martin; Schwaiger, Markus; Wester, Hans-Jürgen; Skerra, Arne

2015-01-01

Although antigen-binding fragments (Fabs) of antibodies constitute established tracers for in vivo radiodiagnostics, their functionality is hampered by a very short circulation half-life. PASylation, the genetic fusion with a long, conformationally disordered amino acid chain comprising Pro, Ala and Ser, provides a convenient way to expand protein size and, consequently, retard renal filtration. Humanized αHER2 and αCD20 Fabs were systematically fused with 100 to 600 PAS residues and produced in E. coli. Cytofluorimetric titration analysis on tumor cell lines confirmed that antigen-binding activities of the parental antibodies were retained. The radio-iodinated PASylated Fabs were studied by positron emission tomography (PET) imaging and biodistribution analysis in mouse tumor xenograft models. While the unmodified αHER2 and αCD20 Fabs showed weak tumor uptake (0.8% and 0.2% ID/g, respectively; 24 h p.i.) tumor-associated radioactivity was boosted with increasing PAS length (up to 9 and 26-fold, respectively), approaching an optimum for Fab-PAS400. Remarkably, 6- and 5-fold higher tumor-to-blood ratios compared with the unmodified Fabs were measured in the biodistribution analysis (48 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200, respectively. These findings were confirmed by PET studies, showing high imaging contrast in line with tumor-to-blood ratios of 12.2 and 5.7 (24 h p.i.) for αHER2 Fab-PAS100 and Fab-PAS200. Even stronger tumor signals were obtained with the corresponding αCD20 Fabs, both in PET imaging and biodistribution analysis, with an uptake of 2.8% ID/g for Fab-PAS100 vs. 0.24% ID/g for the unmodified Fab. Hence, by engineering Fabs via PASylation, plasma half-life can be tailored to significantly improve tracer uptake and tumor contrast, thus optimally matching reagent/target interactions. PMID:25484039

Frontal assessment battery (FAB) performance following traumatic brain injury hospitalized in an acute care setting.

PubMed

Rojas, Natalia; Laguë-Beauvais, Maude; Belisle, Arielle; Lamoureux, Julie; AlSideiri, Ghusn; Marcoux, Judith; Maleki, Mohammed; Alturki, Abdulrahman Y; Anchouche, Sonia; Alquraini, Hanan; Feyz, Mitra; Guise, Elaine de

2018-01-19

The Frontal Assessment Battery (FAB) has been shown to be useful in several clinical settings. The aim of the present study was to examine the performance of patients with traumatic brain injury (TBI) on the FAB and to predict their acute outcome. The FAB was administered to 89 patients with mild (27 = uncomplicated and 39 = complicated) and moderate (n = 23) TBI during hospitalization in an acute care setting. The length of stay in days (LOS), Glasgow Outcome Scale-Revised score (GOSE) and Disability Rating Scale (DRS) score were collected. Results showed no significant differences between the three groups on the FAB score, but age and education were significantly associated with the FAB score. Parietal lesions were associated with lower total FAB score, and with the Similarities, Motor series and Conflicting instructions subscales, while frontal lesions were associated with lower performance on the Motor series and Conflicting instructions subscales. Total FAB score was significantly correlated with all outcome measures, and together the FAB total score and the Glasgow Coma Scale (GCS) score explained 30.8% of the variance in the DRS score. The FAB may be useful clinically to acutely assess frontal and parietal lobe functions at bedside in patients with TBI and, in combination with the GCS score to measure TBI severity, can enable clinicians to predict early outcome.

LC-MS/MS quantification of free and Fab-bound colchicine in plasma, urine and organs following colchicine administration and colchicine-specific Fab fragments treatment in Göttingen minipigs.

PubMed

Fabresse, Nicolas; Allard, Julien; Sardaby, Marine; Thompson, Adrian; Clutton, R Eddie; Eddleston, Michael; Alvarez, Jean-Claude

2017-08-15

Clinical evaluation of a colchicine specific antigen-binding fragment (Fab) in order to treat colchicine poisoning required the development of an accurate method allowing quantification of free and Fab-bound colchicine in plasma and urine, and free colchicine in tissues, to measure colchicine redistribution after Fab administration. Three methods have been developed for this purpose, and validated in plasma, urine and liver: total colchicine was determined after denaturation of Fab by dilution in water and heating; free colchicine was separated from Fab-bound colchicine by filtration with 30KDa micro-filters; tissues were homogenized in a tissue mixer. Deuterated colchicine was used as internal standard. Samples were extracted by liquid-liquid extraction and analyzed with a LC-MS/MS. LOQ were 0.5ng/mL in plasma and urine for free and total colchicine and 5pg/mg in tissues. The methods were linear in the 0.5-100ng/mL range in plasma and urine, and 5-300pg/mg in tissues with determination coefficients>0.99. Precision and accuracy of QC samples presented a CV<9.4%. The methods require only 200μL of sample and allow a high throughput due to short analytical run (2min). These methods were successfully applied to a pig intoxicated with colchicine and treated with colchicine specific Fab fragments. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of Fab' fragments of specific egg yolk antibody (IgY-Fab') against Shewanella putrefaciens on the preservation of refrigerated turbot.

PubMed

Zhang, Qian; Lin, Hong; Sui, Jianxin; Wang, Jingxue; Cao, Limin

2015-01-01

In our previous studies the specific egg yolk antibody (IgY) against Shewanella putrefaciens (one of the specific spoilage organisms for marine products during aerobic chilling storage) demonstrated significant activity to prolong the shelf life of refrigerated fish. The exploitation of the antigen-binding fragment plus the hinge region (IgY-Fab') is now considered a promising method for improving the efficiency of such natural antimicrobial agents. The antimicrobial activity of IgY-Fab' against S. putrefaciens was investigated using refrigerated turbot as samples. By microbial, chemical and sensory tests, it was shown to be able to effectively inhibit bacterial growth and prolong the shelf life of samples, with an efficiency evaluated significantly higher than that of whole IgY with the same molarity. The interaction between IgY agents and S. putrefaciens cells was also investigated, and the IgY-Fab' showed a much greater ability to damage cell membranes than the whole IgY. Compared to whole IgY with the same molarity, IgY-Fab' demonstrated higher and more durable antimicrobial efficiency. Such a result was assumed to be closely related to its structural properties (such as the much lower molecular weight), which may enhance its ability to influence physiological activities of antigen bacteria, especially the property or/and structure of cell membranes. © 2014 Society of Chemical Industry.

Fab is the most efficient format to express functional antibodies by yeast surface display.

PubMed

Sivelle, Coline; Sierocki, Raphaël; Ferreira-Pinto, Kelly; Simon, Stéphanie; Maillere, Bernard; Nozach, Hervé

2018-04-30

Multiple formats are available for engineering of monoclonal antibodies (mAbs) by yeast surface display, but they do not all lead to efficient expression of functional molecules. We therefore expressed four anti-tumor necrosis factor and two anti-IpaD mAbs as single-chain variable fragment (scFv), antigen-binding fragment (Fab) or single-chain Fabs and compared their expression levels and antigen-binding efficiency. Although the scFv and scFab formats are widely used in the literature, 2 of 6 antibodies were either not or weakly expressed. In contrast, all 6 antibodies expressed as Fab revealed strong binding and high affinity, comparable to that of the soluble form. We also demonstrated that the variations in expression did not affect Fab functionality and were due to variations in light chain display and not to misfolded dimers. Our results suggest that Fab is the most versatile format for the engineering of mAbs.

Coagulation parameters in copperhead compared to other Crotalinae envenomation: secondary analysis of the F(ab')2 versus Fab antivenom trial.

PubMed

Gerardo, Charles J; Vissoci, Joao R Nickenig; Brown, Michael W J; Bush, Sean P

2017-02-01

Coagulation derangements in copperhead envenomation are considered less severe than other crotaline envenomations, resulting in recommendations to limit both coagulation testing and antivenom treatment. A prospective, blinded, multicenter, randomized clinical trial comparing the effectiveness of F(ab') 2 versus Fab antivenom in crotaline envenomation patients was completed in 2011. We determined the difference between coagulation parameters in copperhead compared to other crotaline envenomations. We performed a post hoc analysis comparing the coagulation parameters (platelets and fibrinogen) prospectively obtained in the aforementioned trial. All the patients received antivenom in one of three treatment arms [F(ab') 2 with maintenance, F(ab') 2 with placebo maintenance, or Fab with maintenance]. Coagulation parameters were measured at pretreatment baseline, during acute hospitalization, day 5, day 8, and day 15 post-envenomation. Mean platelet count and fibrinogen levels for the copperhead and other crotaline groups were compared. The platelet and fibrinogen point estimates with distribution are presented graphically over time. 122 patients were enrolled in the study. There were 22 patients with copperhead envenomation, 93 with other crotaline envenomations, and 7 that could not be definitively determined. The mean age was 42 (SD 20) years. There was a minor pretreatment difference in mean baseline platelet count between the copperhead group (246 × 109/L 95% CI 215, 277) compared to other crotaline envenomation patients (184 × 109/L 95% CI 167, 202). There was a modest pretreatment difference in mean fibrinogen level between copperhead patients (345 mg/dL 95% CI 277, 415) and other crotaline patients (261mg/dL 95% CI 241, 281). Pretreatment coagulation parameter means were normal and converged post treatment. On average, copperhead envenomations have less severe initial coagulation derangements. However, in mild envenomations, differences in laboratory

Comparison of early myocardial technetium-99m pyrophosphate uptake to early peaking of creatine kinase and creatine kinase-MB as indicators of early reperfusion in acute myocardial infarction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kondo, M.; Yuzuki, Y.; Arai, H.

1987-10-01

The value of technetium-99m pyrophosphate (Tc-99m-PYP) scintigraphy as an indicator of reperfusion 2.8 to 8 hours after the onset of symptoms of acute myocardial infarction was compared with the value of early peak creatine kinase (CK) and CK-MB release within 16 hours after the onset of symptoms. In 29 patients who received thrombolytic therapy, recanalization was seen (group 1) and in 7 it was not (group 2). In 23 patients (79%) in group 1 scintigraphic findings were positive and in all 7 in group 2 they were negative. In 15 patients (52%) in group 1 and 1 patient (14%) inmore » group 2, CK reached its peak level within 16 hours. In 20 patients (69%) in group 1 and 3 (43%) in group 2 the CK-MB level reached a peak within 16 hours. The sensitivity, specificity and predictive accuracy of positive results of early Tc-99m-PYP scintigraphy in predicting the reperfusion were 79%, 100% and 83%. These values are significantly higher than or similar to those of early peaking of CK and CK-MB release. In contrast to measurements of enzyme release, reperfusion data for Tc-99m-PYP scintigraphy are available immediately after thrombolytic therapy. Therefore, early Tc-99m-PYP scintigraphy (3 to 8 hours after onset of symptoms) is valuable as a noninvasive technique for early diagnosis of reperfusion.« less

Near-Atomic Resolution Structure of a Highly Neutralizing Fab Bound to Canine Parvovirus

PubMed Central

Organtini, Lindsey J.; Lee, Hyunwook; Iketani, Sho; Huang, Kai; Ashley, Robert E.; Makhov, Alexander M.; Conway, James F.

2016-01-01

ABSTRACT Canine parvovirus (CPV) is a highly contagious pathogen that causes severe disease in dogs and wildlife. Previously, a panel of neutralizing monoclonal antibodies (MAb) raised against CPV was characterized. An antibody fragment (Fab) of MAb E was found to neutralize the virus at low molar ratios. Using recent advances in cryo-electron microscopy (cryo-EM), we determined the structure of CPV in complex with Fab E to 4.1 Å resolution, which allowed de novo building of the Fab structure. The footprint identified was significantly different from the footprint obtained previously from models fitted into lower-resolution maps. Using single-chain variable fragments, we tested antibody residues that control capsid binding. The near-atomic structure also revealed that Fab binding had caused capsid destabilization in regions containing key residues conferring receptor binding and tropism, which suggests a mechanism for efficient virus neutralization by antibody. Furthermore, a general technical approach to solving the structures of small molecules is demonstrated, as binding the Fab to the capsid allowed us to determine the 50-kDa Fab structure by cryo-EM. IMPORTANCE Using cryo-electron microscopy and new direct electron detector technology, we have solved the 4 Å resolution structure of a Fab molecule bound to a picornavirus capsid. The Fab induced conformational changes in regions of the virus capsid that control receptor binding. The antibody footprint is markedly different from the previous one identified by using a 12 Å structure. This work emphasizes the need for a high-resolution structure to guide mutational analysis and cautions against relying on older low-resolution structures even though they were interpreted with the best methodology available at the time. PMID:27535057

20 CFR 30.319 - May a claimant request reconsideration of a final decision of the FAB?

Code of Federal Regulations, 2010 CFR

2010-04-01

... final decision of the FAB? 30.319 Section 30.319 Employees' Benefits OFFICE OF WORKERS' COMPENSATION... reconsideration of a final decision of the FAB? (a) A claimant may request reconsideration of a final decision of the FAB by filing a written request with the FAB within 30 days from the date of issuance of such...

Selective disulfide reduction for labeling and enhancement of Fab antibody fragments.

PubMed

Kirley, Terence L; Greis, Kenneth D; Norman, Andrew B

2016-11-25

Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab') 2 fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab') 2 fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neither homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications. Copyright © 2016 Elsevier Inc. All rights reserved.

Recurrent coagulopathy and thrombocytopenia in children treated with crotalidae polyvalent immune fab: a case series.

PubMed

Miller, Alexander D; Young, Michael C; DeMott, Megan C; Ly, Binh T; Clark, Richard F

2010-08-01

Recurrent signs and symptoms after initial treatment and control of coagulopathy and thrombocytopenia after American pit viper (crotaline) envenomations have been previously described in patients treated with Crotalidae polyvalent immune Fab antivenom (FabAV). The significance and necessity of treatment of these recurrent abnormalities are uncertain. Our goal was to further characterize recurrent coagulopathy or thrombocytopenia in pediatric patients. All cases presenting to our Toxicology Consult Service, which covers 6 hospitals in a metropolitan area, from May 2007 to April 2008 with recurrent coagulopathy after initial control with FabAV were included and retrospectively reviewed. Four cases of pediatric patients are presented who presented with recurrent coagulopathy and/or thrombocytopenia after initial control with FabAV. The patients were all treated with delayed administration of FabAV with variable results. Blood products administered without concurrent FabAV were of limited use. The laboratory abnormalities took up to 18 days to resolve in one case. One patient developed hemodynamically significant spontaneous bleeding. The cases presented here suggest administration of FabAV may correct delayed coagulopathy associated with crotaline envenomations. The first 3 cases illustrate that in the face of severe derangements in laboratory values, most envenomated patients treated with FabAV do not develop significant bleeding. These cases may respond to additional antivenom alone. However, case 4 illustrates that hemodynamically significant spontaneous bleeding can occur. Until more data are available, readministration of FabAV is a reasonable first-line therapy for delayed coagulopathy associated with crotaline envenomations.

A universal phage display system for the seamless construction of Fab libraries.

PubMed

Nelson, Renae S; Valadon, Philippe

2017-11-01

The construction of Fab phage libraries requires the cloning of domains from both the light and the heavy chain of antibodies. Despite the advent of powerful strategies such as splicing-by-overlap extension PCR, obtaining high quality libraries with excellent coverage remains challenging. Here, we explored the use of type IIS restriction enzymes for the seamless cloning of Fab libraries. We analyzed human, murine and rabbit germline antibody repertoires and identified combinations of restriction enzymes that exhibit very few or no recognition sites in the antibody sequences. We describe three phagemid vectors, pUP-22Hb, pUP-22Mc and pUP-22Rc, which were employed for cloning the Fab repertoire of these hosts using BsmBI and SapI (human) or SapI alone (mouse and rabbit). Using human serum albumin as a model immunization, we built a mouse/human chimeric Fab library and a mouse Fab library in a single step ligation and successfully panned multiple cognate antibodies. The overall process is highly scalable and faster than PCR-based techniques, with a Fab insertion success rate of around 80%. By using carefully chosen overhangs on each end of the antibody domains, this approach paves the way to the universal, sequence- and vector-independent cloning and reformatting of antibody libraries. Copyright © 2017 Elsevier B.V. All rights reserved.

"Fab 13": The Learning Factory.

ERIC Educational Resources Information Center

Crooks, Steven M.; Eucker, Tom R.

2001-01-01

Describes how situated learning theory was employed in the design of Fab 13, a four-day simulation-based learning experience for manufacturing professionals at Intel Corporation. Presents a conceptual framework for understanding situated learning and discusses context, content, anchored instruction, facilitation, scaffolding, collaborating,…

Safety and efficacy of Crotalidae Polyvalent Immune Fab in pediatric crotaline envenomations.

PubMed

Pizon, Anthony F; Riley, Bradley D; LoVecchio, Frank; Gill, Ruqayya

2007-04-01

Since it was approved by the Food and Drug Administration in October 2000, Crotalidae Polyvalent Immune Fab (CroFab) has largely replaced previously used crotaline antivenom. CroFab is more specifically tailored for crotalids of North America and is less allergenic than whole immunoglobulin antivenoms. However, premarketing and postmarketing studies have excluded children. To describe the safety and efficacy of CroFab in pediatric crotaline envenomations. Using admission and billing records, the authors identified all children 13 years of age and younger treated with CroFab at a pediatric hospital between October 2000 and September 2005. Charts were reviewed by two trained, blinded extractors. Data regarding age, signs of envenomation, laboratory values, total antivenom vials used, total vials used to gain control, transfused blood products, signs of acute allergy to antivenom, and any surgical procedures were abstracted. Data were analyzed using descriptive statistics. Twenty-four patients were identified, and their mean age was 7.3 (range, 1.9-13) years. At presentation, all had swelling, 14 (58%) had a prothrombin time >13 seconds, two (8.3%) had a fibrinogen level <150 mg/dL, and three (12.5%) had platelet counts <150,000/mL. The mean number of total antivenom vials used was 12.3 (range, 4-24). Five patients had resolution of swelling, but platelet counts continued to fall despite antivenom treatment. No patient required blood products, debridement of skin, or fasciotomy. There was only one (4.2%) possible acute allergy to CroFab, and there were no deaths. In this pediatric series, CroFab appears safe and effective, despite occasional resistant thrombocytopenia.

Machine learning for fab automated diagnostics

NASA Astrophysics Data System (ADS)

Giollo, Manuel; Lam, Auguste; Gkorou, Dimitra; Liu, Xing Lan; van Haren, Richard

2017-06-01

Process optimization depends largely on field engineer's knowledge and expertise. However, this practice turns out to be less sustainable due to the fab complexity which is continuously increasing in order to support the extreme miniaturization of Integrated Circuits. On the one hand, process optimization and root cause analysis of tools is necessary for a smooth fab operation. On the other hand, the growth in number of wafer processing steps is adding a considerable new source of noise which may have a significant impact at the nanometer scale. This paper explores the ability of historical process data and Machine Learning to support field engineers in production analysis and monitoring. We implement an automated workflow in order to analyze a large volume of information, and build a predictive model of overlay variation. The proposed workflow addresses significant problems that are typical in fab production, like missing measurements, small number of samples, confounding effects due to heterogeneity of data, and subpopulation effects. We evaluate the proposed workflow on a real usecase and we show that it is able to predict overlay excursions observed in Integrated Circuits manufacturing. The chosen design focuses on linear and interpretable models of the wafer history, which highlight the process steps that are causing defective products. This is a fundamental feature for diagnostics, as it supports process engineers in the continuous improvement of the production line.

Anti-fouling properties of Fab' fragments immobilized on silane-based adlayers

NASA Astrophysics Data System (ADS)

Crivianu-Gaita, Victor; Romaschin, Alexander; Thompson, Michael

2015-12-01

Biosensors require surfaces that are highly specific towards the target analyte and that are minimally fouling. However, surface tuning to minimize fouling is a difficult task. The last decade has seen an increase in the use of immobilized antigen-binding antibody fragments (Fab') in biosensors. One Fab' linker compound S-(11-trichlorosilyl-undecanyl)-benzothiosulfonate (TUBTS) and three spacers were used to create the silane-based adlayers. The ultra-high frequency electromagnetic piezoelectric acoustic sensor (EMPAS) was used to gauge the fouling properties of the various surfaces using bovine serum albumin (BSA), goat IgG, and mouse serum. X-ray photoelectron spectroscopy (XPS), contact angle, and atomic force microscopy (AFM) were employed to characterize the surfaces. It was discovered that immobilized oriented Fab' fragments reduced the fouling levels of surfaces up to 80% compared to the surfaces without fragments. An explanation for this phenomenon is that the antibody fragments increase the hydration of the surfaces and aid in the formation of an anti-fouling water barrier. The anti-fouling effect of the Fab' fragments is at its maximum when there is an even distribution of fragments across the surfaces. Finally, using Fab'-covered surfaces, a cancer biomarker was detected from serum, showing the applicability of this work to the field of biodetection.

Late hematologic toxicity following treatment of rattlesnake envenomation with crotalidae polyvalent immune Fab antivenom.

PubMed

Ruha, Anne-Michelle; Curry, Steven C; Albrecht, Clay; Riley, Brad; Pizon, Anthony

2011-01-01

North American rattlesnake envenomations commonly produce defibrination, coagulopathy and/or thrombocytopenia, which may be reversed following treatment with Crotalidae Polyvalent Immune Fab Ovine (FabAV). Despite initial resolution with FabAV, late onset or recurrence of venom-induced hematologic effects may occur. Time at which onset of late hematotoxicity may first be detected is unknown. The purpose of this study was to identify the incidence and time of onset of recurrent or new late hypofibrinogenemia, coagulopathy, or thrombocytopenia in a cohort of rattlesnake envenomation patients seen in outpatient follow-up after treatment with FabAV, and to report hematologic outcomes in these patients. Review of 66 charts of patients with rattlesnake envenomation who were treated with FabAV, and subsequently had outpatient follow-up evaluation at least 48 h after last FabAV, was performed. Demographic information, rattlesnake and bite characteristics, dose and timing of antivenom administration, adverse events, in-patient laboratory values, length of hospital stay, and follow-up laboratory values were collected. The primary outcome parameters were recurrent or delayed onset coagulopathy, hypofibrinogenemia, or thrombocytopenia identified no sooner than 48 h after last dose of FabAV. Prior to control of the envenomation with FabAV, 42 patients (63.6%) experienced hematologic toxicity. At follow-up, 21 patients (32%) were found to have late coagulopathy, hypofibrinogenemia, or thrombocytopenia. Of twenty-three patients (35%) with more than one follow-up visit, fifteen had normal laboratory findings at the first follow-up visit. Five of these 15 patients (8% of total study group; 33% of this subgroup) with normal hematologic studies at first follow-up exhibited late hematologic toxicity at second follow-up. Severe late hematologic toxicity developed in five of 66 (8%) patients. One patient was retreated with FabAV for late severe thrombocytopenia. Recurrent and delayed

Structural Characterisation of FabG from Yersinia pestis, a Key Component of Bacterial Fatty Acid Synthesis.

PubMed

Nanson, Jeffrey D; Forwood, Jade K

2015-01-01

Ketoacyl-acyl carrier protein reductases (FabG) are ubiquitously expressed enzymes that catalyse the reduction of acyl carrier protein (ACP) linked thioesters within the bacterial type II fatty acid synthesis (FASII) pathway. The products of these enzymes, saturated and unsaturated fatty acids, are essential components of the bacterial cell envelope. The FASII reductase enoyl-ACP reductase (FabI) has been the focus of numerous drug discovery efforts, some of which have led to clinical trials, yet few studies have focused on FabG. Like FabI, FabG appears to be essential for survival in many bacteria, similarly indicating the potential of this enzyme as a drug target. FabG enzymes are members of the short-chain alcohol dehydrogenase/reductase (SDR) family, and like other SDRs, exhibit highly conserved secondary and tertiary structures, and contain a number of conserved sequence motifs. Here we describe the crystal structures of FabG from Yersinia pestis (YpFabG), the causative agent of bubonic, pneumonic, and septicaemic plague, and three human pandemics. Y. pestis remains endemic in many parts of North America, South America, Southeast Asia, and Africa, and a threat to human health. YpFabG shares a high degree of structural similarity with bacterial homologues, and the ketoreductase domain of the mammalian fatty acid synthase from both Homo sapiens and Sus scrofa. Structural characterisation of YpFabG, and comparison with other bacterial FabGs and the mammalian fatty acid synthase, provides a strong platform for virtual screening of potential inhibitors, rational drug design, and the development of new antimicrobial agents to combat Y. pestis infections.

Temporal Trends in the Prevalence, Severity, and Localization of Myocardial Ischemia and Necrosis at Myocardial Perfusion Imaging After Myocardial Infarction.

PubMed

Nudi, Francesco; Schillaci, Orazio; Di Belardino, Natale; Versaci, Francesco; Tomai, Fabrizio; Pinto, Annamaria; Neri, Giandomenico; Procaccini, Enrica; Nudi, Alessandro; Frati, Giacomo; Biondi-Zoccai, Giuseppe

2017-10-15

The definition, presentation, and management of myocardial infarction (MI) have changed substantially in the last decade. Whether these changes have impacted on the presence, severity, and localization of necrosis at myocardial perfusion imaging (MPI) has not been appraised to date. Subjects undergoing MPI and reporting a history of clinical MI were shortlisted. We focused on the presence, severity, and localization of necrosis at MPI with a retrospective single-center analysis. A total of 10,476 patients were included, distinguishing 5 groups according to the period in which myocardial perfusion scintigraphy had been performed (2004 to 2005, 2006 to 2007, 2008 to 2009, 2010 to 2011, 2012 to 2013). Trend analysis showed over time a significant worsening in baseline features (e.g., age, diabetes mellitus, and Q waves at electrocardiogram), whereas medical therapy and revascularization were offered with increasing frequency. Over the years, there was also a lower prevalence of normal MPI (from 16.8% to 13.6%) and ischemic MPI (from 35.6% to 32.8%), and a higher prevalence of ischemic and necrotic MPI (from 12.0% to 12.7%) or solely necrotic MPI (from 35.7% to 40.9%, p <0.001). Yet the prevalence of severe ischemia decreased over time from 11.4% to 2.0%, with a similar trend for moderate ischemia (from 15.9% to 11.8%, p <0.001). Similarly sobering results were wound for the prevalence of severe necrosis (from 19.8% to 8.2%) and moderate necrosis (from 8.5% to 7.8%, p = 0.028). These trends were largely confirmed at regional level and after propensity score matching. In conclusion, the outlook of stable patients with previous MI has substantially improved in the last decade, with a decrease in the severity of residual myocardial ischemia and necrosis, despite an apparent worsening in baseline features. Copyright © 2017 Elsevier Inc. All rights reserved.

Crystallization and preliminary X-ray diffraction analysis of FabG from Yersinia pestis.

PubMed

Nanson, Jeffrey David; Forwood, Jade Kenneth

2014-01-01

The type II fatty-acid biosynthesis pathway of bacteria provides enormous potential for antibacterial drug development owing to the structural differences between this and the type I fatty-acid biosynthesis system found in mammals. β-Ketoacyl-ACP reductase (FabG) is responsible for the reduction of the β-ketoacyl group linked to acyl carrier protein (ACP), and is essential for the formation of fatty acids and bacterial survival. Here, the cloning, expression, purification, crystallization and diffraction of FabG from Yersinia pestis (ypFabG), the highly virulent causative agent of plague, are reported. Recombinant FabG was expressed, purified to homogeneity and crystallized via the hanging-drop vapour-diffusion technique. Diffraction data were collected at the Australian Synchrotron to 2.30 Å resolution. The crystal displayed P2(1)2(1)2(1) symmetry, with unit-cell parameters a = 68.22, b = 98.68, c = 169.84 Å, and four ypFabG molecules in the asymmetric unit.

Immediate hypersensitivity reaction associated with the rapid infusion of Crotalidae polyvalent immune Fab (ovine).

PubMed

Holstege, Christopher P; Wu, Jeffrey; Baer, Alexander B

2002-06-01

A 16-year-old boy presented to the emergency department with rapidly progressing extremity pain, edema, and ecchymosis after envenomation by a copperhead. Crotalidae polyvalent immune Fab (ovine) (CroFab; FabAV) was infused. Six vials were placed in 250 mL of normal saline solution, and the infusion was gradually increased. Fifty minutes after beginning, the infusion was increased to 640 mL/h. Within minutes of the rate increase, the patient experienced full-body urticaria, facial edema, voice change, and tachycardia. The infusion was stopped. Hydroxyzine pamoate, famotidine, methylprednisolone, and a 1-L bolus of normal saline solution were administered intravenously. The symptoms abated, and the remaining FabAV was infused at a slower rate without return of this reaction. This immediate hypersensitivity reaction was most likely a rate-related anaphylactoid reaction that has not been previously reported with FabAV.[Holstege CP, Wu J, Baer AB. Immediate hypersensitivity reaction associated with the rapid infusion of Crotalidae polyvalent immune Fab (ovine). Ann Emerg Med. June 2002;39:677-679.

Near-Atomic Resolution Structure of a Highly Neutralizing Fab Bound to Canine Parvovirus.

PubMed

Organtini, Lindsey J; Lee, Hyunwook; Iketani, Sho; Huang, Kai; Ashley, Robert E; Makhov, Alexander M; Conway, James F; Parrish, Colin R; Hafenstein, Susan

2016-11-01

Canine parvovirus (CPV) is a highly contagious pathogen that causes severe disease in dogs and wildlife. Previously, a panel of neutralizing monoclonal antibodies (MAb) raised against CPV was characterized. An antibody fragment (Fab) of MAb E was found to neutralize the virus at low molar ratios. Using recent advances in cryo-electron microscopy (cryo-EM), we determined the structure of CPV in complex with Fab E to 4.1 Å resolution, which allowed de novo building of the Fab structure. The footprint identified was significantly different from the footprint obtained previously from models fitted into lower-resolution maps. Using single-chain variable fragments, we tested antibody residues that control capsid binding. The near-atomic structure also revealed that Fab binding had caused capsid destabilization in regions containing key residues conferring receptor binding and tropism, which suggests a mechanism for efficient virus neutralization by antibody. Furthermore, a general technical approach to solving the structures of small molecules is demonstrated, as binding the Fab to the capsid allowed us to determine the 50-kDa Fab structure by cryo-EM. Using cryo-electron microscopy and new direct electron detector technology, we have solved the 4 Å resolution structure of a Fab molecule bound to a picornavirus capsid. The Fab induced conformational changes in regions of the virus capsid that control receptor binding. The antibody footprint is markedly different from the previous one identified by using a 12 Å structure. This work emphasizes the need for a high-resolution structure to guide mutational analysis and cautions against relying on older low-resolution structures even though they were interpreted with the best methodology available at the time. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Effectiveness of Alpha-toxin Fab Monoclonal Antibody Therapy in Limiting the Pathology of Staphylococcus aureus Keratitis.

PubMed

Caballero, Armando R; Foletti, Davide L; Bierdeman, Michael A; Tang, Aihua; Arana, Angela M; Hasa-Moreno, Adela; Sangalang, Emma Ruth B; O'Callaghan, Richard J

2015-08-01

To investigate the effectiveness of a high-affinity human monoclonal antibody Fab fragment to Staphylococcus aureus alpha-toxin (LTM14 Fab) as therapy for S. aureus keratitis. A single topical drop of the LTM14 Fab antibody to alpha-toxin alone, or in 0.006% benzalkonium chloride (BAK), was applied every 30 min to S. aureus-infected rabbit corneas from 9 to 14 hours post-infection. Erosions and pathology were measured at 15 h post-infection. LTM14 Fab with BAK limited corneal erosions better than LTM14 Fab alone (p = 0.036), and both limited erosions compared to untreated eyes (p ≤ 0.0001). Overall pathology was similar in all groups (p ≥ 0.070), but iritis and chemosis were reduced by treatment (p ≤ 0.036). The high-affinity human monoclonal Fab fragment antibody (LTM14 Fab) to S. aureus alpha-toxin was effective in reducing corneal damage during S. aureus keratitis.

[FAB immunoglobulin fragments. I. The comparative characteristics of the serological and virus-neutralizing properties of a gamma globulin against tick-borne encephalitis and of the FAB fragments isolated from it].

PubMed

Barban, P S; Minaeva, V M; Pantiukhina, A N; Startseva, M G

1976-06-01

A comparative study was made of the serological properties and virus-neutralizing activity of antiencephalitis gamma-globulin and Fab-fragments isolated from it by gel-filtration. Horse immunoglobulins against the autumno-summer tick-borne encephalitis virus could be disintegrated with the aid of papaine to monovalent Fab-fragments which (according to the complement fixation reaction, the test of suppression of the complement fixation, and the HAIT) retained the serological activity whose level was compared with that of the serological activity of gamma-globulin. Fab-fragments possessed a marked virus-neutralizing activity. The mean value of a logarithm of the neutralization index was 2.65 +/- 0.2 for Fab-fragments and 3.74 +/- 0.38 for gamma-globulin (P less than 0.01).

FabAV antivenin use after copperhead snakebite: clinically indicated or knee-jerk reaction?

PubMed

Gale, Stephen C; Peters, Jo Ann; Allen, LaDonna; Creath, Robert; Dombrovskiy, Viktor Y

2016-01-01

Crotalidae Polyvalent Immune Fab (Ovine) (FabAV) antivenin is commonly recommended after pit viper snakebites. Because copperhead envenomations are usually self-limited, some physicians are reluctant to use this costly treatment routinely, while others follow a more liberal approach. We hypothesized that, in practice, only patients with evidence of significant (moderate or severe) copperhead envenomation [those with snakebite severity score (SSS) > 3] receive FabAV and examined a large cohort to determine the relationship between clinical findings and FabAV administration. All data from patients evaluated for copperhead snakebite at a rural tertiary referral center from 5/2002 to 10/2013 were compiled. Demographics, transfer status, antivenin use, and clinical findings were collected; SSS was calculated. The relationships among FabAV use, clinical findings, and SSS were analyzed using t-test, chi-square, and Pearson's coefficient (p < 0.05 was significant). During the study period, 318 patients were treated for copperhead snakebite; 44 (13.8 %) received antivenin. Median dose was four vials (range: 1-10; IQR: 4,6). There were no deaths. Most patients receiving FabAV (63.6 %) were admitted. With regard to demographics and symptoms, only the degree of swelling (moderate vs. none/mild; p < 0.01) and bite location (hand/arm vs. leg: p < 0.0001) were associated with FabAV use. A SSS > 3, indicating moderate or severe envenomation, was only very weakly correlated with antivenin use (r = 0.217; p < 0.0001). The majority of patients with SSS > 3 (65.8 %) did not receive antivenin while most patients who did receive antivenin (70.5 %) had SSS ≤ 3 (indicating mild envenomation). Considerable variation occurs in antivenin administration after copperhead snakebite. Use of FabAV appears poorly correlated with patients' symptoms. This practice may expose patients to the risks of antivenin and increasing costs of medical care without

Imaging Potential Evaluation of Fab Derived from the Anti-EGFRvIII Monoclonal Antibody 4G1.

PubMed

Jing, Shen; He, Yujia; He, Yanqiong; Wang, Liang; Jia, Jianhua; Shan, Xiaomin; Liu, Shuang; Tang, Min; Peng, Zhiping; Liu, Xujie

2018-05-31

As one of the most crucial epidermal growth factor receptor (EGFR) variants, EGFRvIII can be detected in various tumors but rarely in normal tissues, making it an ideal target for prognosis, diagnosis or immune therapy. The recently developed anti-EGFRvIII monoclonal antibody (mAb), 4G1, has been validated as a promising molecular probe to detect EGFRvIII expression in tumors by single-photon emission computed tomography/computed tomography imaging. To overcome shortcomings associated with the whole antibody, including long-term retention, circulation and enhanced permeability and retention effects, the Fab fragment of 4G1 (Fab-4G1) was generated, labeled with 131 I and evaluated in vitro and in vivo to test its potential application in molecular imaging. Whole mAb 4G1 was first digested by immobilized ficin and then purified through a protein A column to generate the Fab fragment, Fab-4G1. Next, SDS-PAGE, Western blot, indirect fluorescence assay, flow cytometry and enzyme-linked immunosorbent assay were performed to verify molecular weight, specificity and affinity of Fab-4G1. Finally, biodistribution planar gamma imaging was performed by injection of 131 I-labeled Fab-4G1 into xenografted EGFRvIII-overexpressed tumors in nude mice. Parallel studies were also performed with intact 4G1. The molecular weight of Fab was determined to be 35-40 kDa by SDS-PAGE. In vitro tests confirmed both intact 4G1 and Fab-4G1 specifically bound EGFRvIII but not wild-type EGFR, and Fab-4G1 showed decreased affinity. Compared to 131 I-4G1, biodistribution studies showed lower tumor uptake of 131 I-Fab-4G1 at all time points, but much faster elimination in all normal organs. As for planar gamma imaging, 131 I-Fab-4G1 and 31 I-4G1 showed similar imaging effect at 2 h after injection of tracer, while 131 I-Fab-4G1 was eliminated more quickly with time, suggesting radiolabeled Fab-4G1 could be potentially used for imaging of EGFRvIII-positive tumors at early time points. Radiolabeled

Mapping of Fab-1:VEGF Interface Using Carboxyl Group Footprinting Mass Spectrometry

NASA Astrophysics Data System (ADS)

Wecksler, Aaron T.; Kalo, Matt S.; Deperalta, Galahad

2015-12-01

A proof-of-concept study was performed to demonstrate that carboxyl group footprinting, a relatively simple, bench-top method, has utility for first-pass analysis to determine epitope regions of therapeutic mAb:antigen complexes. The binding interface of vascular endothelial growth factor (VEGF) and the Fab portion of a neutralizing antibody (Fab-1) was analyzed using carboxyl group footprinting with glycine ethyl ester (GEE) labeling. Tryptic peptides involved in the binding interface between VEGF and Fab-1 were identified by determining the specific GEE-labeled residues that exhibited a reduction in the rate of labeling after complex formation. A significant reduction in the rate of GEE labeling was observed for E93 in the VEGF tryptic peptide V5, and D28 and E57 in the Fab-1 tryptic peptides HC2 and HC4, respectively. Results from the carboxyl group footprinting were compared with the binding interface identified from a previously characterized crystal structure (PDB: 1BJ1). All of these residues are located at the Fab-1:VEGF interface according to the crystal structure, demonstrating the potential utility of carboxyl group footprinting with GEE labeling for mapping epitopes.

Development of tools to study personal weight control strategies: OxFAB taxonomy

PubMed Central

Aveyard, Paul; Koshiaris, Constantinos; Jebb, Susan A.

2016-01-01

Objective To describe the development of the Oxford Food and Activity Behaviors (OxFAB) taxonomy and questionnaire to explore the cognitive and behavioral strategies used by individuals during weight management attempts. Methods The taxonomy was constructed through a qualitative analysis of existing resources and a review of existing behavior change taxonomies and theories. The taxonomy was translated into a questionnaire to identify strategies used by individuals. Think‐aloud interviews were conducted to test the face/concept validity of the questionnaire, and test–retest reliability was assessed in a sample of 138 participants. Results The OxFAB taxonomy consists of 117 strategies grouped into 23 domains. Compared to taxonomies used to describe interventions, around half of the domains and strategies identified are unique to the OxFAB taxonomy. The OxFAB questionnaire consists of 117 questions, one for each strategy from the taxonomy. Test–retest resulted in a mean PABAK score of 0.61 (SD 0.15). Questions were revised where appropriate. Conclusions The OxFAB taxonomy and questionnaire provide a conceptual framework to identify the cognitive and behavioral strategies used by individuals during attempts at weight control. PMID:26748902

Management of Tissue Loss After Agkistrodon Snakebite: Appropriate Use of Crotalidae-Fab Antivenin.

PubMed

Larson, Kenneth W; Schaefer, Keith R; Austin, Cindy; Norton, Rhy; Finley, Phillip J

2016-01-01

Although initially created for the treatment of rattlesnake (genus: Crotalus) bites, Crotalidae-Fab antivenin is used to treat many different pit viper envenomations. However, the efficacy of Crotalidae-Fab in preventing tissue loss from copperhead (Agkistrodon contortrix) or cottonmouth (Agkistrodon piscivorus) snakebites remains unclear. Recent reports show that Agkistrodon-related bites rarely require treatment beyond simple observation and pain control. The purpose of this study was to examine the amount of tissue loss in patients who received Crotalidae-Fab compared with those who did not after an Agkistrodon bite. After institutional review board approval, a retrospective study was completed at a Level 1 trauma center. Between 2009 and 2013, a total of 57 snakebites were identified. Of the 57 bites, the snake species was documented in 36 cases including 31 copperheads, 1 cottonmouth, and 4 rattlesnakes. The other 21 bites were from unknown or nonvenomous species. Of the 32 Agkistrodon-related bites, 15 patients received Crotalidae-Fab (average of 3 vials administered) and 17 did not receive Crotalidae-Fab. None of the 32 patients, regardless of treatment option, had tissue loss or required surgical interventions. Only 1 patient received Crotalidae-Fab and debridement of a vesicle associated with the bite. No clinically significant differences were observed between the groups. These findings support previous literature that failed to show added benefit of Crotalidae-Fab treatment for Agkistrodon bites beyond patient comfort and pain control. Evaluation of current protocols for Agkistrodon envenomations is warranted. Snakebite wound education in trauma physicians and nurses may decrease unnecessary use of antivenom medication.

Contribution of Antibody Hydrodynamic Size to Vitreal Clearance Revealed through Rabbit Studies Using a Species-Matched Fab.

PubMed

Shatz, Whitney; Hass, Philip E; Mathieu, Mary; Kim, Hok Seon; Leach, Kim; Zhou, Michelle; Crawford, Yongping; Shen, Amy; Wang, Kathryn; Chang, Debby P; Maia, Mauricio; Crowell, Susan R; Dickmann, Leslie; Scheer, Justin M; Kelley, Robert F

2016-09-06

We have developed a tool Fab fragment of a rabbit monoclonal antibody that is useful for early evaluation in rabbit models of technologies for long acting delivery (LAD) of proteins to the eye. Using this Fab we show that vitreal clearance can be slowed through increased hydrodynamic size. Fab (G10rabFab) and Fab' (G10rabFab') fragments of a rabbit monoclonal antibody (G10rabIgG) were expressed in Chinese hamster ovary (CHO) cells and purified using antigen-based affinity chromatography. G10rabFab retains antigen-binding upon thermal stress (37 °C) for 8 weeks in phosphate-buffered saline (PBS) and can be detected in rabbit tissues using an antigen-based ELISA. Hydrodynamic radius, measured using quasi-elastic light scattering (QELS), was increased through site-specific modification of the G10rabFab' free cysteine with linear methoxy-polyethylene glycol(PEG)-maleimide of 20000 or 40000 molecular weight. Pharmacokinetic studies upon intravitreal dosing in New Zealand white rabbits were conducted on the G10rabFab and PEGylated G10rabFab'. Results of single and multidose pharmacokinetic experiments yield reproducible results and a vitreal half-life for G10rabFab of 3.2 days. Clearance from the eye is slowed through increased hydrodynamic size, with vitreal half-life showing a linear dependence on hydrodynamic radius (RH). A linear dependence of vitreal half-life on RH suggests that molecule diffusivity makes an important contribution to vitreal clearance. A method for prediction of vitreal half-life from RH measurements is proposed.

Site-specific fab fragment biotinylation at the conserved nucleotide binding site for enhanced Ebola detection.

PubMed

Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

2015-07-01

The nucleotide binding site (NBS) is a highly conserved region between the variable light and heavy chains at the Fab domains of all antibodies, and a small molecule that we identified, indole-3-butyric acid (IBA), binds specifically to this site. Fab fragment, with its small size and simple production methods compared to intact antibody, is good candidate for use in miniaturized diagnostic devices and targeted therapeutic applications. However, commonly used modification techniques are not well suited for Fab fragments as they are often more delicate than intact antibodies. Fab fragments are of particular interest for sensor surface functionalization but immobilization results in damage to the antigen binding site and greatly reduced activity due to their truncated size that allows only a small area that can bind to surfaces without impeding antigen binding. In this study, we describe an NBS-UV photocrosslinking functionalization method (UV-NBS(Biotin) in which a Fab fragment is site-specifically biotinylated with an IBA-EG11-Biotin linker via UV energy exposure (1 J/cm(2)) without affecting its antigen binding activity. This study demonstrates successful immobilization of biotinylated Ebola detecting Fab fragment (KZ52 Fab fragment) via the UV-NBS(Biotin) method yielding 1031-fold and 2-fold better antigen detection sensitivity compared to commonly used immobilization methods: direct physical adsorption and NHS-Biotin functionalization, respectively. Utilization of the UV-NBS(Biotin) method for site-specific conjugation to Fab fragment represents a proof of concept use of Fab fragment for various diagnostic and therapeutic applications with numerous fluorescent probes, affinity molecules and peptides. © 2015 Wiley Periodicals, Inc.

X-ray studies of recombinant anti-testosterone Fab fragments: the use of PEG 3350 in crystallization.

PubMed

Valjakka, J; Hemminki, A; Teerinen, T; Takkinen, K; Rouvinen, J

2000-02-01

Recombinant anti-testosterone wild-type Fab fragment and mutant Fab fragments with high binding selectivity developed by protein engineering have been crystallized with and without ligands. Crystals of these Fab fragments were obtained by the vapour-diffusion technique at room temperature using solutions of PEG 3350 with various biological buffers and with a wide pH range. So far, five data sets have been collected from crystals of three Fab-antigen complexes and from two uncomplexed Fab fragments, with resolutions ranging from 2.10 to 3.1 A. Crystallization conditions for Fab fragments were found by using modifications of the low ionic strength PEG 3350 series. Suitable concentrations of PEG 400, MPD and glycerol solutions for use as cryoprotectants in PEG 3350 solutions have been determined. One useful observation was that PEG 3350 is able to work alone as a cryoprotectant. The screening protocol used requires a smaller amount of protein material to achieve auspicious pre-crystals than previously. Results support the claim that PEG 3350 is more suitable for the crystallization of Fab fragments than higher molecular weight PEGs.

Validating a faster method for reconstitution of Crotalidae Polyvalent Immune Fab (ovine).

PubMed

Gerring, David; King, Thomas R; Branton, Richard

2013-07-01

Reconstitution of CroFab(®) (Crotalidae Polyvalent Immune Fab [ovine]) lyophilized drug product was previously performed using 10 mL sterile water for injection followed by up to 36 min of gentle swirling of the vial. CroFab has been clinically demonstrated to be most effective when administered within 6 h of snake envenomation, and improved clinical outcomes are correlated with quicker timing of administration. An alternate reconstitution method was devised, using 18 mL 0.9% saline with manual inversion, with the goal of shortening reconstitution time while maintaining a high quality, efficacious product. An analytical study was designed to compare the physicochemical properties of 3 separate batches of CroFab when reconstituted using the standard procedure (10 mL WFI with gentle swirling) and a modified rapid procedure using 18 mL 0.9% saline and manual inversion. The physical and chemical characteristics of the same 3 batches were assessed using various analytic methodologies associated with routine quality control release testing. In addition further analytical methodologies were applied in order to elucidate possible structural changes that may be induced by the changed reconstitution procedure. Batches A, B, and C required mean reconstitution times of 25 min 51 s using the label method and 3 min 07 s (a 88.0% mean decrease) using the modified method. Physicochemical characteristics (color and clarity, pH, purity, protein content, potency) were found to be highly comparable. Characterization assays (dynamic light scattering, analytical ultracentrifugation, LC-MS, SDS-PAGE and circular dichroism spectroscopy were also all found to be comparable between methods. When comparing CroFab batches that were reconstituted using the labeled and modified methods, the physicochemical and biological (potency) characteristics of CroFab were not significantly changed when challenged by the various standard analytical methodologies applied in routine quality control analysis

Fast conversion of scFv to Fab antibodies using type IIs restriction enzymes.

PubMed

Sanmark, Hanna; Huovinen, Tuomas; Matikka, Tero; Pettersson, Tiina; Lahti, Maria; Lamminmäki, Urpo

2015-11-01

Single chain variable fragment (scFv) antibody libraries are widely used for developing novel bioaffinity reagents, although Fab or IgG molecules are the preferred antibody formats in many final applications. Therefore, rapid conversion methods for combining multiple DNA fragments are needed to attach constant domains to the scFv derived variable domains. In this study we describe a fast and easy cloning method for the conversion of single framework scFv fragments to Fab fragments using type IIS restriction enzymes. All cloning steps excluding plating of the Fab transformants can be done in 96 well plates and the procedure can be completed in one working day. The concept was tested by converting 69 scFv clones into Fab format on 96 well plates, which resulted in 93% success rate. The method is particularly useful as a high-throughput tool for the conversion of the chosen scFv clones into Fab molecules in order to analyze them as early as possible, as the conversion can significantly affect the binding properties of the chosen clones. Copyright © 2015 Elsevier B.V. All rights reserved.

Reference metrology in a research fab: the NIST clean calibrations thrust

NASA Astrophysics Data System (ADS)

Dixson, Ronald; Fu, Joe; Orji, Ndubuisi; Renegar, Thomas; Zheng, Alan; Vorburger, Theodore; Hilton, Al; Cangemi, Marc; Chen, Lei; Hernandez, Mike; Hajdaj, Russell; Bishop, Michael; Cordes, Aaron

2009-03-01

In 2004, the National Institute of Standards and Technology (NIST) commissioned the Advanced Measurement Laboratory (AML) - a state-of-the-art, five-wing laboratory complex for leading edge NIST research. The NIST NanoFab - a 1765 m2 (19,000 ft2) clean room with 743 m2 (8000 ft2) of class 100 space - is the anchor of this facility and an integral component of the new Center for Nanoscale Science and Technology (CNST) at NIST. Although the CNST/NanoFab is a nanotechnology research facility with a different strategic focus than a current high volume semiconductor fab, metrology tools still play an important role in the nanofabrication research conducted here. Some of the metrology tools available to users of the NanoFab include stylus profiling, scanning electron microscopy (SEM), and atomic force microscopy (AFM). Since 2001, NIST has collaborated with SEMATECH to implement a reference measurement system (RMS) using critical dimension atomic force microscopy (CD-AFM). NIST brought metrology expertise to the table and SEMATECH provided access to leading edge metrology tools in their clean room facility in Austin. Now, in the newly launched "clean calibrations" thrust at NIST, we are implementing the reference metrology paradigm on several tools in the CNST/NanoFab. Initially, we have focused on calibration, monitoring, and uncertainty analysis for a three-tool set consisting of a stylus profiler, an SEM, and an AFM. Our larger goal is the development of new and supplemental calibrations and standards that will benefit from the Class 100 environment available in the NanoFab and offering our customers calibration options that do not require exposing their samples to less clean environments. Toward this end, we have completed a preliminary evaluation of the performance of these instruments. The results of these evaluations suggest that the achievable uncertainties are generally consistent with our measurement goals.

QSAR models for prediction of chromatographic behavior of homologous Fab variants.

PubMed

Robinson, Julie R; Karkov, Hanne S; Woo, James A; Krogh, Berit O; Cramer, Steven M

2017-06-01

While quantitative structure activity relationship (QSAR) models have been employed successfully for the prediction of small model protein chromatographic behavior, there have been few reports to date on the use of this methodology for larger, more complex proteins. Recently our group generated focused libraries of antibody Fab fragment variants with different combinations of surface hydrophobicities and electrostatic potentials, and demonstrated that the unique selectivities of multimodal resins can be exploited to separate these Fab variants. In this work, results from linear salt gradient experiments with these Fabs were employed to develop QSAR models for six chromatographic systems, including multimodal (Capto MMC, Nuvia cPrime, and two novel ligand prototypes), hydrophobic interaction chromatography (HIC; Capto Phenyl), and cation exchange (CEX; CM Sepharose FF) resins. The models utilized newly developed "local descriptors" to quantify changes around point mutations in the Fab libraries as well as novel cluster descriptors recently introduced by our group. Subsequent rounds of feature selection and linearized machine learning algorithms were used to generate robust, well-validated models with high training set correlations (R 2  > 0.70) that were well suited for predicting elution salt concentrations in the various systems. The developed models then were used to predict the retention of a deamidated Fab and isotype variants, with varying success. The results represent the first successful utilization of QSAR for the prediction of chromatographic behavior of complex proteins such as Fab fragments in multimodal chromatographic systems. The framework presented here can be employed to facilitate process development for the purification of biological products from product-related impurities by in silico screening of resin alternatives. Biotechnol. Bioeng. 2017;114: 1231-1240. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Production and characterization of anti-human IgG F(ab')2 antibody fragment.

PubMed

Valedkarimi, Zahra; Nasiri, Hadi; Aghebati-Maleki, Leili; Abdolalizadeh, Jalal; Esparvarinha, Mojghan; Majidi, Jafar

2018-04-10

In present study an optimized protocol for the separation of antibodies into antigen-binding fragments F(ab')2 using pepsin digestion was investigated. The production of these fragments is a consequential step in the development of medical research, treatment and diagnosis. For production of polyclonal antibody rabbit received antigen in four steps. The rabbit serum at 1/128000 dilution showed high absorbance in reaction with human IgG at the designed ELISA method. Rabbit IgG was purified by Ion-Exchange Chromatography (IEC) method. Purity was assessed by SDS-PAGE method. In non-reduced condition only one band was seen in about 150 kDa MW position and in reduced form, two bands were seen in 50 and 25 kDa MW positions. Rabbit IgG was digested by pepsin enzyme. The antibody fragments solution was applied to Gel filtration column to isolate the F(ab')2. Non-reduced SDS-PAGE for determining the purity of F(ab')2 fragment resulted in one band in 100 kDa corresponds to F(ab')2 fragment and a band in 150 kDa MW position corresponds to undigested IgG antibodies. The activities of FITC conjugated F(ab')2 fragment and commercial ones were compared using flowcytometry method. The activity results implied that the FITC conjugated- anti human F(ab')2 fragment worked as efficiently as the commercial one.

Fab glycosylation of immunoglobulin G does not associate with improvement of rheumatoid arthritis during pregnancy.

PubMed

Bondt, Albert; Wuhrer, Manfred; Kuijper, T Martijn; Hazes, Johanna M W; Dolhain, Radboud J E M

2016-11-25

Changes in immunoglobulin G (IgG) constant domain (Fc) glycosylation are associated with changes in rheumatoid arthritis (RA) disease activity in response to pregnancy. Here, we sought to determine whether the same holds true for variable domain (Fab) glycosylation. IgGs were captured from RA and control sera obtained before (RA only), during and after pregnancy, followed by Fc and Fab separation, glycan release, and mass spectrometric detection. In parallel, glycans from intact IgG were analysed. The data was used to calculate glycosylation traits, and to estimate the level of Fab glycosylation. The overall level of Fab glycosylation was increased in RA patients compared to controls, while no differences in Fab glycosylation patterns were found. For the Fc and intact IgG (Total) previously observed differences in galactosylation and bisection were confirmed. Furthermore, increased galactosylation of Fc and Total were associated with lower disease activity and autoantibody positivity. In addition, the change in Fc galactosylation associated with the change in disease activity during pregnancy and after delivery, while this was not the case for Fab. In contrast to changes in Fc glycosylation, changes in Fab glycosylation are not associated with improvement of RA during pregnancy and arthritis flare after delivery.

Crystal structure and substrate specificity of the [beta]-ketoacyl-acyl carrier protein synthase III (FabH) from Staphylococcus aureus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qiu, Xiayang; Choudhry, Anthony E.; Janson, Cheryl A.

{beta}-Ketoacyl-ACP synthase III (FabH), an essential enzyme for bacterial viability, catalyzes the initiation of fatty acid elongation by condensing malonyl-ACP with acetyl-CoA. We have determined the crystal structure of FabH from Staphylococcus aureus, a Gram-positive human pathogen, to 2 {angstrom} resolution. Although the overall structure of S. aureus FabH is similar to that of Escherichia coli FabH, the primer binding pocket in S. aureus FabH is significantly larger than that present in E. coli FabH. The structural differences, which agree with kinetic parameters, provide explanation for the observed varying substrate specificity for E. coli and S. aureus FabH. The rankmore » order of activity of S. aureus FabH with various acyl-CoA primers was as follows: isobutyryl- > hexanoyl- > butyryl- > isovaleryl- >> acetyl-CoA. The availability of crystal structure may aid in designing potent, selective inhibitors of S. aureus FabH.« less

Acute hypersensitivity reactions associated with administration of crotalidae polyvalent immune Fab antivenom.

PubMed

Cannon, Robert; Ruha, Anne-Michelle; Kashani, John

2008-04-01

Acute hypersensitivity reactions are well known to occur with the administration of the Antivenin (Crotalidae) Polyvalent (Wyeth Laboratories, Marietta, PA). Crotalidae polyvalent immune Fab (ovine) (CroFab; FabAV, Protherics, Inc., Brentwood, TN) was introduced in 2001, and early studies reported a hypersensitivity reaction rate up to 19%. We describe the incidence of acute hypersensitivity reactions to FabAV in patients bitten by rattlesnakes. This was a nonconcurrent observational cohort study, with data obtained by chart review of all patients admitted to our service for rattlesnake bites from July 2000 to June 2004. The study was conducted at an urban Level I trauma center and urban children's hospital. All patients treated with FabAV were included. Those who received no antivenom or who were treated with Antivenin (Crotalidae) Polyvalent were excluded. The main outcome variable was whether an acute hypersensitivity reaction developed. Ninety-three patients were included in the review (72 male and 21 female patients). The mean age was 34.5 years (range 16 months to 91 years), and the mean dose of antivenom was 12 vials (range 4 to 32 vials). The incidence of acute hypersensitivity reactions was 5 of 93, or 5.4%. Four patients developed a mild reaction that was easily treated and were able to finish the full course of antivenom. Only 1 patient developed a reaction that prevented further antivenom administration. FabAV appears to be associated with a lower incidence of acute hypersensitivity than initially reported. Most reactions are mild and easily treated and do not preclude further dosing of antivenom.

Isolation of human anti-serum albumin Fab antibodies with an extended serum-half life.

PubMed

Kang, Hyeon-Ju; Kim, Hye-Jin; Cha, Sang-Hoon

2016-01-01

The serum albumin (SA) has been exploited to generate long-acting biotherapeutics by taking advantage of the FcRn-mediated recycling mechanism in a direct or an indirect way. Since Fab fragments have been proven to be clinically safe for human usage, we assumed that human anti-SA Fab antibodies could have a great potential as a carrier molecule to extend the serum half-life of therapeutic proteins. We, herein, had attempted to isolate anti-SA Fab antibodies from HuDVFab-8L antibody library via a phage display technology, and identified eight discrete human Fab antibodies. One of the Fab antibodies, SL335, showed the strongest binding reactivity to human SA with nM range of affinity at both pH 6 and pH 7.4, and cross-reacted to SAs from various species including rat, mouse, canine and monkey. The in vivo pharmacokinetic assay using a rat model indicated that SL335 has approximately 10 fold longer serum half-life and 26 to 44-fold increase in AUC0 → ∞ compared to the negative control Fab molecule in both intravenous and subcutaneous administrations. Knowing that Fabs have proven to be safe in clinics for a long time, SL335 seems to have a great potential in generating long-acting protein drugs by tagging effector molecules with either chemical conjugation or genetic fusion. Copyright © 2015 Elsevier B.V. All rights reserved.

NT-pro-BNP is associated with inducible myocardial ischemia in mildly symptomatic type 2 diabetic patients.

PubMed

Wiersma, Jacobijne J; van der Zee, P Marc; van Straalen, Jan P; Fischer, Johan C; van Eck-Smit, Berthe L F; Tijssen, Jan G P; Trip, Mieke D; Piek, Jan J; Verberne, Hein J

2010-11-19

Baseline levels of N-terminal fragment of the brain natriuretic peptide prohormone (NT-pro-BNP) are associated with myocardial ischemia in non-diabetic patients with stable angina pectoris. A total of 281 patients with diabetes mellitus type 2 and stable angina pectoris underwent myocardial perfusion scintigraphy (MPS). Myocardial ischemia on MPS was present in 140 (50%) patients. These ischemic patients had significantly higher NT-pro-BNP levels compared with patients without ischemia: 183 pg/ml (64-324 pg/ml) vs. 88 pg/ml (34-207 pg/ml), respectively (p<0.001). In addition, NT-pro-BNP ≥180 pg/ml was an independent predictor of the presence of myocardial ischemia (OR 2.36, 95%CI 1.40-3.97, p=0.001). Possible confounding factors such as age and creatinine clearance were of no influence on the predictive value in this specific patient population. These findings strengthen the idea that NT-pro-BNP may be of value in the early detection of diabetic patients with hemodynamic significant coronary artery disease. Copyright © 2009 Elsevier Ireland Ltd. All rights reserved.

Development of tools to study personal weight control strategies: OxFAB taxonomy.

PubMed

Hartmann-Boyce, Jamie; Aveyard, Paul; Koshiaris, Constantinos; Jebb, Susan A

2016-02-01

To describe the development of the Oxford Food and Activity Behaviors (OxFAB) taxonomy and questionnaire to explore the cognitive and behavioral strategies used by individuals during weight management attempts. The taxonomy was constructed through a qualitative analysis of existing resources and a review of existing behavior change taxonomies and theories. The taxonomy was translated into a questionnaire to identify strategies used by individuals. Think-aloud interviews were conducted to test the face/concept validity of the questionnaire, and test-retest reliability was assessed in a sample of 138 participants. The OxFAB taxonomy consists of 117 strategies grouped into 23 domains. Compared to taxonomies used to describe interventions, around half of the domains and strategies identified are unique to the OxFAB taxonomy. The OxFAB questionnaire consists of 117 questions, one for each strategy from the taxonomy. Test-retest resulted in a mean PABAK score of 0.61 (SD 0.15). Questions were revised where appropriate. The OxFAB taxonomy and questionnaire provide a conceptual framework to identify the cognitive and behavioral strategies used by individuals during attempts at weight control. © 2016 The Authors. Obesity published by Wiley Periodicals, Inc. on behalf of The Obesity Society (TOS).

Immediate and delayed allergic reactions to Crotalidae polyvalent immune Fab (ovine) antivenom.

PubMed

Clark, Richard F; McKinney, Patrick E; Chase, Peter B; Walter, Frank G

2002-06-01

Allergic reactions are the most commonly reported adverse events after administration of antivenoms. Conventional horse serum-based crotalid antivenom used in the United States (Antivenin [Crotalidae] polyvalent) can lead to both immediate and delayed hypersensitivity reactions. Crotalidae polyvalent immune Fab (ovine) (CroFab; FabAV) has recently been approved for use in the United States. Experience from premarketing trials of this product and in the administration of other types of Fab, such as in digoxin poisoning, has demonstrated these fragments to be safe and effective, with a low incidence of sequella; however, allergic reactions can occur when any animal-protein derivatives are administered to human subjects. We report in detail the nature and course of allergic reactions that occurred in 4 patients treated with FabAV. Cases of anaphylaxis, acute urticaria, angioedema, and delayed serum sickness are described. All reactions were easily treated with some combination of antihistamines, epinephrine, and steroids, with prompt resolution of signs and symptoms enabling further dosing of antivenom as required. Several of these cases may have resulted from batches of antivenom contaminated with Fc fragments. The overall incidence of immediate and delayed allergic reactions to this product appears so far to be lower than that reported with conventional whole-immunoglobulin G (IgG) antivenom, but postmarketing surveillance is warranted.

Effect of polyethylene glycol conjugation on conformational and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab').

PubMed

Roque, Cristopher; Sheung, Anthony; Rahman, Nausheen; Ausar, S Fernando

2015-02-02

We have investigated the effects of site specific "hinge" polyethylene glycol conjugation (PEGylation) on thermal, pH, and colloidal stability of a monoclonal antibody antigen-binding fragment (Fab') using a variety of biophysical techniques. The results obtained by circular dichroism (CD), ultraviolet (UV) absorbance, and fluorescence spectroscopy suggested that the physical stability of the Fab' is maximized at pH 6-7 with no apparent differences due to PEGylation. Temperature-induced aggregation experiments revealed that PEGylation was able to increase the transition temperature, as well as prevent the formation of visible and subvisible aggregates. Statistical comparison of the three-index empirical phase diagram (EPD) revealed significant differences in thermal and pH stability signatures between Fab' and PEG-Fab'. Upon mechanical stress, micro-flow imaging (MFI) and measurement of the optical density at 360 nm showed that the PEG-Fab' had significantly higher resistance to surface-induced aggregation compared to the Fab'. Analysis of the interaction parameter, kD, indicated repulsive intermolecular forces for PEG-Fab' and attractive forces for Fab'. In conclusion, PEGylation appears to protect Fab' against thermal and mechanical stress-induced aggregation, likely due to a steric hindrance mechanism.

Myocardial perfusion abnormalities in asymptomatic patients with systemic lupus erythematosus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hosenpud, J.D.; Montanaro, A.; Hart, M.V.

1984-08-01

Accelerated coronary artery disease and myocardial infarction in young patients with systemic lupus erythematosus is well documented; however, the prevalence of coronary involvement is unknown. Accordingly, 26 patients with systemic lupus were selected irrespective of previous cardiac history to undergo exercise thallium-201 cardiac scintigraphy. Segmental perfusion abnormalities were present in 10 of the 26 studies (38.5 percent). Five patients had reversible defects suggesting ischemia, four patients had persistent defects consistent with scar, and one patient had both reversible and persistent defects in two areas. There was no correlation between positive thallium results and duration of disease, amount of corticosteroid treatment,more » major organ system involvement or age. Only a history of pericarditis appeared to be associated with positive thallium-201 results (p less than 0.05). It is concluded that segmental myocardial perfusion abnormalities are common in patients with systemic lupus erythematosus. Whether this reflects large-vessel coronary disease or small-vessel abnormalities remains to be determined.« less

Crotalidae polyvalent immune Fab: a guide to its use in North American crotaline envenomation.

PubMed

Keating, Gillian M; Lyseng-Williamson, Katherine A

2012-08-01

Intravenous crotalidae polyvalent immune Fab (CroFab(®)) is effective in patients with minimal, moderate or severe crotaline envenomation, halting the progression of local venom effects and ameliorating haematological and other systemic abnormalities of envenomation. Despite treatment, patients may experience delayed-onset or recurrent venom effects (e.g. coagulopathy). Intravenous crotalidae polyvalent immune Fab is generally well tolerated, with acute hypersensitivity reactions being the most commonly occurring adverse event.

20 CFR 30.316 - How does the FAB issue a final decision on a claim?

Code of Federal Regulations, 2010 CFR

2010-04-01

... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false How does the FAB issue a final decision on a... Adjudicatory Process Hearings and Final Decisions on Claims § 30.316 How does the FAB issue a final decision on... waives any objections to all or part of the recommended decision, the FAB may issue a final decision...

Reorienting the Fab Domains of Trastuzumab Results in Potent HER2 Activators

PubMed Central

Scheer, Justin M.; Sandoval, Wendy; Elliott, J. Michael; Shao, Lily; Luis, Elizabeth; Lewin-Koh, Sock-Cheng; Schaefer, Gabriele; Vandlen, Richard

2012-01-01

The structure of the Fab region of antibodies is critical to their function. By introducing single cysteine substitutions into various positions of the heavy and light chains of the Fab region of trastuzumab, a potent antagonist of HER2, and using thiol chemistry to link the different Fabs together, we produced a variety of monospecific F(ab′)2-like molecules with activities spanning from activation to inhibition of breast tumor cell growth. These isomers (or bis-Fabs) of trastuzumab, with varying relative spatial arrangements between the Fv-regions, were able to either promote or inhibit cell-signaling activities through the PI3K/AKT and MAPK pathways. A quantitative phosphorylation mapping of HER2 indicated that the agonistic isomers produced a distinct phosphorylation pattern associated with activation. This study suggests that antibody geometric isomers, found both in nature and during synthetic antibody development, can have profoundly different biological activities independent of their affinities for their target molecules. PMID:23284778

Scatterometry on pelliclized masks: an option for wafer fabs

NASA Astrophysics Data System (ADS)

Gallagher, Emily; Benson, Craig; Higuchi, Masaru; Okumoto, Yasuhiro; Kwon, Michael; Yedur, Sanjay; Li, Shifang; Lee, Sangbong; Tabet, Milad

2007-03-01

Optical scatterometry-based metrology is now widely used in wafer fabs for lithography, etch, and CMP applications. This acceptance of a new metrology method occurred despite the abundance of wellestablished CD-SEM and AFM methods. It was driven by the desire to make measurements faster and with a lower cost of ownership. Over the last year, scatterometry has also been introduced in advanced mask shops for mask measurements. Binary and phase shift masks have been successfully measured at all desired points during photomask production before the pellicle is mounted. There is a significant benefit to measuring masks with the pellicle in place. From the wafer fab's perspective, through-pellicle metrology would verify mask effects on the same features that are characterized on wafer. On-site mask verification would enable quality control and trouble-shooting without returning the mask to a mask house. Another potential application is monitoring changes to mask films once the mask has been delivered to the fab (haze, oxide growth, etc.). Similar opportunities apply to the mask metrologist receiving line returns from a wafer fab. The ability to make line-return measurements without risking defect introduction is clearly attractive. This paper will evaluate the feasibility of collecting scatterometry data on pelliclized masks. We explore the effects of several different pellicle types on scatterometry measurements made with broadband light in the range of 320-780 nm. The complexity introduced by the pellicles' optical behavior will be studied.

75 FR 21353 - Intel Corporation, Fab 20 Division, Including On-Site Leased Workers From Volt Technical...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-23

... DEPARTMENT OF LABOR Employment and Training Administration [TA-W-73,642] Intel Corporation, Fab 20... of Intel Corporation, Fab 20 Division, including on-site leased workers of Volt Technical Resources... Precision, Inc. were employed on-site at the Hillsboro, Oregon location of Intel Corporation, Fab 20...

High-Affinity Recombinant Antibody Fragments (Fabs) Can Be Applied in Peptide Enrichment Immuno-MRM Assays

PubMed Central

2015-01-01

High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays. PMID:24568200

High-affinity recombinant antibody fragments (Fabs) can be applied in peptide enrichment immuno-MRM assays.

PubMed

Whiteaker, Jeffrey R; Zhao, Lei; Frisch, Christian; Ylera, Francisco; Harth, Stefan; Knappik, Achim; Paulovich, Amanda G

2014-04-04

High-affinity antibodies binding to linear peptides in solution are a prerequisite for performing immuno-MRM, an emerging technology for protein quantitation with high precision and specificity using peptide immunoaffinity enrichment coupled to stable isotope dilution and targeted mass spectrometry. Recombinant antibodies can be generated from appropriate libraries in high-throughput in an automated laboratory and thus may offer advantages over conventional monoclonal antibodies. However, recombinant antibodies are typically obtained as fragments (Fab or scFv) expressed from E. coli, and it is not known whether these antibody formats are compatible with the established protocols and whether the affinities necessary for immunocapture of small linear peptides can be achieved with this technology. Hence, we performed a feasibility study to ask: (a) whether it is feasible to isolate high-affinity Fabs to small linear antigens and (b) whether it is feasible to incorporate antibody fragments into robust, quantitative immuno-MRM assays. We describe successful isolation of high-affinity Fab fragments against short (tryptic) peptides from a human combinatorial Fab library. We analytically characterize three immuno-MRM assays using recombinant Fabs, full-length IgGs constructed from these Fabs, or traditional monoclonals. We show that the antibody fragments show similar performance compared with traditional mouse- or rabbit-derived monoclonal antibodies. The data establish feasibility of isolating and incorporating high-affinity Fabs into peptide immuno-MRM assays.

'Zipbody' leucine zipper-fused Fab in E. coli in vitro and in vivo expression systems.

PubMed

Ojima-Kato, Teruyo; Fukui, Kansuke; Yamamoto, Hiroaki; Hashimura, Dai; Miyake, Shiro; Hirakawa, Yuki; Yamasaki, Tomomi; Kojima, Takaaki; Nakano, Hideo

2016-04-01

A small antibody fragment, fragment of antigen binding (Fab), is favorable for various immunological assays. However, production efficiency of active Fab in microorganisms depends considerably on the clones. In this study, leucine zipper-peptide pairs that dimerize in parallel (ACID-p1 (LZA)/BASE-p1 (LZB) or c-Jun/c-Fos) were fused to the C-terminus of heavy chain (Hc, VH-CH1) and light chain (Lc, VL-CL), respectively, to accelerate the association of Hc and Lc to form Fab in Escherichia coli in vivo and in vitro expression systems. The leucine zipper-fused Fab named 'Zipbody' was constructed using anti-E. coli O157 monoclonal antibody obtained from mouse hybridoma and produced in both in vitro and in vivo expression systems in an active form, whereas Fab without the leucine zipper fusion was not. Similarly, Zipbody of rabbit monoclonal antibody produced in in vitro expression showed significant activity. The purified, mouse Zipbody produced in the E. coli strain Shuffle T7 Express had specificity toward the antigen; in bio-layer interferometry analysis, the KD value was measured to be 1.5-2.0 × 10(-8) M. These results indicate that leucine zipper fusion to Fab C-termini markedly enhances active Fab formation in E. coli. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

3D printing in social education: Eki-Fab and student PBL

NASA Astrophysics Data System (ADS)

Makino, Masato; Saito, Azusa; Kodama, Mai; Takamatsu, Kyuuichiro; Tamate, Hideaki; Sakai, Kazuyuki; Wada, Masato; Khosla, Ajit; Kawakami, Masaru; Furukawa, Hidemitsu

2017-04-01

Additive manufacturing or 3D printer is one of the most innovative material processing methods. We are considering that human resources for 3D printing would be needed in the future. To educate the abilities of the digital fabrication, we have the public digital fabrication space "Eki-Fab" for junior and high school students and Project Based Learning (PBL) class for undergraduate students. Eki-Fab is held on every Saturday at the Yonezawa train station. In the "Eki-Fab", anybody can study the utilizing of 3D printer and modeling technics under the instruction of staff in Yamagata University. In the PBL class, we have the class every Thursday. The students get the techniques of the digital fabrication through the PBL.

The MUT056399 Inhibitor of FabI Is a New Antistaphylococcal Compound▿

PubMed Central

Escaich, S.; Prouvensier, L.; Saccomani, M.; Durant, L.; Oxoby, M.; Gerusz, V.; Moreau, F.; Vongsouthi, V.; Maher, Kirsty; Morrissey, Ian; Soulama-Mouze, C.

2011-01-01

MUT056399 is a highly potent new inhibitor of the FabI enzyme of both Staphylococcus aureus and Escherichia coli. In vitro, MUT056399 was very active against S. aureus strains, including methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. aureus (MRSA), linezolid-resistant, and multidrug-resistant strains, with MIC90s between 0.03 and 0.12 μg/ml. MUT056399 was also active against coagulase-negative staphylococci, with MIC90s between 0.12 and 4 μg/ml. The antibacterial spectrum is consistent with specific FabI inhibition with no activity against bacteria using FabK but activity against FabI-containing Gram-negative bacilli. In vitro, resistant clones of S. aureus were obtained at a low frequency. All of the resistant clones analyzed were found to contain mutations in the fabI gene. In vivo, MUT056399, administered subcutaneously, protected mice from a lethal systemic infection induced by MSSA, MRSA, and vancomycin-intermediate S. aureus strains (50% effective doses ranging from 19.3 mg/kg/day to 49.6 mg/kg/day). In the nonneutropenic murine thigh infection model, the same treatment with MUT056399 reduced the bacterial multiplication of MSSA and MRSA in the thighs of immunocompetent mice. These properties support MUT056399 as a very promising candidate for a novel drug to treat severe staphylococcal infections. PMID:21825292

The Female Athlete Body (FAB) study: Rationale, design, and baseline characteristics.

PubMed

Stewart, Tiffany M; Pollard, Tarryn; Hildebrandt, Tom; Beyl, Robbie; Wesley, Nicole; Kilpela, Lisa Smith; Becker, Carolyn Black

2017-09-01

Eating Disorders (EDs) are serious psychiatric illnesses marked by psychiatric comorbidity, medical complications, and functional impairment. Research indicates that female athletes are often at greater risk for developing ED pathology versus non-athlete females. The Female Athlete Body (FAB) study is a three-site, randomized controlled trial (RCT) designed to assess the efficacy of a behavioral ED prevention program for female collegiate athletes when implemented by community providers. This paper describes the design, intervention, and participant baseline characteristics. Future papers will discuss outcomes. Female collegiate athletes (N=481) aged 17-21 were randomized by site, team, and sport type to either FAB or a waitlist control group. FAB consisted of three sessions (1.3h each) of a behavioral ED prevention program. Assessments were conducted at baseline (pre-intervention), post-intervention (3weeks), and six-, 12-, and 18-month follow-ups. This study achieved 96% (N=481) of target recruitment (N=500). Few group differences emerged at baseline. Total sample analyses revealed moderately low baseline instances of ED symptoms and clinical cases. Health risks associated with EDs necessitate interventions for female athletes. The FAB study is the largest existing RCT for female athletes aimed at both reduction of ED risk factors and ED prevention. The methods presented and population recruited for this study represent an ideal intervention for assessing the effects of FAB on both the aforementioned outcomes. We anticipate that findings of this study (reported in future papers) will make a significant contribution to the ED risk factor reduction and prevention literature. Copyright © 2017 Elsevier Inc. All rights reserved.

Selective disulfide reduction for labeling and enhancement of Fab antibody fragments

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kirley, Terence L., E-mail: terry.kirley@uc.edu; Greis, Kenneth D.; Norman, Andrew B.

Many methods have been developed for chemical labeling and enhancement of the properties of antibodies and their common fragments, including the Fab and F(ab’){sub 2} fragments. Somewhat selective reduction of some antibody disulfide bonds has been previously achieved, yielding antibodies and antibody fragments that can be labeled at defined sites, enhancing their utility and properties. Selective reduction of the two hinge disulfide bonds present in F(ab’){sub 2} fragments using mild reduction has been useful. However, such reduction is often not quantitative and results in the reduction of multiple disulfide bonds, and therefore subsequent multiple labeling or conjugation sites are neithermore » homogenous nor stoichiometric. Here, a simple and efficient selective reduction of the single disulfide bond linking the partial heavy chain and the intact light chain which compose the Fab fragment is accomplished utilizing tris(2-carboxyethyl)phosphine (TCEP) immobilized on agarose beads. The resultant reduced cysteine residues were labeled with several cysteine-selective fluorescent reagents, as well as by cysteine-directed PEGylation. These two cysteine residues can also be re-ligated by means of a bifunctional cysteine cross-linking agent, dibromobimane, thereby both restoring a covalent linkage between the heavy and light chains at this site, far removed from the antigen binding site, and also introducing a fluorescent probe. There are many other research and clinical uses for these selectively partially reduced Fab fragments, including biotinylation, toxin and drug conjugation, and incorporation of radioisotopes, and this technique enables simple generation of very useful Fab fragment derivatives with many potential applications. - Highlights: • TCEP agarose is effective for selective reduction of a single Fab disulfide bond. • This disulfide is solvent accessible and distant from the antigen binding site. • A variety of buffers of varying pHs can be

Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes

PubMed Central

Ericson, Megan E.; Frank, Matthew W.

2016-01-01

Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes. Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. monocytogenes. PMID:27736774

Enoyl-Acyl Carrier Protein Reductase I (FabI) Is Essential for the Intracellular Growth of Listeria monocytogenes.

PubMed

Yao, Jiangwei; Ericson, Megan E; Frank, Matthew W; Rock, Charles O

2016-12-01

Enoyl-acyl carrier protein reductase catalyzes the last step in each elongation cycle of type II bacterial fatty acid synthesis and is a key regulatory protein in bacterial fatty acid synthesis. Genes of the facultative intracellular pathogen Listeria monocytogenes encode two functional enoyl-acyl carrier protein isoforms based on their ability to complement the temperature-sensitive growth phenotype of Escherichia coli strain JP1111 [fabI(Ts)]. The FabI isoform was inactivated by the FabI selective inhibitor AFN-1252, but the FabK isoform was not affected by the drug, as expected. Inhibition of FabI by AFN-1252 decreased endogenous fatty acid synthesis by 80% and lowered the growth rate of L. monocytogenes in laboratory medium. Robust exogenous fatty acid incorporation was not detected in L. monocytogenes unless the pathway was partially inactivated by AFN-1252 treatment. However, supplementation with exogenous fatty acids did not restore normal growth in the presence of AFN-1252. FabI inactivation prevented the intracellular growth of L. monocytogenes, showing that neither FabK nor the incorporation of host cellular fatty acids was sufficient to support the intracellular growth of L. monocytogenes Our results show that FabI is the primary enoyl-acyl carrier protein reductase of type II bacterial fatty acid synthesis and is essential for the intracellular growth of L. monocytogenes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

In vitro Fab display: a cell-free system for IgG discovery

PubMed Central

Stafford, Ryan L.; Matsumoto, Marissa L.; Yin, Gang; Cai, Qi; Fung, Juan Jose; Stephenson, Heather; Gill, Avinash; You, Monica; Lin, Shwu-Hwa; Wang, Willie D.; Masikat, Mary Rose; Li, Xiaofan; Penta, Kalyani; Steiner, Alex R.; Baliga, Ramesh; Murray, Christopher J.; Thanos, Christopher D.; Hallam, Trevor J.; Sato, Aaron K.

2014-01-01

Selection technologies such as ribosome display enable the rapid discovery of novel antibody fragments entirely in vitro. It has been assumed that the open nature of the cell-free reactions used in these technologies limits selections to single-chain protein fragments. We present a simple approach for the selection of multi-chain proteins, such as antibody Fab fragments, using ribosome display. Specifically, we show that a two-chain trastuzumab (Herceptin) Fab domain can be displayed in a format which tethers either the heavy or light chain to the ribosome while retaining functional antigen binding. Then, we constructed synthetic Fab HC and LC libraries and performed test selections against carcinoembryonic antigen (CEA) and vascular endothelial growth factor (VEGF). The Fab selection output was reformatted into full-length immunoglobulin Gs (IgGs) and directly expressed at high levels in an optimized cell-free system for immediate screening, purification and characterization. Several novel IgGs were identified using this cell-free platform that bind to purified CEA, CEA positive cells and VEGF. PMID:24586053

Crotalidae polyvalent immune Fab (ovine) antivenom is efficacious for envenomations by Southern Pacific rattlesnakes (Crotalus helleri).

PubMed

Bush, Sean P; Green, Steven M; Moynihan, James A; Hayes, William K; Cardwell, Michael D

2002-12-01

Southern Pacific rattlesnake (Crotalus helleri ) venom is not 1 of the 4 venoms used to produce Crotalidae polyvalent immune Fab (ovine) (CroFab; FabAV). There is currently no published clinical experience regarding the efficacy of this new antivenom for confirmed C helleri envenomation, and animal data suggest greatly diminished efficacy. We assessed the efficacy of FabAV for patients with confirmed C helleri envenomation. We conducted a prospective observational study of 23 consecutive rattlesnake envenomations that were treated with FabAV at our center. Patients were excluded if the species of snake could not be confirmed, if FabAV antivenom was not given, or if Antivenin (Crotalidae) polyvalent (equine) was given. We collected serial physical examination and laboratory data over a 24-hour period to serially evaluate the severity score and performed follow-up to evaluate delayed reactions. There were 15 patients who received FabAV and had the species of rattlesnake confirmed (9 C helleri, 4 C scutulatus scutulatus, 1 C mitchellii pyrrhus, 1 C ruber ruber ). C helleri envenomations demonstrated similar improvement in serial snakebite severity scores to those of other species. Three patients treated with scheduled dosing had recurrence of progressive swelling (2 C helleri and 1 C mitchellii pyrrhus ) during the 24-hour study period. We observed similar improvement in FabAV-treated patients with C helleri envenomation compared with those of other species and conclude that this treatment in standard doses appears efficacious for bites by this species. Progressive swelling may recur despite scheduled dosing.

Differences in substrate specificity of V. cholerae FabH enzymes suggest new approaches for the development of novel antibiotics and biofuels.

PubMed

Hou, Jing; Zheng, Heping; Tzou, Wen-Shyong; Cooper, David R; Chruszcz, Maksymilian; Chordia, Mahendra D; Kwon, Keehwan; Grabowski, Marek; Minor, Wladek

2018-06-19

Vibrio cholerae, the causative pathogen of the life-threatening infection cholera, encodes two copies of β-ketoacyl-ACP synthase III (vcFabH1 and vcFabH2). vcFabH1 and vcFabH2 are pathogenic proteins associated with fatty acid synthesis, lipid metabolism, and potential applications in biofuel production. Our biochemical assays characterize vcFabH1 as exhibiting specificity for acetyl-CoA and CoA thioesters with short acyl chains, similar to that observed for FabH homologs found in most Gram-negative bacteria. vcFabH2 prefers medium chain-length acyl-CoA thioesters, particularly octanoyl-CoA, which is a pattern of specificity rarely seen in bacteria. Structural characterization of one vcFabH and six vcFabH2 structures determined in either apo-form or in complex with acetyl-CoA/octanoyl-CoA indicate that the substrate binding pockets of vcFabH1 and vcFabH2 are of different sizes, accounting for variations in substrate chain-length specificity. An unusual and unique feature of vcFabH2 is its C-terminal fragment that interacts with both the substrate-entrance loop and the dimer interface of the enzyme. Our discovery of the pattern of substrate specificity of both vcFabH1 and vcFabH2 can potentially aid the development of novel antibacterial agents against V. cholerae. Additionally, the distinctive substrate preference of FabH2 in V. cholerae and related facultative anaerobes conceivably make it an attractive component of genetically engineered bacteria used for commercial biofuel production. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Use of CroFab antivenin in the management of a very young pediatric copperhead envenomation.

PubMed

Trinh, Hai H; Hack, Jason B

2005-08-01

The use of crotalid Fab antivenin (CroFab) in the treatment of snake envenomations in the pediatric population is still an underexplored area. There are very limited data to confirm the efficacy and safety of dosing children the same as adults and even less information available to evaluate this antivenin use in copperhead snake bites in children. We report the first use of crotalid Fab antivenin in an adult dose for a copperhead snake envenomation in a 2-year-old child. She had rapid resolution of symptoms with no adverse effects. The report serves to increase the literature supporting the current dosing recommendations of crotalid Fab antivenin in very young pediatric patients evidenced by its effectiveness in this patient.

Cyclization strategies of meditopes: affinity and diffraction studies of meditope–Fab complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bzymek, Krzysztof P.; Ma, Yuelong; Avery, Kendra A.

An overview of cyclization strategies of a Fab-binding peptide to maximize affinity. Recently, a unique binding site for a cyclic 12-residue peptide was discovered within a cavity formed by the light and heavy chains of the cetuximab Fab domain. In order to better understand the interactions that drive this unique complex, a number of variants including the residues within the meditope peptide and the antibody, as well as the cyclization region of the meditope peptide, were created. Here, multiple crystal structures of meditope peptides incorporating different cyclization strategies bound to the central cavity of the cetuximab Fab domain are presented.more » The affinity of each cyclic derivative for the Fab was determined by surface plasmon resonance and correlated to structural differences. Overall, it was observed that the disulfide bond used to cyclize the peptide favorably packs against a hydrophobic ‘pocket’ and that amidation and acetylation of the original disulfide meditope increased the overall affinity ∼2.3-fold. Conversely, replacing the terminal cysteines with serines and thus creating a linear peptide reduced the affinity over 50-fold, with much of this difference being reflected in a decrease in the on-rate. Other cyclization methods, including the formation of a lactam, reduced the affinity but not to the extent of the linear peptide. Collectively, the structural and kinetic data presented here indicate that small perturbations introduced by different cyclization strategies can significantly affect the affinity of the meditope–Fab complex.« less

Production and purification of polyclonal antibody against F(ab')2 fragment of human immunoglobulin G

PubMed Central

Nasiri, Hadi; Valedkarimi, Zahra; Aghebati-Maleki, Leili; Abdolalizadeh, Jalal; Kazemi, Tohid; Esparvarinha, Mojghan; Majidi, Jafar

2017-01-01

Antibodies are essential tools of biomedical and biochemical researches. Polyclonal antibodies are produced against different epitopes of antigens. Purified F(ab')2 can be used for animal’s immunization to produce polyclonal antibodies. Human immunoglobulin G (IgG) was purified by ion exchange chromatography method. In all stages verification method of the purified antibodies was sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified IgG was digested by pepsin enzyme and F(ab')2 fragment was purified by gel filtration separation method. For production of polyclonal antibody, rabbit was immunized by purified F(ab')2 and antibody production was investigated by enzyme-linked immunosorbent assay. Purified anti-IgG F(ab')2 was conjugated with fluorescein isothiocyanate. Ion exchange chromatography purification yielded 38 mg of human IgG antibody. The results of SDS-PAGE in reduced and non-reduced conditions showed bands with 25-30 kDa molecular weight (MW) and 50-kDa respectively and a distinct band with 150 kDa MW. The results of non-reduced SDS-PAGE for determining the purity of F(ab')2 fragment showed one band in 90 kDa and a band in 150 kDa MW position. Purification by Ion exchange chromatography method resulted about 12 mg rabbit polyclonal antibody. Flow cytometry showed generated polyclonal antibody had an acceptable activity compared to commercial antibody. Taking together, purified IgG F(ab')2 and polyclonal anti-IgG F(ab')2 are useful tools in biomedical and biochemical researches and diagnostic kits. PMID:29326789

Production and purification of polyclonal antibody against F(ab')2 fragment of human immunoglobulin G.

PubMed

Nasiri, Hadi; Valedkarimi, Zahra; Aghebati-Maleki, Leili; Abdolalizadeh, Jalal; Kazemi, Tohid; Esparvarinha, Mojghan; Majidi, Jafar

2017-01-01

Antibodies are essential tools of biomedical and biochemical researches. Polyclonal antibodies are produced against different epitopes of antigens. Purified F(ab') 2 can be used for animal's immunization to produce polyclonal antibodies. Human immunoglobulin G (IgG) was purified by ion exchange chromatography method. In all stages verification method of the purified antibodies was sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified IgG was digested by pepsin enzyme and F(ab') 2 fragment was purified by gel filtration separation method. For production of polyclonal antibody, rabbit was immunized by purified F(ab') 2 and antibody production was investigated by enzyme-linked immunosorbent assay. Purified anti-IgG F(ab') 2 was conjugated with fluorescein isothiocyanate. Ion exchange chromatography purification yielded 38 mg of human IgG antibody. The results of SDS-PAGE in reduced and non-reduced conditions showed bands with 25-30 kDa molecular weight (MW) and 50-kDa respectively and a distinct band with 150 kDa MW. The results of non-reduced SDS-PAGE for determining the purity of F(ab') 2 fragment showed one band in 90 kDa and a band in 150 kDa MW position. Purification by Ion exchange chromatography method resulted about 12 mg rabbit polyclonal antibody. Flow cytometry showed generated polyclonal antibody had an acceptable activity compared to commercial antibody. Taking together, purified IgG F(ab') 2 and polyclonal anti-IgG F(ab') 2 are useful tools in biomedical and biochemical researches and diagnostic kits.

Investigation of protein selectivity in multimodal chromatography using in silico designed Fab fragment variants.

PubMed

Karkov, Hanne Sophie; Krogh, Berit Olsen; Woo, James; Parimal, Siddharth; Ahmadian, Haleh; Cramer, Steven M

2015-11-01

In this study, a unique set of antibody Fab fragments was designed in silico and produced to examine the relationship between protein surface properties and selectivity in multimodal chromatographic systems. We hypothesized that multimodal ligands containing both hydrophobic and charged moieties would interact strongly with protein surface regions where charged groups and hydrophobic patches were in close spatial proximity. Protein surface property characterization tools were employed to identify the potential multimodal ligand binding regions on the Fab fragment of a humanized antibody and to evaluate the impact of mutations on surface charge and hydrophobicity. Twenty Fab variants were generated by site-directed mutagenesis, recombinant expression, and affinity purification. Column gradient experiments were carried out with the Fab variants in multimodal, cation-exchange, and hydrophobic interaction chromatographic systems. The results clearly indicated that selectivity in the multimodal system was different from the other chromatographic modes examined. Column retention data for the reduced charge Fab variants identified a binding site comprising light chain CDR1 as the main electrostatic interaction site for the multimodal and cation-exchange ligands. Furthermore, the multimodal ligand binding was enhanced by additional hydrophobic contributions as evident from the results obtained with hydrophobic Fab variants. The use of in silico protein surface property analyses combined with molecular biology techniques, protein expression, and chromatographic evaluations represents a previously undescribed and powerful approach for investigating multimodal selectivity with complex biomolecules. © 2015 Wiley Periodicals, Inc.

20 CFR 30.908 - How will the FAB evaluate new medical evidence submitted to challenge the impairment...

Code of Federal Regulations, 2010 CFR

2010-04-01

... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false How will the FAB evaluate new medical... Medical Evidence of Impairment § 30.908 How will the FAB evaluate new medical evidence submitted to... impairment evaluation that differs from the impairment evaluation relied upon by the district office, the FAB...

β-Hydroxyacyl-acyl Carrier Protein Dehydratase (FabZ) from Francisella tularensis and Yersinia pestis : Structure Determination, Enzymatic Characterization, and Cross-Inhibition Studies

DOE PAGES

McGillick, Brian E.; Kumaran, Desigan; Vieni, Casey; ...

2016-01-28

The bacterial system for fatty acid biosynthesis (FAS) contains several enzymes whose sequence and structure are highly conserved across a vast array of pathogens. Coupled with their low homology and difference in organization compared to the equivalent system in humans, this makes the FAS pathway an excellent target for antimicrobial drug development. To this end, we have cloned, expressed, and purified the β-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from both Francisella tularensis (FtFabZ) and Yersinia pestis (YpFabZ). We also solved the crystal structures and performed an enzymatic characterization of both enzymes and several mutant forms of YpFabZ. In addition, we havemore » discovered two novel inhibitors of FabZ, mangostin and stictic acid, which show similar potencies against both YpFabZ and FtFabZ. Lastly, we selected several compounds from the literature that have been shown to be active against single homologues of FabZ and tested them against both YpFabZ and FtFabZ. Our results have revealed clues as to which scaffolds are likely to lead to broad-spectrum antimicrobials targeted against FabZ as well as modifications to existing FabZ inhibitors that may improve potency.« less

Pre- and Posttransplant IgA Anti-Fab Antibodies to Predict Long-term Kidney Graft Survival.

PubMed

Amirzargar, M A; Amirzargar, A; Basiri, A; Hajilooi, M; Roshanaei, G; Rajabi, G; Solgi, G

2015-05-01

Immunologic factors are reliable markers for allograft monitoring, because of their seminal role in rejection process. One of these factors is the immunoglobulin (Ig)A anti-Fab of the IgG antibody. This study aimed to evaluate the predictive value of pre- and posttransplant levels of this marker for kidney allograft function and survival. Sera samples of 59 living unrelated donor kidney recipients were collected before and after transplantation (days 7, 14, and 30) and investigated for IgA anti-Fab of IgG antibody levels using enzyme-linked immunosorbent assay in relation with allograft outcome. Among 59 patients, 15 cases (25%) including 10 with acute rejection and 5 with chronic rejection episodes showed graft failure during a mean of 5 years of follow-up. High posttransplant levels of IgA anti-Fab antibodies were observed more frequently in patients with stable graft function (SGF) compared with patients with graft failure (P = 2 × 10(-6)). None of patients with acute or chronic rejection episodes had high levels of IgA anti-Fab antibodies at day 30 posttransplant compared with the SGF group (P = 10(-6) and P = .01, respectively). In addition, high levels of IgA anti-Fab antibody correlated with lesser concentration of serum creatinine at 1 month posttransplantation (P = .01). Five-year graft survival was associated with high levels of pre- and posttransplant IgA anti-Fab antibodies (P = .02 and P = .003, respectively). Our findings indicate the protective effect of higher levels of IgA anti-Fab antibodies regarding to kidney allograft outcomes and long-term graft survival. Copyright © 2015 Elsevier Inc. All rights reserved.

1,2-Dithiole-3-Ones as Potent Inhibitors of the Bacterial 3-Ketoacyl Acyl Carrier Protein Synthase III (FabH)

PubMed Central

He, Xin; Reeve, Anne McElwee; Desai, Umesh R.; Kellogg, Glen E.; Reynolds, Kevin A.

2004-01-01

The enzyme FabH catalyzes the initial step of fatty acid biosynthesis via a type II dissociated fatty acid synthase. The pivotal role of this essential enzyme, combined with its unique structural features and ubiquitous occurrence in bacteria, has made it an attractive new target for the development of antibacterial and antiparasitic compounds. We have searched the National Cancer Institute database for compounds bearing structural similarities to thiolactomycin, a natural product which exhibits a weak activity against FabH. This search has yielded several substituted 1,2-dithiole-3-ones that are potent inhibitors of FabH from both Escherichia coli (ecFabH) and Staphylococcus aureus (saFabH). The most potent inhibitor was 4,5-dichloro-1,2-dithiole-3-one, which had 50% inhibitory concentration (IC50) values of 2 μM (ecFabH) and 0.16 μM (saFabH). The corresponding 3-thione analog exhibited comparable activities. Analogs in which the 4-chloro substituent was replaced with a phenyl group were also potent inhibitors, albeit somewhat less effectively (IC50 values of 5.7 and 0.98 μM for ecFabH and saFabH, respectively). All of the 5-chlorinated inhibitors were most effective when they were preincubated with FabH in the absence of substrates. The resulting enzyme-inhibitor complex did not readily regain activity after excess inhibitor was removed, suggesting that a slow dissociation occurs. In stark contrast, a series of inhibitors in which the 5-chloro substituent was replaced with the isosteric and isoelectronic trifluoromethyl group were poorer inhibitors (IC50 values typically ranging from 25 to >100 μM for both ecFabH and saFabH), did not require a preincubation period for maximal activity, and generated an enzyme-inhibitor complex which readily dissociated. Possible modes of binding of 5-chloro-1,2-dithiole-3-ones and 5-chloro-1,2-dithiole-3-thiones with FabH which account for the role of the 5-chloro substituent were considered. PMID:15273125

20 CFR 30.320 - Can a claim be reopened after the FAB has issued a final decision?

Code of Federal Regulations, 2010 CFR

2010-04-01

... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false Can a claim be reopened after the FAB has... AMENDED Adjudicatory Process Reopening Claims § 30.320 Can a claim be reopened after the FAB has issued a final decision? (a) At any time after the FAB has issued a final decision pursuant to § 30.316, and...

3-Substituted Indole Inhibitors Against Francisella tularensis FabI Identified by Structure-Based Virtual Screening

DTIC Science & Technology

2013-07-01

FabI, but share low sequence identity and are poorly inhibited by triclosan.25,26 S. pneumoniae and P. aeruginosa contain FabK,24 and Vibrio cholerae,27...with 0.2 mM IPTG. The cells were harvested after an overnight induction period at 17 °C. The cells were lysed and sonicated and loaded onto a nickel...of enoyl- (acyl-carrier protein) reductase, FabV, from Vibrio fischeri. Acta Crystallogr., Sect. F: Struct. Biol. Cryst. Commun. 2012, 68, 78−80. (27

A new helper phage for improved monovalent display of Fab molecules.

PubMed

Beaber, John W; Tam, Eric M; Lao, Llewelyn S; Rondon, Isaac J

2012-02-28

Phage display technology is a powerful tool for the identification of novel antibodies for drug discovery. Phage display libraries have been constructed with massive diversity, but their use may be hindered by limited antibody display levels when rescued with the M13KO7 helper phage. Variants of M13KO7 have been constructed previously that increase the levels of display of rescued phage, but all produce phage that display multiple copies of the antibody fragment on their surface and have reduced titer and infectivity. In this study, we describe a new helper phage, XP5, which increased the display level of Fab molecules more than two-fold compared to phage rescued with M13KO7. XP5 uses a combination of ribosome binding site spacing alterations and rare codon clusters to reduce the expression of pIII from the helper phage. This reduction in pIII expression leads to an increase in the incorporation of pIII-Fab fusions during phage rescue. The rescued phage displayed a single copy of the Fab molecule, preventing any avidity effects during the selection process. This also suggests that the percentage of the population of phage displaying a Fab molecule is increased when rescued with XP5. Additionally, the phage titers and infectivity are comparable to libraries rescued with M13KO7. After two rounds of panning we observed a nearly 5-fold increase in the number of antigen binding Fab molecules compared to panning conducted with the same library rescued with M13KO7. The nature of the mutations in XP5 makes it a universal substitute for M13KO7 in pIII-based phage display, compatible with most phagemids and bacterial strains. Copyright © 2011 Elsevier B.V. All rights reserved.

Crotalidae polyvalent immune Fab (ovine) antivenom is effective in the neutralization of South American viperidae venoms in a murine model.

PubMed

Richardson, William H; Tanen, David A; Tong, Tri C; Betten, David P; Carstairs, Shaun D; Williams, Saralyn R; Cantrell, Frank L; Clark, Richard F

2005-06-01

Crotalidae polyvalent immune Fab (ovine) (CroFab; FabAV) is used in the treatment of symptomatic crotaline envenomations in North America. Unlike Antivenin (Crotalidae) Polyvalent, which is approved for treatment of crotaline envenomation in North and South America, FabAV is manufactured using only venoms from crotaline snakes native to the United States. This study was designed to evaluate the efficacy of FabAV in the neutralization of venom from 2 South American crotaline snakes: Crotalus durissus terrificus (tropical rattlesnake) and Bothrops atrox (fer-de-lance). A randomized, blinded, placebo-controlled murine model of intraperitoneal venom injection was used. Venom potency was determined in preliminary median lethal dose (LD 50) dosing studies. Study animals were then divided into 7 groups: (1) C durissus terrificus venom (Sigma-Aldrich Co.)+FabAV, (2) C durissus terrificus venom (Sigma-Aldrich Co.)+0.9% normal saline solution, (3) C durissus terrificus venom (Biotoxins Inc.)+FabAV, (4) C durissus terrificus venom (Biotoxins Inc.)+normal saline solution, (5) B atrox venom+FabAV, (6) B atrox venom+normal saline solution, and (7) FabAV+normal saline solution. Twice the estimated LD 50 was the chosen venom dose, and the amount of FabAV injected was 10 times the amount needed for venom neutralization. Statistical analysis included Fisher's exact test and log-rank testing to compare survival rates and times. The venom LD 50 was found in preliminary studies to be 0.9 mg/kg and 1.35 mg/kg for the C durissus terrificus venom obtained from Sigma-Aldrich Co. and Biotoxins Inc., respectively. The LD 50 for B atrox venom was 5.0 mg/kg. All animals receiving venom only and saline solution died. Animals receiving FabAV together with either venom survived to the end of the 24-hour observation period ( P <.001). Comparison of survival times between groups demonstrated a significant difference in time to death between venom-only control groups and the FabAV+venom groups (P

Biodistribution of charged F(ab')2 photoimmunoconjugates in a xenograft model of ovarian cancer.

PubMed

Duska, L R; Hamblin, M R; Bamberg, M P; Hasan, T

1997-01-01

The effect of charge modification of photoimmunoconjugates (PICs) on their biodistribution in a xenograft model of ovarian cancer was investigated. Chlorin(e6)c(e6) was attached site specifically to the F(ab')2 fragment of the murine monoclonal antibody OC125, directed against human ovarian cancer cells, via poly-1-lysine linkers carrying cationic or anionic charges. Preservation of immunoreactivity was checked by enzyme-linked immunosorbent assay (ELISA). PICs were radiolabelled with 125I and compared with non-specific rabbit IgG PICs after intraperitoneal (i.p.) injection into nude mice. Samples were taken from normal organs and tumour at 3 h and 24 h. Tumour to normal 125I ratios showed that the cationic OC125F(ab')2 PIC had the highest tumour selectivity. Ratios for c(e6) were uniformly higher than for 125I, indicating that c(e6) became separated from 125I. OC125F(ab')2 gave highest tissue values of 125I, followed by cationic OC125F(ab')2 PIC; other species were much lower. The amounts of c(e6) delivered per gram of tumour were much higher for cationic OC125F(ab')2 PIC than for other species. The results indicate that cationic charge stimulates the endocytosis and lysosomal degradation of the OC125F(ab')2-pl-c(e6) that has bound to the i.p. tumour. Positively charged PICs may have applications in the i.p. photoimmunotherapy of minimal residual ovarian cancer.

Biodistribution of charged F(ab')2 photoimmunoconjugates in a xenograft model of ovarian cancer.

PubMed Central

Duska, L. R.; Hamblin, M. R.; Bamberg, M. P.; Hasan, T.

1997-01-01

The effect of charge modification of photoimmunoconjugates (PICs) on their biodistribution in a xenograft model of ovarian cancer was investigated. Chlorin(e6)c(e6) was attached site specifically to the F(ab')2 fragment of the murine monoclonal antibody OC125, directed against human ovarian cancer cells, via poly-1-lysine linkers carrying cationic or anionic charges. Preservation of immunoreactivity was checked by enzyme-linked immunosorbent assay (ELISA). PICs were radiolabelled with 125I and compared with non-specific rabbit IgG PICs after intraperitoneal (i.p.) injection into nude mice. Samples were taken from normal organs and tumour at 3 h and 24 h. Tumour to normal 125I ratios showed that the cationic OC125F(ab')2 PIC had the highest tumour selectivity. Ratios for c(e6) were uniformly higher than for 125I, indicating that c(e6) became separated from 125I. OC125F(ab')2 gave highest tissue values of 125I, followed by cationic OC125F(ab')2 PIC; other species were much lower. The amounts of c(e6) delivered per gram of tumour were much higher for cationic OC125F(ab')2 PIC than for other species. The results indicate that cationic charge stimulates the endocytosis and lysosomal degradation of the OC125F(ab')2-pl-c(e6) that has bound to the i.p. tumour. Positively charged PICs may have applications in the i.p. photoimmunotherapy of minimal residual ovarian cancer. PMID:9062404

Generation and characterization of high affinity humanized fab against hepatitis B surface antigen.

PubMed

Tiwari, Ashutosh; Dutta, Durgashree; Khanna, Navin; Acharya, Subrat K; Sinha, Subrata

2009-09-01

5S is a mouse monoclonal IgG1 that binds to the 'a' epitope of the Hepatitis B surface antigen (HBsAg) and tested positive in an in vitro test for virus neutralization. We have earlier reported the generation of humanized single chain variable fragment (scFv) from the same. In this article we report the generation of a recombinant Fab molecule by fusing humanized variable domains of 5S with the constant domains of human IgG1. The humanized Fab expressed in E. coli and subsequently purified, retained a high binding affinity (K(D) = 3.63 nmol/L) to HBsAg and bound to the same epitope of HBsAg as the parent molecule. The humanized Fab also maintained antigen binding in the presence of various destabilizing agents like 3 M NaCl, 30% DMSO, 8 M urea, and extreme pH. This high affinity humanized Fab provides a basis for the development of therapeutic molecules that can be safely utilized for the prophylaxis and treatment for Hepatitis B infection.

Effect of DNA sequence of Fab fragment on yield characteristics and cell growth of E. coli.

PubMed

Kulmala, Antti; Huovinen, Tuomas; Lamminmäki, Urpo

2017-06-19

Codon usage is one of the factors influencing recombinant protein expression. We were interested in the codon usage of an antibody Fab fragment gene exhibiting extreme toxicity in the E. coli host. The toxic synthetic human Fab gene contained domains optimized by the "one amino acid-one codon" method. We redesigned five segments of the Fab gene with a "codon harmonization" method described by Angov et al. and studied the effects of these changes on cell viability, Fab yield and display on filamentous phage using different vectors and bacterial strains. The harmonization considerably reduced toxicity, increased Fab expression from negligible levels to 10 mg/l, and restored the display on phage. Testing the impact of the individual redesigned segments revealed that the most significant effects were conferred by changes in the constant domain of the light chain. For some of the Fab gene variants, we also observed striking differences in protein yields when cloned from a chloramphenicol resistant vector into an identical vector, except with ampicillin resistance. In conclusion, our results show that the expression of a heterodimeric secretory protein can be improved by harmonizing selected DNA segments by synonymous codons and reveal additional complexity involved in heterologous protein expression.

Effectiveness of delayed use of crotalidae polyvalent immune Fab (ovine) antivenom.

PubMed

Bebarta, Vikhyat; Dart, Richard C

2004-01-01

Traditionally, horse-serum-based antivenom has been used in the United States for North American crotaline snake evenomation. Crotalidae polyvalent immune Fab (ovine) was approved in 2000 for use in mild to moderate envenomations. The manufacture recommends use within 6 h of envenomation. Published postmarketing retrospective reports describe its use up to 9 h after envenomation. We describe a case of effective use of FabAV 52 h after envenomation with resultant correction of coagulopathy and mild improvement of local symptoms.

Dipyridamole stress myocardial perfusion by computed tomography in patients with left bundle branch block.

PubMed

Cabeda, Estêvan Vieira; Falcão, Andréa Maria Gomes; Soares, José; Rochitte, Carlos Eduardo; Nomura, César Higa; Ávila, Luiz Francisco Rodrigues; Parga, José Rodrigues

2015-12-01

Functional tests have limited accuracy for identifying myocardial ischemia in patients with left bundle branch block (LBBB). To assess the diagnostic accuracy of dipyridamole-stress myocardial computed tomography perfusion (CTP) by 320-detector CT in patients with LBBB using invasive quantitative coronary angiography (QCA) (stenosis ≥ 70%) as reference; to investigate the advantage of adding CTP to coronary computed tomography angiography (CTA) and compare the results with those of single photon emission computed tomography (SPECT) myocardial perfusion scintigraphy. Thirty patients with LBBB who had undergone SPECT for the investigation of coronary artery disease were referred for stress tomography. Independent examiners performed per-patient and per-coronary territory assessments. All patients gave written informed consent to participate in the study that was approved by the institution's ethics committee. The patients' mean age was 62 ± 10 years. The mean dose of radiation for the tomography protocol was 9.3 ± 4.6 mSv. With regard to CTP, the per-patient values for sensitivity, specificity, positive and negative predictive values, and accuracy were 86%, 81%, 80%, 87%, and 83%, respectively (p = 0.001). The per-territory values were 63%, 86%, 65%, 84%, and 79%, respectively (p < 0.001). In both analyses, the addition of CTP to CTA achieved higher diagnostic accuracy for detecting myocardial ischemia than SPECT (p < 0.001). The use of the stress tomography protocol is feasible and has good diagnostic accuracy for assessing myocardial ischemia in patients with LBBB.

Crotaline Fab antivenom appears to be effective in cases of severe North American pit viper envenomation: An integrative review

PubMed Central

Lavonas, Eric J; Schaeffer, Tammi H; Kokko, Jamie; Mlynarchek, Sara L; Bogdan, Gregory M

2009-01-01

Background In 2000, the United States Food and Drug Administration approved Crotalidae Polyvalent Immune Fab (Ovine) (hereafter, FabAV), "for the management of patients with minimal to moderate North American Crotalid envenomation." Because whole-IgG pit viper antivenom is no longer available in the United States, FabAV is currently the only specific treatment option available to United States clinicians treating snakebite victims of any severity. No clinical trial data are available concerning the effectiveness of FabAV for treatment of severe snakebite, but several published articles describe its use in this setting. Methods We performed a comprehensive review of the English-language medical literature to identify all publications (1996 to July, 2008) containing data about the administration of FabAV. Two trained reviewers separately extracted case-level data concerning the administration of FabAV to patients with severe envenomation by North American crotaline snakes to a standardized form. Descriptive statistics were used. In addition, we hand-searched the US National Poison Data System reports for the years 2000–2006 to identify and describe any reports of death that occurred after FabAV administration. Results The literature review found 147 unique publications regarding FabAV. Twenty-four evaluable cases of severe human envenomation treated with FabAV were identified in 19 publications. Seven cases were described in five cohort studies, and 17 cases were described in 14 single patient case reports or non-cohort case series. Sixty-five specific severe venom effects were reported in these 24 patients, of which 50 effects (77%) improved or resolved after FabAV therapy. Initial control of all severe venom effects was achieved in 12 patients (50%). The rate at which initial control was achieved was significantly higher among patients reported in the cohort series than in the case series and non-cohort reports (100% vs. 29%, P = 0.005). The median dose of Fab

The Ubx Polycomb response element bypasses an unpaired Fab-8 insulator via cis transvection in Drosophila.

PubMed

Lu, Danfeng; Li, Zhuoran; Li, Lingling; Yang, Liping; Chen, Guijun; Yang, Deying; Zhang, Yue; Singh, Vikrant; Smith, Sheryl; Xiao, Yu; Wang, Erlin; Ye, Yunshuang; Zhang, Wei; Zhou, Lei; Rong, Yikang; Zhou, Jumin

2018-01-01

Chromatin insulators or boundary elements protect genes from regulatory activities from neighboring genes or chromatin domains. In the Drosophila Abdominal-B (Abd-B) locus, the deletion of such elements, such as Frontabdominal-7 (Fab-7) or Fab-8 led to dominant gain of function phenotypes, presumably due to the loss of chromatin barriers. Homologous chromosomes are paired in Drosophila, creating a number of pairing dependent phenomena including transvection, and whether transvection may affect the function of Polycomb response elements (PREs) and thus contribute to the phenotypes are not known. Here, we studied the chromatin barrier activity of Fab-8 and how it is affected by the zygosity of the transgene, and found that Fab-8 is able to block the silencing effect of the Ubx PRE on the DsRed reporter gene in a CTCF binding sites dependent manner. However, the blocking also depends on the zygosity of the transgene in that the barrier activity is present when the transgene is homozygous, but absent when the transgene is heterozygous. To analyze this effect, we performed chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) experiments on homozygous transgenic embryos, and found that H3K27me3 and H3K9me3 marks are restricted by Fab-8, but they spread beyond Fab-8 into the DsRed gene when the two CTCF binding sites within Fab-8 were mutated. Consistent with this, the mutation reduced H3K4me3 and RNA Pol II binding to the DsRed gene, and consequently, DsRed expression. Importantly, in heterozygous embryos, Fab-8 is unable to prevent the spread of H3K27me3 and H3K9me3 marks from crossing Fab-8 into DsRed, suggesting an insulator bypass. These results suggest that in the Abd-B locus, deletion of the insulator in one copy of the chromosome could lead to the loss of insulator activity on the homologous chromosome, and in other loci where chromosomal deletion created hemizygous regions of the genome, the chromatin barrier could be compromised. This study highlights

A parallel panning scheme used for selection of a GluA4-specific Fab targeting the ligand-binding domain.

PubMed

Clausen, Rasmus P; Mohr, Andreas Ø; Riise, Erik; Jensen, Anders A; Gill, Avinash; Madden, Dean R; Kastrup, Jette S; Skottrup, Peter D

2016-11-01

A method for development of murine Fab fragments towards extracellular domains of a surface receptor is presented. The GluA4 ionotropic glutamate receptor is used as a model system. Recombinant GluA4 ectodomain comprising both the N-terminal domain (NTD) and the ligand-binding domain (LBD) in one molecule was used for immunization. A Fab-phage library was constructed and a parallel panning approach enabled selection of murine Fab fragments towards either intact ectodomain or the isolated LBD of the GluA4 receptor. One LBD-Fab (FabL9) showed exclusive selectivity for the GluA4 LBD, over a panel of LBDs from GluA2, GluK1, GluK2 and GluD2. Soluble FabL9 was produced in amounts suitable for characterization. Competitive ELISA and rat-brain immunoprecipitation experiments confirmed that the FabL9 epitope is conserved in the LBD and in the intact native receptor. By an alignment of GluA2 and GluA4, the likely binding epitope for FabL9 was predicted. This study demonstrates a simple approach for development of antibody fragments towards specific sub-domains of a large ligand-gated ion channel, and this method could be utilized for all multi-domain surface receptors where antibody domain-selectivity may be desirable. Furthermore, we present for the first time a GluA4 subtype-specific murine Fab fragment targeting the LBD of the receptor. Copyright © 2016 Elsevier B.V. All rights reserved.

fabH deletion increases DHA production in Escherichia coli expressing Pfa genes.

PubMed

Giner-Robles, Laura; Lázaro, Beatriz; de la Cruz, Fernando; Moncalián, Gabriel

2018-06-08

Some marine bacteria, such as Moritella marina, produce the nutraceutical docosahexaenoic acid (DHA) thanks to a specific enzymatic complex called Pfa synthase. Escherichia coli heterologously expressing the pfa gene cluster from M. marina also produces DHA. The aim of this study was to find genetic or metabolic conditions to increase DHA production in E. coli. First, we analysed the effect of the antibiotic cerulenin, showing that DHA production increased twofold. Then, we tested a series of single gene knockout mutations affecting fatty acid biosynthesis, in order to optimize the synthesis of DHA. The most effective mutant, fabH, showed a threefold increase compared to wild type strain. The combination of cerulenin inhibition and fabH deletion rendered a 6.5-fold improvement compared to control strain. Both strategies seem to have the same mechanism of action, in which fatty acid synthesis via the canonical pathway (fab pathway) is affected in its first catalytic step, which allows the substrates to be used by the heterologous pathway to synthesize DHA. DHA-producing E. coli strain that carries a fabH gene deletion boosts DHA production by tuning down the competing canonical biosynthesis pathway. Our approach can be used for optimization of DHA production in different organisms.

Trastuzumab- and Fab' fragment-modified curcumin PEG-PLGA nanoparticles: preparation and evaluation in vitro and in vivo.

PubMed

Duan, Dongyu; Wang, Aiping; Ni, Ling; Zhang, Liping; Yan, Xiuju; Jiang, Ying; Mu, Hongjie; Wu, Zimei; Sun, Kaoxiang; Li, Youxin

2018-01-01

Nanoparticles (NPs) modified with bio-ligands represent a promising strategy for active targeted drug delivery to tumour. However, many targeted ligands, such as trastuzumab (TMAB), have high molecular weight, limiting their application for targeting. In this study, we prepared Fab' (antigen-binding fragments cut from TMAB)-modified NPs (Fab'-NPs) with curcumin (Cur) as a model drug for more effective targeting of human epidermal growth factor receptor 2 (HER2/ErbB2/Neu), which is overexpressed on breast cancer cells. The release kinetics was conducted by dialysis bags. The ability to kill HER2-overexpressing BT-474 cells of Fab'-Cur-NPs compared with TMAB-Cur-NPs was conducted by cytotoxicity experiments. Qualitative and quantitative cell uptake studies using coumarin-6 (fluorescent probe)-loaded NPs were performed by fluorescence microscopy and flow cytometry. Pharmacokinetics and biodistribution experiments in vivo were assessed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The release kinetics showed that both Fab'-Cur-NPs and TMAB-Cur-NPs provided continuous, slow release of curcumin for 72 h, with no significant difference. In vitro cytotoxicity experiments showed that Fab'-Cur-NPs manifested prominent ability to kill HER2-overexpressing BT-474 cells compared with TMAB-Cur-NPs. Qualitative and quantitative cell uptake studies indicated that the accumulation of Fab'-NPs was greater than that of TMAB-NPs in BT-474 (HER2+) cells; However, there was no significant difference in MDA-MB-231 (HER2-) cells. Pharmacokinetics and biodistribution experiments in vivo demonstrated that the half-life (t1/2) and area under the blood concentration-time curve (AUC0-t) of Fab'-Cur-NPs increased 5.30-fold and 1.76-fold relative to those of TMAB-Cur-NPs, respectively. Furthermore, the tumor accumulation of Fab'-Cur-NPs was higher than that of TMAB-Cur-NPs. Fab' fragment has greater capacity than the intact antibody to achieve tumor targeting through NP

Two decades of pharmacovigilance and clinical experience with highly purified rabies immunoglobulin F(ab')2 fragments.

PubMed

Reveneau, Elisa; Cottin, Pascale; Rasuli, Anvar

2017-03-01

Rabies is a worldwide zoonotic viral disease with no specific treatment once symptoms occur; manifest disease is almost always fatal. WHO recommendations for exposed individuals include immediate attention to the wound and use of rabies immunoglobulin and/or vaccine for post-exposure prophylaxis (PEP). Here, we provide an overview of the clinical experience with a highly purified preparation of F(ab') 2 fragments from equine rabies immunoglobulin (F(ab') 2 pERIG; Favirab TM ) in rabies PEP. Areas covered: Our review comprises a retrospective analysis of adverse event reports in the Sanofi Pasteur global pharmacovigilance database for F(ab') 2 pERIG, including adverse event reports from eight Sanofi Pasteur-sponsored clinical trials and post-market surveillance data collected between 1995 and 2014. The general safety profile of F(ab') 2 pERIG is discussed, as are the occurrence of rare anaphylactic reactions, and suspected intervention failure. Expert commentary: Over 20 years of clinical development and post-licensure experience has established the safety and effectiveness of F(ab') 2 pERIG (Favirab TM ) in rabies PEP.

Generating and Purifying Fab Fragments from Human and Mouse IgG Using the Bacterial Enzymes IdeS, SpeB and Kgp.

PubMed

Sjögren, Jonathan; Andersson, Linda; Mejàre, Malin; Olsson, Fredrik

2017-01-01

Fab fragments are valuable research tools in various areas of science including applications in imaging, binding studies, removal of Fc-mediated effector functions, mass spectrometry, infection biology, and many others. The enzymatic tools for the generation of Fab fragments have been discovered through basic research within the field of molecular bacterial pathogenesis. Today, these enzymes are widely applied as research tools and in this chapter, we describe methodologies based on bacterial enzymes to generate Fab fragments from both human and mouse IgG. For all human IgG subclasses, the IdeS enzyme from Streptococcus pyogenes has been applied to generate F(ab')2 fragments that subsequently can be reduced under mild conditions to generate a homogenous pool of Fab' fragments. The enzyme Kgp from Porphyromonas gingivalis has been applied to generate intact Fab fragments from human IgG1 and the Fab fragments can be purified using a CH1-specific affinity resin. The SpeB protease, also from S. pyogenes, is able to digest mouse IgGs and has been applied to digest antibodies and Fab fragments can be purified on light chain affinity resins. In this chapter, we describe methodologies that can be used to obtain Fab fragments from human and mouse IgG using bacterial proteases.

Antibody Fab display and selection through fusion to the pIX coat protein of filamentous phage.

PubMed

Tornetta, Mark; Baker, Scott; Whitaker, Brian; Lu, Jin; Chen, Qiang; Pisors, Eileen; Shi, Lei; Luo, Jinquan; Sweet, Raymond; Tsui, Ping

2010-08-31

Fab antibody display on filamentous phage is widely applied to de novo antibody discovery and engineering. Here we describe a phagemid system for the efficient display and affinity selection of Fabs through linkage to the minor coat protein pIX. Display was successful by fusion of either Fd or Lc through a short linker to the amino terminus of pIX and co-expression of the counter Lc or Fd as a secreted, soluble fragment. Assembly of functional Fab was confirmed by demonstration of antigen-specific binding using antibodies of known specificity. Phage displaying a Fab specific for RSV-F protein with Fd linked to pIX showed efficient, antigen-specific enrichment when mixed with phage displaying a different specificity. The functionality of this system for antibody engineering was evaluated in an optimization study. A RSV-F protein specific antibody with an affinity of about 2nM was randomized at 4 positions in light chain CDR1. Three rounds of selection with decreasing antigen concentration yielded Fabs with an affinity improvement up to 70-fold and showed a general correlation between enrichment frequency and affinity. We conclude that the pIX coat protein complements other display systems in filamentous phage as an efficient vehicle for low copy display and selection of Fab proteins. 2010 Elsevier B.V. All rights reserved.

Anti-digoxin Fab variants generated by phage display.

PubMed

Murata, Viviane Midori; Schmidt, Mariana Costa Braga; Kalil, Jorge; Tsuruta, Lilian Rumi; Moro, Ana Maria

2013-06-01

Digoxin is a pharmaceutical used in the control of cardiac dysfunction. Its therapeutic window is narrow, with effect dosage very close to the toxic dosage. To counteract the toxic effect, polyclonal Fab fragments are commercially available. Our study is based on a monoclonal anti-digoxin antibody, which would provide a product with a specific potency and more precise dosage for the detoxification of patients under digoxin treatment. Phage display technology was used to select variants with high affinity. From an anti-digoxin hybridoma, RNA was extracted for subsequent cDNA synthesis. Specific primers were used for the LC and Fd amplifications, then cloned sequentially in a phagemid vector (pComb3X) for the combinatorial Fab library construction. Clones were selected for their ability to bind to digoxin-BSA. The presence of light and heavy chains was checked, randomly selected clones then sequenced and induced to produce soluble Fabs, and subsequently analyzed for anti-digoxin expression. Out of ten clones randomly chosen, six resulted positive expression of the product. The sequencing of these revealed two identical clones and one presenting a pseudogene in the LC. Four clones presenting variations in the framework1 showed binding to digoxin-BSA by ELISA and western blotting. The specific binding was further confirmed by Biacore(®), which allowed ranking of the clones. The development of these clones allowed the selection of variants with higher affinity than the original version.

Improving Crotalidae polyvalent immune Fab reconstitution times.

PubMed

Quan, Asia N; Quan, Dan; Curry, Steven C

2010-06-01

Crotalidae polyvalent immune Fab (CroFab) is used to treat rattlesnake envenomations in the United States. Time to infusion may be a critical factor in the treatment of these bites. Per manufacturer's instructions, 10 mL of sterile water for injection (SWI) and hand swirling are recommended for reconstitution. We wondered whether completely filling vials with 25 mL of SWI would result in shorter reconstitution times than using 10-mL volumes and how hand mixing compared to mechanical agitation of vials or leaving vials undisturbed. Six sets of 5 vials were filled with either 10 mL or 25 mL. Three mixing techniques were used as follows: undisturbed; agitation with a mechanical agitator; and continuous hand rolling and inverting of vials. Dissolution was determined by observation and time to complete dissolution for each vial. Nonparametric 2-tailed P values were calculated. Filling vials completely with 25 mL resulted in quicker dissolution than using 10-mL volumes, regardless of mixing method (2-tailed P = .024). Mixing by hand was shorter than other methods (P < .001). Reconstitution with 25 mL and hand mixing resulted in the shortest dissolution times (median, 1.1 minutes; range, 0.9-1.3 minutes). This appeared clinically important because dissolution times using 10 mL and mechanical rocking of vials (median, 26.4 minutes) or leaving vials undisturbed (median, 33.6 minutes) was several-fold longer. Hand mixing after filling vials completely with 25 mL results in shorter dissolution times than using 10 mL or other methods of mixing and is recommended, especially when preparing initial doses of CroFab. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Tumour imaging by the detection of fibrin clots in tumour stroma using an anti-fibrin Fab fragment.

PubMed

Obonai, Toshifumi; Fuchigami, Hirobumi; Furuya, Fumiaki; Kozuka, Naoyuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

2016-03-24

The diagnosis of early and aggressive types of cancer is important for providing effective cancer therapy. Cancer-induced fibrin clots exist only within lesions. Previously, we developed a monoclonal antibody (clone 102-10) that recognizes insoluble fibrin but not fibrinogen or soluble fibrin and confirmed that fibrin clots form continuously in various cancers. Here, we describe the development of a Fab fragment probe of clone 102-10 for tumour imaging. The distribution of 102-10 Fab was investigated in genetically engineered mice bearing pancreatic ductal adenocarcinoma (PDAC), and its effect on blood coagulation was examined. Immunohistochemical and ex vivo imaging revealed that 102-10 Fab was distributed selectively in fibrin clots in PDAC tumours 3 h after injection and that it disappeared from the body after 24 h. 102-10 Fab had no influence on blood coagulation or fibrinolysis. Tumour imaging using anti-fibrin Fab may provide a safe and effective method for the diagnosis of invasive cancers by detecting fibrin clots in tumour stroma.

PET Imaging of Abdominal Aortic Aneurysm with 64Cu-Labeled Anti-CD105 Antibody Fab Fragment.

PubMed

Shi, Sixiang; Orbay, Hakan; Yang, Yunan; Graves, Stephen A; Nayak, Tapas R; Hong, Hao; Hernandez, Reinier; Luo, Haiming; Goel, Shreya; Theuer, Charles P; Nickles, Robert J; Cai, Weibo

2015-06-01

The critical challenge in abdominal aortic aneurysm (AAA) research is the accurate diagnosis and assessment of AAA progression. Angiogenesis is a pathologic hallmark of AAA, and CD105 is highly expressed on newly formed vessels. Our goal was to use (64)Cu-labeled anti-CD105 antibody Fab fragment for noninvasive assessment of angiogenesis in the aortic wall in a murine model of AAA. Fab fragment of TRC105, a mAb that specifically binds to CD105, was generated by enzymatic papain digestion and conjugated to NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) for (64)Cu labeling. The binding affinity/specificity of NOTA-TRC105-Fab was evaluated by flow cytometry and various ex vivo studies. BALB/c mice were anesthetized and treated with calcium phosphate to induce AAA and underwent weekly PET scans using (64)Cu-NOTA-TRC105-Fab. Biodistribution and autoradiography studies were also performed to confirm the accuracy of PET results. NOTA-TRC105-Fab exhibited high purity and specifically bound to CD105 in vitro. Uptake of (64)Cu-NOTA-TRC105-Fab increased from a control level of 3.4 ± 0.1 to 9.5 ± 0.4 percentage injected dose per gram (%ID/g) at 6 h after injection on day 5 and decreased to 7.2 ± 1.4 %ID/g on day 12, which correlated well with biodistribution and autoradiography studies (i.e., much higher tracer uptake in AAA than normal aorta). Of note, enhanced AAA contrast was achieved, due to the minimal background in the abdominal area of mice. Degradation of elastic fibers and highly expressed CD105 were observed in ex vivo studies. (64)Cu-NOTA-TRC105-Fab cleared rapidly through the kidneys, which enabled noninvasive PET imaging of the aorta with enhanced contrast and showed increased angiogenesis (CD105 expression) during AAA. (64)Cu-NOTA-TRC105-Fab PET may potentially be used for future diagnosis and prognosis of AAA. © 2015 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Spherical cows in the sky with fab four

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kaloper, Nemanja; Sandora, McCullen, E-mail: kaloper@physics.ucdavis.edu, E-mail: mesandora@ucdavis.edu

2014-05-01

We explore spherically symmetric static solutions in a subclass of unitary scalar-tensor theories of gravity, called the 'Fab Four' models. The weak field large distance solutions may be phenomenologically viable, but only if the Gauss-Bonnet term is negligible. Only in this limit will the Vainshtein mechanism work consistently. Further, classical constraints and unitarity bounds constrain the models quite tightly. Nevertheless, in the limits where the range of individual terms at large scales is respectively Kinetic Braiding, Horndeski, and Gauss-Bonnet, the horizon scale effects may occur while the theory satisfies Solar system constraints and, marginally, unitarity bounds. On the other hand,more » to bring the cutoff down to below a millimeter constrains all the couplings scales such that 'Fab Fours' can't be heard outside of the Solar system.« less

Myocardial Injury without Electrocardiographic Changes after a Suicide Attempt by an Overdose of Glimepiride and Zolpidem: A Case Report and Literature Review.

PubMed

Chou, Shengpu; Ayabe, Seiji; Sekine, Nobuo

2015-01-01

A 40-year-old diabetic man was admitted to our hospital for poor glycemic control. During hospitalization, he took 42 mg glimepiride and 50 mg zolpidem as a suicide attempt. The following day, the creatine kinase-MB fraction and troponin I levels were elevated to 112 IU/L and 8.77 ng/mL, respectively, without any electrocardiographic abnormalities. The patient recovered completely without any complications. Four weeks later, coronary computed tomography angiography and myocardial perfusion scintigraphy revealed moderate one-vessel coronary disease without the evidence of myocardial ischemia or old infarction. Cardiac-specific markers must be considered in sulfonylurea-induced hypoglycemic patients, particularly when the patient is unconscious and does not exhibit any clinical manifestations.

A novel human Fab antibody for Trop2 inhibits breast cancer growth in vitro and in vivo.

PubMed

Lin, Hong; Zhang, Huiling; Wang, Jun; Lu, Meiping; Zheng, Feng; Wang, Changjun; Tang, Xiaojun; Xu, Ning; Chen, Renjie; Zhang, Dawei; Zhao, Ping; Zhu, Jin; Mao, Yuan; Feng, Zhenqing

2014-03-01

Human trophoblastic cell surface antigen 2 (Trop2) has been suggested as an oncogene, which is associated with the different types of tumors. In this study, a human Fab antibody against Trop2 extracellular domain was isolated from phage library by phage display technology, and characterized by ELISA, FACS, fluorescence staining and Western blotting analysis. MTT, apoptosis assay and wound healing assay were employed to evaluate the inhibitory effects of Trop2 Fab on breast cancer cell growth in vitro, while tumor-xenograft model was employed to evaluate the inhibitory effects on breast cancer growth in vivo. The results showed that Trop2 Fab inhibited the proliferation, induced the apoptosis and suspended the migration of MDA-MB-231 cells in a dose dependent manner. The expression caspase-3 was activated, and the expression of Bcl-2 was reduced while that of Bax was elevated in MDA-MB-231 cells by treating with Trop2 Fab. In addition, Trop2 Fab inhibited the growth of breast cancer xenografts and the expression of Bcl-2 was reduced while that of Bax was elevated in xenografts. Trop2 Fab, which was isolated successfully in this research, is a promising therapeutic agent for the treatment of Trop2 expressing breast cancer. © 2013 UICC.

Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments.

PubMed

Ellis, Mark; Patel, Pareshkumar; Edon, Marjory; Ramage, Walter; Dickinson, Robert; Humphreys, David P

2017-01-01

Humanized Fab' fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab' yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab' expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab', DsbC had the most significant impact, increasing humanized Fab' production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored "wild type" cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD 600 105), 40 h post Fab' induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab' fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212-220, 2017. © 2016 American Institute of Chemical Engineers.

Functionally Approached Body (FAB) Strategies for Young Children Who Have Behavioral and Sensory Processing Challenges

ERIC Educational Resources Information Center

Pagano, John

2005-01-01

Functionally Approached Body (FAB) Strategies offer a clinical approach to help parents of young children with behavioral and sensory processing strategies. This article introduces the FAB Strategies, clinical strategies developed by the author for understanding and addressing young children's behavioral and sensory processing challenges. The FAB…

Utilisation of Crotalidae polyvalent immune fab (ovine) for Viperidae envenomations in children.

PubMed

Johnson, P N; McGoodwin, L; Banner, W

2008-12-01

Snakebite envenomations occur in 45,000 patients in the USA annually and are associated with morbidity especially in children and the elderly. Crotalidae polyvalent immune fab (ovine; FabAV) is a polyvalent antivenom derived from sheep for crotalid envenomations. Limited clinical trials are available in paediatric patients. A literature search using MEDLINE (1950-February 2008), International Pharmaceutical Abstracts (1970-February 2008), EMBASE (1988-February 2008) and Cochrane Library (1996-June 2008) was conducted using key words including: antivenom OR snakebites OR children OR Crotalid OR envenomations. All English-language articles were identified from data sources. Pertinent studies pertaining to FabAV in children and adolescents with crotalid envenomations were included for analysis. Ten papers were included for review, representing 47 children. Initial doses ranging from 2 to 18 g were administered and initial control was achieved in most children. Maintenance dosing was continued in 63.8% (30/47) of patients; 4.3% (2/47) of patients had episodes of venom recurrence. Adverse events were noted in 8.5% of children (4/47) when pooled for data analysis. FabAV appears to be a safe and effective agent for children with crotalid envenomations. Clinicians should adapt dosing recommendations used for adults until future large, well-designed trials can confirm the efficacy and safety from observation studies and case reports.

Fab-dsFv: A bispecific antibody format with extended serum half-life through albumin binding.

PubMed

Davé, Emma; Adams, Ralph; Zaccheo, Oliver; Carrington, Bruce; Compson, Joanne E; Dugdale, Sarah; Airey, Michael; Malcolm, Sarah; Hailu, Hanna; Wild, Gavin; Turner, Alison; Heads, James; Sarkar, Kaushik; Ventom, Andrew; Marshall, Diane; Jairaj, Mark; Kopotsha, Tim; Christodoulou, Louis; Zamacona, Miren; Lawson, Alastair D; Heywood, Sam; Humphreys, David P

2016-10-01

An antibody format, termed Fab-dsFv, has been designed for clinical indications that require monovalent target binding in the absence of direct Fc receptor (FcR) binding while retaining substantial serum presence. The variable fragment (Fv) domain of a humanized albumin-binding antibody was fused to the C-termini of Fab constant domains, such that the VL and VH domains were individually connected to the Cκ and CH1 domains by peptide linkers, respectively. The anti-albumin Fv was selected for properties thought to be desirable to ensure a durable serum half-life mediated via FcRn. The Fv domain was further stabilized by an inter-domain disulfide bond. The bispecific format was shown to be thermodynamically and biophysically stable, and retained good affinity and efficacy to both antigens simultaneously. In in vivo studies, the serum half-life of Fab-dsFv, 2.6 d in mice and 7.9 d in cynomolgus monkeys, was equivalent to Fab'-PEG.

Fab-dsFv: A bispecific antibody format with extended serum half-life through albumin binding

PubMed Central

Davé, Emma; Adams, Ralph; Zaccheo, Oliver; Carrington, Bruce; Compson, Joanne E.; Dugdale, Sarah; Airey, Michael; Malcolm, Sarah; Hailu, Hanna; Wild, Gavin; Turner, Alison; Heads, James; Sarkar, Kaushik; Ventom, Andrew; Marshall, Diane; Jairaj, Mark; Kopotsha, Tim; Christodoulou, Louis; Zamacona, Miren; Lawson, Alastair D.; Heywood, Sam; Humphreys, David P.

2016-01-01

ABSTRACT An antibody format, termed Fab-dsFv, has been designed for clinical indications that require monovalent target binding in the absence of direct Fc receptor (FcR) binding while retaining substantial serum presence. The variable fragment (Fv) domain of a humanized albumin-binding antibody was fused to the C-termini of Fab constant domains, such that the VL and VH domains were individually connected to the Cκ and CH1 domains by peptide linkers, respectively. The anti-albumin Fv was selected for properties thought to be desirable to ensure a durable serum half-life mediated via FcRn. The Fv domain was further stabilized by an inter-domain disulfide bond. The bispecific format was shown to be thermodynamically and biophysically stable, and retained good affinity and efficacy to both antigens simultaneously. In in vivo studies, the serum half-life of Fab-dsFv, 2.6 d in mice and 7.9 d in cynomolgus monkeys, was equivalent to Fab'-PEG. PMID:27532598

Alteration of the fatty acid profile of Streptomyces coelicolor by replacement of the initiation enzyme 3-ketoacyl acyl carrier protein synthase III (FabH).

PubMed

Li, Yongli; Florova, Galina; Reynolds, Kevin A

2005-06-01

The first elongation step of fatty acid biosynthesis by a type II dissociated fatty acid synthases is catalyzed by 3-ketoacyl-acyl carrier protein (ACP) synthase III (KASIII, FabH). This enzyme, encoded by the fabH gene, catalyzes a decarboxylative condensation between an acyl coenzyme A (CoA) primer and malonyl-ACP. In organisms such as Escherichia coli, which generate only straight-chain fatty acids (SCFAs), FabH has a substrate preference for acetyl-CoA. In streptomycetes and other organisms which produce a mixture of both SCFAs and branched-chain fatty acids (BCFAs), FabH has been shown to utilize straight- and branched-chain acyl-CoA substrates. We report herein the generation of a Streptomyces coelicolor mutant (YL/ecFabH) in which the chromosomal copy of the fabH gene has been replaced and the essential process of fatty acid biosynthesis is initiated by plasmid-based expression of the E. coli FabH (bearing only 35% amino acid identity to the Streptomyces enzyme). The YL/ecFabH mutant produces predominantly SCFAs (86%). In contrast, BCFAs predominate (approximately 70%) in both the S. coelicolor parental strain and S. coelicolor YL/sgFabH (a deltafabH mutant carrying a plasmid expressing the Streptomyces glaucescens FabH). These results provide the first unequivocal evidence that the substrate specificity of FabH observed in vitro is a determinant of the fatty acid made in an organism. The YL/ecFabH strain grows significantly slower on both solid and liquid media. The levels of FabH activity in cell extracts of YL/ecFabH were also significantly lower than those in cell extracts of YL/sgFabH, suggesting that a decreased rate of fatty acid synthesis may account for the observed decreased growth rate. The production of low levels of BCFAs in YL/ecFabH suggests either that the E. coli FabH is more tolerant of different acyl-CoAs substrates than previously thought or that there is an additional pathway for initiation of BCFA biosynthesis in Streptomyces coelicolor.

Crystallization of the Fab from a human monoclonal antibody against gp 41 of human immunodeficiency virus type I

NASA Technical Reports Server (NTRS)

Casale, Elena; He, Xiao-Min; Snyder, Robert S.; Carter, Daniel C.; Wenisch, Elisabeth; Jungbauer, Alois; Tauer, Christa; Ruker, Florian; Righetti, Pier Giorgio

1990-01-01

A monoclonal IgG antibody directed against gp 41 from the human immunodeficiency virus (HIV-1) has been crystallized in both intact and Fab forms. Crystals of the intact antibody grow as tetragonal-like prisms too small for conventional X-ray analysis. However, the Fab portion of the antibody produces suitable platelike crystals which belong to the space group P2(1)2(1)2(1) with unit cell constants of a = 66.5 A, b = 74.3 A, and c = 105.3 A. There is one molecule of Fab in the asymmetric unit. The Fab crystals show diffraction to d-spacings less than 3.0 A.

Structure of FabH and factors affecting the distribution of branched fatty acids in Micrococcus luteus.

PubMed

Pereira, Jose H; Goh, Ee-Been; Keasling, Jay D; Beller, Harry R; Adams, Paul D

2012-10-01

Micrococcus luteus is a Gram-positive bacterium that produces iso- and anteiso-branched alkenes by the head-to-head condensation of fatty-acid thioesters [coenzyme A (CoA) or acyl carrier protein (ACP)]; this activity is of interest for the production of advanced biofuels. In an effort to better understand the control of the formation of branched fatty acids in M. luteus, the structure of FabH (MlFabH) was determined. FabH, or β-ketoacyl-ACP synthase III, catalyzes the initial step of fatty-acid biosynthesis: the condensation of malonyl-ACP with an acyl-CoA. Analysis of the MlFabH structure provides insights into its substrate selectivity with regard to length and branching of the acyl-CoA. The most structurally divergent region of FabH is the L9 loop region located at the dimer interface, which is involved in the formation of the acyl-binding channel and thus limits the substrate-channel size. The residue Phe336, which is positioned near the catalytic triad, appears to play a major role in branched-substrate selectivity. In addition to structural studies of MlFabH, transcriptional studies of M. luteus were also performed, focusing on the increase in the ratio of anteiso:iso-branched alkenes that was observed during the transition from early to late stationary phase. Gene-expression microarray analysis identified two genes involved in leucine and isoleucine metabolism that may explain this transition.

Frontal Assessment Battery (FAB) is a simple tool for detecting executive deficits in chronic cannabis users.

PubMed

Fontes, Maria Alice; Bolla, Karen I; Cunha, Paulo Jannuzzi; Almeida, Priscila Previato; Jungerman, Flávia; Laranjeira, Ronaldo Ramos; Bressan, Rodrigo A; Lacerda, Acioly L T

2011-06-01

Cannabis is the most used illicit drug in the world, and its use has been associated with prefrontal cortex (PFC) dysfunction, including deficits in executive functions (EF). Considering that EF may influence treatment outcome, it would be interesting to have a brief neuropsychological battery to assess EF in chronic cannabis users (CCU). In the present study, the Frontal Assessment Battery (FAB), a brief, easy to use neuropsychological instrument aimed to evaluate EF, was used to evaluate cognitive functioning of CCU. We evaluated 107 abstinent CCU with the FAB and compared with 44 controls matched for age, estimated IQ, and years of education. CCU performed poorly as compared to controls (FAB total score = 16.53 vs. 17.09, p < .05). CCU had also a poor performance in the Motor Programming subtest (2.47 vs. 2.73, p < .05). This study examined effects of cannabis in executive functioning and showed evidence that the FAB is sensitive to detect EF deficits in early abstinent chronic cannabis users. Clinical significance of these findings remains to be investigated in further longitudinal studies. FAB may be useful as a screening instrument to evaluate the necessity for a complete neuropsychological assessment in this population.

Nuclear Scintigraphy in Practice: Gastrointestinal Motility.

PubMed

Solnes, Lilja B; Sheikhbahaei, Sara; Ziessman, Harvey A

2018-05-29

The purpose of this article is to describe the clinical utility of state-of-theart gastrointestinal transit scintigraphy, including the standardized esophageal transit, solid and liquid gastric emptying, small-bowel transit, colon transit, and whole-gut transit scintigraphy, with an emphasis on procedure performance. Radionuclide gastrointestinal motility studies are noninvasive, quantitative, and physiologic diagnostic tools for evaluating patients with gastrointestinal complaints.

Tumour imaging by the detection of fibrin clots in tumour stroma using an anti-fibrin Fab fragment

PubMed Central

Obonai, Toshifumi; Fuchigami, Hirobumi; Furuya, Fumiaki; Kozuka, Naoyuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

2016-01-01

The diagnosis of early and aggressive types of cancer is important for providing effective cancer therapy. Cancer-induced fibrin clots exist only within lesions. Previously, we developed a monoclonal antibody (clone 102-10) that recognizes insoluble fibrin but not fibrinogen or soluble fibrin and confirmed that fibrin clots form continuously in various cancers. Here, we describe the development of a Fab fragment probe of clone 102-10 for tumour imaging. The distribution of 102-10 Fab was investigated in genetically engineered mice bearing pancreatic ductal adenocarcinoma (PDAC), and its effect on blood coagulation was examined. Immunohistochemical and ex vivo imaging revealed that 102-10 Fab was distributed selectively in fibrin clots in PDAC tumours 3 h after injection and that it disappeared from the body after 24 h. 102-10 Fab had no influence on blood coagulation or fibrinolysis. Tumour imaging using anti-fibrin Fab may provide a safe and effective method for the diagnosis of invasive cancers by detecting fibrin clots in tumour stroma. PMID:27009516

Extending green technology innovations to enable greener fabs

NASA Astrophysics Data System (ADS)

Takahisa, Kenji; Yoo, Young Sun; Fukuda, Hitomi; Minegishi, Yuji; Enami, Tatsuo

2015-03-01

Semiconductor manufacturing industry has growing concerns over future environmental impacts as fabs expand and new generations of equipment become more powerful. Especially rare gases supply and price are one of prime concerns for operation of high volume manufacturing (HVM) fabs. Over the past year it has come to our attention that Helium and Neon gas supplies could be unstable and become a threat to HVM fabs. To address these concerns, Gigaphoton has implemented various green technologies under its EcoPhoton program. One of the initiatives is GigaTwin deep ultraviolet (DUV) lithography laser design which enables highly efficient and stable operation. Under this design laser systems run with 50% less electric energy and gas consumption compared to conventional laser designs. In 2014 we have developed two technologies to further reduce electric energy and gas efficiency. The electric energy reduction technology is called eGRYCOS (enhanced Gigaphoton Recycled Chamber Operation System), and it reduces electric energy by 15% without compromising any of laser performances. eGRYCOS system has a sophisticated gas flow design so that we can reduce cross-flow-fan rotation speed. The gas reduction technology is called eTGM (enhanced Total gas Manager) and it improves gas management system optimizing the gas injection and exhaust amount based on laser performances, resulting in 50% gas savings. The next steps in our roadmap technologies are indicated and we call for potential partners to work with us based on OPEN INNOVATION concept to successfully develop faster and better solutions in all possible areas where green innovation may exist.

Improving Assessment of Work Related Mental Health Function Using the Work Disability Functional Assessment Battery (WD-FAB).

PubMed

Marfeo, Elizabeth E; Ni, Pengsheng; McDonough, Christine; Peterik, Kara; Marino, Molly; Meterko, Mark; Rasch, Elizabeth K; Chan, Leighton; Brandt, Diane; Jette, Alan M

2018-03-01

Purpose To improve the mental health component of the Work Disability Functional Assessment Battery (WD-FAB), developed for the US Social Security Administration's (SSA) disability determination process. Specifically our goal was to expand the WD-FAB scales of mood & emotions, resilience, social interactions, and behavioral control to improve the depth and breadth of the current scales and expand the content coverage to include aspects of cognition & communication function. Methods Data were collected from a random, stratified sample of 1695 claimants applying for the SSA work disability benefits, and a general population sample of 2025 working age adults. 169 new items were developed to replenish the WD-FAB scales and analyzed using factor analysis and item response theory (IRT) analysis to construct unidimensional scales. We conducted computer adaptive test (CAT) simulations to examine the psychometric properties of the WD-FAB. Results Analyses supported the inclusion of four mental health subdomains: Cognition & Communication (68 items), Self-Regulation (34 items), Resilience & Sociability (29 items) and Mood & Emotions (34 items). All scales yielded acceptable psychometric properties. Conclusions IRT methods were effective in expanding the WD-FAB to assess mental health function. The WD-FAB has the potential to enhance work disability assessment both within the context of the SSA disability programs as well as other clinical and vocational rehabilitation settings.

Investigation of optimal acquisition time of myocardial perfusion scintigraphy using cardiac focusing-collimator

NASA Astrophysics Data System (ADS)

Niwa, Arisa; Abe, Shinji; Fujita, Naotoshi; Kono, Hidetaka; Odagawa, Tetsuro; Fujita, Yusuke; Tsuchiya, Saki; Kato, Katsuhiko

2015-03-01

Recently myocardial perfusion SPECT imaging acquired using the cardiac focusing-collimator (CF) has been developed in the field of nuclear cardiology. Previously we have investigated the basic characteristics of CF using physical phantoms. This study was aimed at determining the acquisition time for CF that enables to acquire the SPECT images equivalent to those acquired by the conventional method in 201TlCl myocardial perfusion SPECT. In this study, Siemens Symbia T6 was used by setting the torso phantom equipped with the cardiac, pulmonary, and hepatic components. 201TlCl solution were filled in the left ventricular (LV) myocardium and liver. Each of CF, the low energy high resolution collimator (LEHR), and the low medium energy general purpose collimator (LMEGP) was set on the SPECT equipment. Data acquisitions were made by regarding the center of the phantom as the center of the heart in CF at various acquisition times. Acquired data were reconstructed, and the polar maps were created from the reconstructed images. Coefficient of variation (CV) was calculated as the mean counts determined on the polar maps with their standard deviations. When CF was used, CV was lower at longer acquisition times. CV calculated from the polar maps acquired using CF at 2.83 min of acquisition time was equivalent to CV calculated from those acquired using LEHR in a 180°acquisition range at 20 min of acquisition time.

Prognostic Usefulness of Cardiac Stress Test Modalities in Patients With Type 2 Diabetes Mellitus Who Underwent Myocardial Perfusion Scintigraphy (from the Basel Asymptomatic High-Risk Diabetics' Outcome Trial).

PubMed

Caobelli, Federico; Haaf, Philip; Chronis, Joannis; Haenny, Gianluca; Brinkert, Miriam; Pfisterer, Matthias E; Zellweger, Michael J

2017-10-01

Our study aimed to assess predictors of the stress test technique used and to evaluate the impact of exercise level achieved on risk stratification in patients with asymptomatic type 2 diabetes without a previous coronary artery disease. Little is known whether co-morbidities of these patients predict the stress technique and whether physical performance provides risk stratification: 400 patients underwent clinical evaluation and myocardial perfusion scintigraphy (MPS) using physical or pharmacological stress. Physical patients were divided into 2 groups: achieving <6 and ≥6 METs, respectively. The mean follow-up time was 2 years. Major cardiac events (MACEs) included myocardial infarction and/or cardiac death. Independent predictors of pharmacological stress were a body mass index of >30 kg/m 2 (hazard ratio 1.076, 95% confidence interval 1.027 to 1.127, p = 0.002) and a peripheral arterial disease (hazard ratio 2.888, 95% confidence interval 1.446 to 5.769, p = 0.003). Pharmacological patients had more MACE than physical patients (3.2% vs 1.0%, p = 0.03). Patients achieving <6 METs had a similar MACE rate as pharmacological patients (3.0% vs 3.2%, p = not significant) and more MACE than patients achieving ≥6 METs (3.0% vs 0.4%, p = 0.01). In patients achieving <6 METs and in pharmacological patients, MPS added an incremental prognostic value to pretest information (p values for global chi-square 0.012 and 0.04, respectively). In high-risk asymptomatic diabetic patients, co-morbidities were predictive of the stress technique used. Pharmacological patients had more MACE, similar to those unable to achieve 6 METs. MPS provided an incremental prognostic value in pharmacological patients and in patients with <6METs. In contrast, patients who were able to achieve ≥6 METs were at low risk and do not need further risk stratification. Copyright © 2017 Elsevier Inc. All rights reserved.

ImmunoPET of tissue factor expression in triple-negative breast cancer with a radiolabeled antibody Fab fragment.

PubMed

Shi, Sixiang; Hong, Hao; Orbay, Hakan; Graves, Stephen A; Yang, Yunan; Ohman, Jakob D; Liu, Bai; Nickles, Robert J; Wong, Hing C; Cai, Weibo

2015-07-01

To date, there is no effective therapy for triple-negative breast cancer (TNBC), which has a dismal clinical outcome. Upregulation of tissue factor (TF) expression leads to increased patient morbidity and mortality in many solid tumor types, including TNBC. Our goal was to employ the Fab fragment of ALT-836, a chimeric anti-human TF mAb, for PET imaging of TNBC, which can be used to guide future TNBC therapy. ALT-836-Fab was generated by enzymatic papain digestion. SDS-PAGE and FACS studies were performed to evaluate the integrity and TF binding affinity of ALT-836-Fab before NOTA conjugation and (64)Cu-labeling. Serial PET imaging and biodistribution studies were carried out to evaluate the tumor targeting efficacy and pharmacokinetics in the MDA-MB-231 TNBC model, which expresses high levels of TF on the tumor cells. Blocking studies, histological assessment, as well as RT-PCR were performed to confirm TF specificity of (64)Cu-NOTA-ALT-836-Fab. ALT-836-Fab was produced with high purity, which exhibited superb TF binding affinity and specificity. Serial PET imaging revealed rapid and persistent tumor uptake of (64)Cu-NOTA-ALT-836-Fab (5.1 ± 0.5 %ID/g at 24 h post-injection; n = 4) and high tumor/muscle ratio (7.0 ± 1.2 at 24 h post-injection; n = 4), several-fold higher than that of the blocking group and tumor models that do not express significant level of TF, which was confirmed by biodistribution studies. TF specificity of the tracer was also validated by histology and RT-PCR. (64)Cu-NOTA-ALT-836-Fab exhibited prominent tissue factor targeting efficiency in MDA-MB-231 TNBC model. The use of a Fab fragment led to fast tumor uptake and good tissue/muscle ratio, which may be translated into same-day immunoPET imaging in the clinical setting to improve TNBC patient management.

Use of blood-pool imaging in evaluation of diffuse activity patterns in technetium-99m pyrophosphate myocardial scintigraphy.

PubMed

Cowley, M J; Mantle, J A; Rogers, W J; Russell, R O; Rackley, C E; Logic, J R

1979-06-01

It has been suggested that diffuse Tc-99m pyrophosphate precordial activity may be due to persistent blood-pool activity in routine delayed views during myocardial imaging. To answer this question, we reviewed myocardial scintigrams recorded 60--90 min following the injection of 12--15 mCi of Tc-99m pyrophosphate for the presence of diffuse precordial activity, and compared these with early images of the blood pool in 265 patients. Diffuse activity in the delayed images was identified in 48 patients: in 20 with acute myocardial infarction and in 28 with no evidence of it. Comparison of these routine delayed images with early views of the blood pool revealed two types of patterns. In patients with acute infarction, 95% had delayed images that were distinguishable from blood pool either because the activity was smaller than the early blood pool, or by the presence of localized activity superimposed on diffuse activity identical to blood pool. In those without infarction, 93% had activity distribution in routine delayed views matching that in the early blood-pool images. The usefulness of the diffuse TcPPi precordial activity in myocardial infarction is improved when early blood-pool imaging is used to exclude persistence of blood-pool activity as its cause. Moreover, it does not require additional amounts of radioactivity nor complex computer processing, a feature that may be of value in the community hospital using the technique to "rule out" infarction 24--72 hr after onset of suggestive symptoms.

Bile Reflux Scintigraphy After Mini-Gastric Bypass.

PubMed

Saarinen, Tuure; Räsänen, Jari; Salo, Jarmo; Loimaala, Antti; Pitkonen, Miia; Leivonen, Marja; Juuti, Anne

2017-08-01

Significant weight-loss and diabetes remission have been reported after mini-gastric bypass (MGB). Concern has been raised regarding postoperative bile reflux (BR), but it has not been demonstrated in previous studies. We set out to find out if BR is evident in hepatobiliary scintigraphy after MGB. Nine consecutive patients, seven with type 2 diabetes, underwent MGB (15 cm gastric tube, 250-275 cm biliary limb) at our institution with a 12-month follow-up, with none lost to follow-up. Then, 10.7 months (8.6-13.0) after MGB, all patients underwent hepatobiliary scintigraphy and a reflux symptom questionnaire (GerdQ) was filled out. A gastroscopy with biopsies was done for all patients with a bile-reflux-positive scintigraphy. Mean age at operation was 56 years (41-65) and preoperative BMI 43.1 kg/m 2 (34.2-54.6). Mean %EWL was 83.9 (49.5-128.3) at 12 months. Four patients reached diabetes remission and two became insulin-independent. Hepatobiliary scintigraphy showed a transient BR into the gastric tube for five patients. Bile tracer was found in the gastric tube at 23-58 min after the tracer injection and highest activity was 8% (1-8%) at 58 min. Bile tracer was not found in the esophagus of any of the patients. One patient with a positive scintigraphy in the gastric tube required re-operation. Two patients with reflux symptoms had a negative scintigraphy. Our results indicate that transient bile reflux is common after MGB in the gastric tube, but not in the esophagus. The clinical relevance of bile reflux needs further studies.

Structural characterization of POM6 Fab and mouse prion protein complex identifies key regions for prions conformational conversion.

PubMed

Baral, Pravas Kumar; Swayampakula, Mridula; Aguzzi, Adriano; James, Michael N G

2018-05-01

Conversion of the cellular prion protein PrP C into its pathogenic isoform PrP S c is the hallmark of prion diseases, fatal neurodegenerative diseases affecting many mammalian species including humans. Anti-prion monoclonal antibodies can arrest the progression of prion diseases by stabilizing the cellular form of the prion protein. Here, we present the crystal structure of the POM6 Fab fragment, in complex with the mouse prion protein (moPrP). The prion epitope of POM6 is in close proximity to the epitope recognized by the purportedly toxic antibody fragment, POM1 Fab also complexed with moPrP. The POM6 Fab recognizes a larger binding interface indicating a likely stronger binding compared to POM1. POM6 and POM1 exhibit distinct biological responses. Structural comparisons of the bound mouse prion proteins from the POM6 Fab:moPrP and POM1 Fab:moPrP complexes reveal several key regions of the prion protein that might be involved in initiating mis-folding events. The structural data of moPrP:POM6 Fab complex are available in the PDB under the accession number www.rcsb.org/pdb/search/structidSearch.do?structureId=6AQ7. © 2018 Federation of European Biochemical Societies.

Double mutation of cell wall proteins CspB and PBP1a increases secretion of the antibody Fab fragment from Corynebacterium glutamicum

PubMed Central

2014-01-01

Background Among other advantages, recombinant antibody-binding fragments (Fabs) hold great clinical and commercial potential, owing to their efficient tissue penetration compared to that of full-length IgGs. Although production of recombinant Fab using microbial expression systems has been reported, yields of active Fab have not been satisfactory. We recently developed the Corynebacterium glutamicum protein expression system (CORYNEX®) and demonstrated improved yield and purity for some applications, although the system has not been applied to Fab production. Results The Fab fragment of human anti-HER2 was successfully secreted by the CORYNEX® system using the conventional C. glutamicum strain YDK010, but the productivity was very low. To improve the secretion efficiency, we investigated the effects of deleting cell wall-related genes. Fab secretion was increased 5.2 times by deletion of pbp1a, encoding one of the penicillin-binding proteins (PBP1a), mediating cell wall peptidoglycan (PG) synthesis. However, this Δpbp1a mutation did not improve Fab secretion in the wild-type ATCC13869 strain. Because YDK010 carries a mutation in the cspB gene encoding a surface (S)-layer protein, we evaluated the effect of ΔcspB mutation on Fab secretion from ATCC13869. The Δpbp1a mutation showed a positive effect on Fab secretion only in combination with the ΔcspB mutation. The ΔcspBΔpbp1a double mutant showed much greater sensitivity to lysozyme than either single mutant or the wild-type strain, suggesting that these mutations reduced cell wall resistance to protein secretion. Conclusion There are at least two crucial permeability barriers to Fab secretion in the cell surface structure of C. glutamicum, the PG layer, and the S-layer. The ΔcspBΔpbp1a double mutant allows efficient Fab production using the CORYNEX® system. PMID:24731213

Topical ocular treatment with monoclonal antibody Fab fragments targeting Japanese cedar pollen Cry j 1 inhibits Japanese cedar pollen-induced allergic conjunctivitis in mice.

PubMed

Mizutani, Nobuaki; Nabe, Takeshi; Yoshino, Shin

2017-03-05

Fab fragments (Fabs) of antibodies having the ability only to bind to specific allergens lack effector functions due to the absence of the Fc portion. In the present study, we examined whether IgG1 monoclonal antibody (mAb) Fabs targeting Japanese cedar pollen (JCP) Cry j 1 were able to regulate JCP-induced allergic conjunctivitis in mice. BALB/c mice actively sensitized with JCP were repeatedly challenged by topical administration of JCP eye drops. Fabs prepared by the digestion of anti-JCP IgG1 mAbs (P1-3 and P1-8) with papain were applied to the eye 15min before the JCP challenges followed by measurement of the clinical conjunctivitis score. In the in vitro experiments, P1-3 and P1-8 showed specific binding to JCP Cry j 1. Furthermore, intact P1-3 binding to Cry j 1 was inhibited by P1-3 Fabs, but not P1-8 Fabs; additionally, P1-8 Fabs, but not P1-3 Fabs, suppressed the intact P1-8 binding, suggesting that the epitopes of Cry j 1 recognized by P1-3 and P1-8 were different. Topical ocular treatment with P1-3 Fabs or P1-8 Fabs was followed by marked suppression of JCP-induced conjunctivitis (P<0.01). In histological evaluation, P1-8 Fabs showed a reduction in eosinophil infiltration in the conjunctiva (P<0.01). These results demonstrated that topical ocular treatment with IgG1 mAb Fabs to Cry j 1 was effective in suppressing JCP-induced allergic conjunctivitis in mice. Furthermore, it suggests the possibility that some epitopes recognized by Fabs could be used as a tool to regulate allergic conjunctivitis. Copyright © 2017 Elsevier B.V. All rights reserved.

Complexes of Neutralizing and Non-Neutralizing Affinity Matured Fabs with a Mimetic of the Internal Trimeric Coiled-Coil of HIV-1 gp41

PubMed Central

Gustchina, Elena; Li, Mi; Ghirlando, Rodolfo; Schuck, Peter; Louis, John M.; Pierson, Jason; Rao, Prashant; Subramaniam, Sriram; Gustchina, Alla; Clore, G. Marius; Wlodawer, Alexander

2013-01-01

A series of mini-antibodies (monovalent and bivalent Fabs) targeting the conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 has been previously constructed and reported. Crystal structures of two closely related monovalent Fabs, one (Fab 8066) broadly neutralizing across a wide panel of HIV-1 subtype B and C viruses, and the other (Fab 8062) non-neutralizing, representing the extremes of this series, were previously solved as complexes with 5-Helix, a gp41 pre-hairpin intermediate mimetic. Binding of these Fabs to covalently stabilized chimeric trimers of N-peptides of HIV-1 gp41 (named (CCIZN36)3 or 3-H) has now been investigated using X-ray crystallography, cryo-electron microscopy, and a variety of biophysical methods. Crystal structures of the complexes between 3-H and Fab 8066 and Fab 8062 were determined at 2.8 and 3.0 Å resolution, respectively. Although the structures of the complexes with the neutralizing Fab 8066 and its non-neutralizing counterpart Fab 8062 were generally similar, small differences between them could be correlated with the biological properties of these antibodies. The conformations of the corresponding CDRs of each antibody in the complexes with 3-H and 5-Helix are very similar. The adaptation to a different target upon complex formation is predominantly achieved by changes in the structure of the trimer of N-HR helices, as well as by adjustment of the orientation of the Fab molecule relative to the N-HR in the complex, via rigid-body movement. The structural data presented here indicate that binding of three Fabs 8062 with high affinity requires more significant changes in the structure of the N-HR trimer compared to binding of Fab 8066. A comparative analysis of the structures of Fabs complexed to different gp41 intermediate mimetics allows further evaluation of biological relevance for generation of neutralizing antibodies, as well as provides novel structural insights into immunogen design. PMID

Impact of surfactants on the target recognition of Fab-conjugated PLGA nanoparticles.

PubMed

Kennedy, Patrick J; Perreira, Ines; Ferreira, Daniel; Nestor, Marika; Oliveira, Carla; Granja, Pedro L; Sarmento, Bruno

2018-06-01

Targeted drug delivery with nanoparticles (NPs) requires proper surface ligand presentation and availability. Surfactants are often used as stabilizers in the production of targeted NPs. Here, we evaluated the impact of surfactants on ligand functionalization and downstream molecular recognition. Our model system consisted of fluorescent poly(lactic-co-glycolic acid) (PLGA) NPs that were nanoprecipitated in one of a small panel of commonly-used surfactants followed by equivalent washes and conjugation of an engineered Fab antibody fragment. Size, polydispersity index and zeta potential were determined by dynamic light scattering and laser Doppler anemometry, and Fab presence on the NPs was assessed by enzyme-linked immunosorbent assay. Most importantly, Fab-decorated NP binding to the cell surface receptor was monitored by fluorescence-activated cell sorting. 2% polyvinyl alcohol, 1% sodium cholate, 0.5% Pluronic F127 (F127) and 2% Tween-80 were initially tested. Of the four surfactants tested, PLGA NPs in 0.5% F127 and 2% Tween-80 had the highest cell binding. These two surfactants were then retested in two different concentrations, 0.5% and 2%. The Fab-decorated PLGA NPs in 2% F127 had the highest cell binding. This study highlights the impact of common surfactants and their concentrations on the downstream targeting of ligand-decorated NPs. Similar principles should be applied in the development of future targeted nanosystems where surfactants are employed. Copyright © 2018 Elsevier B.V. All rights reserved.

Subcutaneous Crotaline Fab antivenom for the treatment of rattlesnake envenomation in a porcine model.

PubMed

Offerman, Steven R; Barry, J David; Richardson, William H; Tong, Tri; Tanen, Dave; Bush, Sean P; Clark, Richard F

2009-01-01

This study was designed to investigate whether the local, subcutaneous injection of Crotaline Fab antivenom (CroFab) at the rattlesnake envenomation site would result in less extremity edema when compared to intravenous (i.v.) antivenom infusion alone. This is a randomized, three-arm laboratory experiment using a porcine model. Each animal was anesthetized, intubated, and maintained on mechanical ventilation. About 6 mg/kg of Crotalus atrox venom was injected subcutaneously at the hock of the right hind leg. Animals were then randomized to immediately receive subcutaneous and i.v. antivenom (SC/IV), i.v. antivenom only, or saline control. SC/IV animals received two vials of CroFab subcutaneously at the envenomation site and two vials intravenously. IV animals received four vials of CroFab intravenously. Limb edema was tracked by serial circumference and volumetric measurements over an 8-h period. Limb circumference was measured at four pre-determined locations hourly. Limb volume was measured by a water displacement method at baseline, 4, and 8 h. Twenty-six animals were randomized to the three treatment groups. The SC/IV and IV arms included nine animals each. Two animals in the SC/IV group died suddenly during the study, leaving seven animals for data analysis. There were eight controls. Increasing limb edema was observed in all groups. No differences were detected in limb circumferences or limb volumes between control and either treatment arms. In this porcine model of crotaline envenomation, no differences in limb edema were found between animals treated with SC/IV or IV CroFab when compared to saline controls.

Effect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in shaken E. coli cultures.

PubMed

Ukkonen, Kaisa; Veijola, Johanna; Vasala, Antti; Neubauer, Peter

2013-07-29

Fab antibody fragments in E. coli are usually directed to the oxidizing periplasmic space for correct folding. From periplasm Fab fragments may further leak into extracellular medium. Information on the cultivation parameters affecting this leakage is scarce, and the unpredictable nature of Fab leakage is problematic regarding consistent product recovery. To elucidate the effects of cultivation conditions, we investigated Fab expression and accumulation into either periplasm or medium in E. coli K-12 and E. coli BL21 when grown in different types of media and under different aeration conditions. Small-scale Fab expression demonstrated significant differences in yield and ratio of periplasmic to extracellular Fab between different culture media and host strains. Expression in a medium with fed-batch-like glucose feeding provided highest total and extracellular yields in both strains. Unexpectedly, cultivation in baffled shake flasks at 150 rpm shaking speed resulted in higher yield and accumulation of Fabs into culture medium as compared to cultivation at 250 rpm. In the fed-batch medium, extracellular fraction in E. coli K-12 increased from 2-17% of total Fab at 250 rpm up to 75% at 150 rpm. This was partly due to increased lysis, but also leakage from intact cells increased at the lower shaking speed. Total Fab yield in E. coli BL21 in glycerol-based autoinduction medium was 5 to 9-fold higher at the lower shaking speed, and the extracellular fraction increased from ≤ 10% to 20-90%. The effect of aeration on Fab localization was reproduced in multiwell plate by variation of culture volume. Yield and leakage of Fab fragments are dependent on expression strain, culture medium, aeration rate, and the combination of these parameters. Maximum productivity in fed-batch-like conditions and in autoinduction medium is achieved under sufficiently oxygen-limited conditions, and lower aeration also promotes increased Fab accumulation into extracellular medium. These

Effect of culture medium, host strain and oxygen transfer on recombinant Fab antibody fragment yield and leakage to medium in shaken E. coli cultures

PubMed Central

2013-01-01

Background Fab antibody fragments in E. coli are usually directed to the oxidizing periplasmic space for correct folding. From periplasm Fab fragments may further leak into extracellular medium. Information on the cultivation parameters affecting this leakage is scarce, and the unpredictable nature of Fab leakage is problematic regarding consistent product recovery. To elucidate the effects of cultivation conditions, we investigated Fab expression and accumulation into either periplasm or medium in E. coli K-12 and E. coli BL21 when grown in different types of media and under different aeration conditions. Results Small-scale Fab expression demonstrated significant differences in yield and ratio of periplasmic to extracellular Fab between different culture media and host strains. Expression in a medium with fed-batch-like glucose feeding provided highest total and extracellular yields in both strains. Unexpectedly, cultivation in baffled shake flasks at 150 rpm shaking speed resulted in higher yield and accumulation of Fabs into culture medium as compared to cultivation at 250 rpm. In the fed-batch medium, extracellular fraction in E. coli K-12 increased from 2-17% of total Fab at 250 rpm up to 75% at 150 rpm. This was partly due to increased lysis, but also leakage from intact cells increased at the lower shaking speed. Total Fab yield in E. coli BL21 in glycerol-based autoinduction medium was 5 to 9-fold higher at the lower shaking speed, and the extracellular fraction increased from ≤ 10% to 20-90%. The effect of aeration on Fab localization was reproduced in multiwell plate by variation of culture volume. Conclusions Yield and leakage of Fab fragments are dependent on expression strain, culture medium, aeration rate, and the combination of these parameters. Maximum productivity in fed-batch-like conditions and in autoinduction medium is achieved under sufficiently oxygen-limited conditions, and lower aeration also promotes increased Fab accumulation into

75 FR 9438 - Samsung Austin Semiconductor, LLC, DRAM Fab 1, a Subsidiary of Samsung Electronics Corporation...

Federal Register 2010, 2011, 2012, 2013, 2014

2010-03-02

... Semiconductor, LLC, DRAM Fab 1, a Subsidiary of Samsung Electronics Corporation, Including On-Site Leased... Semiconductor, LLC, a subsidiary of Samsung Electronics Corporation, DRAM Fab 1, including on-site leased.... The workers are engaged in activities related to the production of DRAM chips for use in electronics...

Structure of Rotavirus Outer-Layer Protein VP7 Bound with a Neutralizing Fab

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aoki, Scott T.; Settembre, Ethan C.; Trask, Shane D.

2009-06-17

Rotavirus outer-layer protein VP7 is a principal target of protective antibodies. Removal of free calcium ions (Ca{sup 2+}) dissociates VP7 trimers into monomers, releasing VP7 from the virion, and initiates penetration-inducing conformational changes in the other outer-layer protein, VP4. We report the crystal structure at 3.4 angstrom resolution of VP7 bound with the Fab fragment of a neutralizing monoclonal antibody. The Fab binds across the outer surface of the intersubunit contact, which contains two Ca{sup 2+} sites. Mutations that escape neutralization by other antibodies suggest that the same region bears the epitopes of most neutralizing antibodies. The monovalent Fab ismore » sufficient to neutralize infectivity. We propose that neutralizing antibodies against VP7 act by stabilizing the trimer, thereby inhibiting the uncoating trigger for VP4 rearrangement. A disulfide-linked trimer is a potential subunit immunogen.« less

Improving Fab' fragment retention in an autonucleolytic Escherichia coli strain by swapping periplasmic nuclease translocation signal from OmpA to DsbA.

PubMed

Schofield, Desmond M; Sirka, Ernestas; Keshavarz-Moore, Eli; Ward, John M; Nesbeth, Darren N

2017-12-01

To reduce unwanted Fab' leakage from an autonucleolytic Escherichia coli strain, which co-expresses OmpA-signalled Staphylococcal nuclease and Fab' fragment in the periplasm, by substituting in Serratial nuclease and the DsbA periplasm translocation signal as alternatives. We attempted to genetically fuse a nuclease from Serratia marcescens to the OmpA signal peptide but plasmid construction failed, possibly due to toxicity of the resultant nuclease. Combining Serratial nuclease to the DsbA signal peptide was successful. The strain co-expressing this nuclease and periplasmic Fab' grew in complex media and exhibited nuclease activity detectable by DNAse agar plate but its growth in defined medium was retarded. Fab' coexpression with Staphylococcal nuclease fused to the DsbA signal peptide resulted in cells exhibiting nuclease activity and growth in defined medium. In cultivation to high cell density in a 5 l bioreactor, DsbA-fused Staphylococcal nuclease co-expression coincided with reduced Fab' leakage relative to the original autonucleolytic Fab' strain with OmpA-fused staphylococcal nuclease. We successfully rescued Fab' leakage back to acceptable levels and established a basis for future investigation of the linkage between periplasmic nuclease expression and leakage of co-expressed periplasmic Fab' fragment to the surrounding growth media.

Hibernating myocardium, morphological studies on intraoperatory myocardial biopsies and on chronic ischemia experimental model.

PubMed

Laky, D; Parascan, Liliana

2007-01-01

Hibernating myocardium represent a prolonged but potentially reversible myocardial contractile dysfunction, an incomplete adaptation caused by chronic myocardial ischemia and persisting at least until blood flow restored. The purpose of this study was to investigate the morphological changes and weather relations exist among function, metabolism and structure in left ventricular hibernating myocardium. Material and methods. Experimental study is making on 12 dogs incomplete coronary obstruction during six weeks for morphologic studies of ischemic zones. On 48 patients with coronary stenosis myocardial biopsies was effectuated during aorto-coronarian bypass graft. On 60 patients with valvular disease associated with segmental coronary atherosclerotic obstructions during surgical interventions on a effectuated repeatedly biopsies from ischemic zones. Dyskinetic ischemic areas was identified by angiography, scintigraphy, low dose dobutamine echography to identify the cells viability. On myocardial biopsies various histological, histoenzymological, immunohistochemical and ultrastructural methods were performed. The morphological cardiomyocytic changes can summarized: loss of myofilaments, accumulation of glycogen, small mitochondria with reversible lesions, decrease of smooth reticulum, absence of T tubules, depression of titin in puncted pattern, loss of cardiotonin, disorganization of cytoskeleton, dispersed nuclear heterochromatin, embryofetal dedifferentiation, and persistence of viability. Extracellular matrix is enlarged with early matrix protein such fibronectin, tenascin, fibroblasts. In experimental material the morphological changes present similarities with the human biopsies, but intermixed with postinfarction scar tissue. Redifferentiation of hibernanting cells end remodeling of extracellular matrix is possible after quigle revascularization through aorto-coronary bypass grafts.

The fatty acid biosynthesis enzyme FabI plays a key role in the development of liver-stage malarial parasites.

PubMed

Yu, Min; Kumar, T R Santha; Nkrumah, Louis J; Coppi, Alida; Retzlaff, Silke; Li, Celeste D; Kelly, Brendan J; Moura, Pedro A; Lakshmanan, Viswanathan; Freundlich, Joel S; Valderramos, Juan-Carlos; Vilcheze, Catherine; Siedner, Mark; Tsai, Jennifer H-C; Falkard, Brie; Sidhu, Amar Bir Singh; Purcell, Lisa A; Gratraud, Paul; Kremer, Laurent; Waters, Andrew P; Schiehser, Guy; Jacobus, David P; Janse, Chris J; Ager, Arba; Jacobs, William R; Sacchettini, James C; Heussler, Volker; Sinnis, Photini; Fidock, David A

2008-12-11

The fatty acid synthesis type II pathway has received considerable interest as a candidate therapeutic target in Plasmodium falciparum asexual blood-stage infections. This apicoplast-resident pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of triclosan, an inhibitor of bacterial FabI. Disruption of fabI in P. falciparum or the rodent parasite P. berghei does not impede blood-stage growth. In contrast, mosquito-derived, FabI-deficient P. berghei sporozoites are markedly less infective for mice and typically fail to complete liver-stage development in vitro. This defect is characterized by an inability to form intrahepatic merosomes that normally initiate blood-stage infections. These data illuminate key differences between liver- and blood-stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions.

Intranasal exposure to monoclonal antibody Fab fragments to Japanese cedar pollen Cry j1 suppresses Japanese cedar pollen‐induced allergic rhinitis

PubMed Central

Mizutani, N

2016-01-01

Background and Purpose Fab fragments (Fabs) of antibodies have the ability to bind to specific allergens but lack the Fc portion that exerts effector functions via binding to receptors including FcεR1 on mast cells. In the present study, we investigated whether intranasal administration of the effector function‐lacking Fabs of a monoclonal antibody IgG1 (mAb, P1‐8) to the major allergen Cry j1 of Japanese cedar pollen (JCP) suppressed JCP‐induced allergic rhinitis in mice. Experimental Approach Balb/c mice sensitized with JCP on days 0 and 14 were challenged intranasally with the pollen on days 28, 29, 30 and 35. Fabs prepared by the digestion of P1‐8 with papain were also administered intranasally 15 min before each JCP challenge. Key Results Intranasal administration of P1‐8 Fabs was followed by marked suppression of sneezing and nasal rubbing in mice with JCP‐induced allergic rhinitis. The suppression of these allergic symptoms by P1‐8 Fabs was associated with decreases in mast cells and eosinophils and decreased hyperplasia of goblet cells in the nasal mucosa. Conclusions and Implications These results demonstrated that intranasal exposure to P1‐8 Fabs was effective in suppressing JCP‐induced allergic rhinitis in mice, suggesting that allergen‐specific mAb Fabs might be used as a tool to regulate allergic pollinosis. PMID:26895546

Recombinant Human Respiratory Syncytial Virus (RSV) Monoclonal Antibody Fab is Effective Therapeutically when Introduced Directly into the Lungs of RSV-Infected Mice

NASA Astrophysics Data System (ADS)

Crowe, James E., Jr.; Murphy, Brian R.; Chanock, Robert M.; Williamson, R. Anthony; Barbas, Carlos F., III; Burton, Dennis R.

1994-02-01

Previously, recombinant human respiratory syncytial virus (RSV) monoclonal antibody Fabs were generated by antigen selection from random combinatorial libraries displayed at the tip of filamentous phage. Two such Fabs, which exhibited high binding affinity for RSV F glycoprotein (a major protective antigen), were evaluated for therapeutic efficacy in infected mice just before or at the time of peak virus replication in the lungs. Fab 19, which neutralized RSV infectivity with high efficiency in tissue culture, was effective therapeutically when delivered directly into the lungs by intranasal instillation under anesthesia. In contrast, RSV Fab 126, which failed to neutralize virus in cell culture, did not exhibit a therapeutic effect under these conditions. The amount of Fab 19 required to effect a 5000- to 12,000-fold reduction in titer of RSV in the lungs within 24 hr was rather small. In four separate experiments, a single instillation of 12.9-50 μg of RSV Fab 19 was sufficient to achieve such a reduction in pulmonary virus in a 25g mouse. The use of Fabs instead of the whole immunoglobulin molecules from which they are derived reduced the protein content of a therapeutic dose. This is important because the protein load that can be delivered effectively into the lungs is limited. The therapeutic effect of a single treatment with Fab 19 was not sustained, so that a rebound in pulmonary virus titer occurred on the 2nd day after treatment. This rebound in pulmonary RSV titer could be prevented by treating infected mice with a single dose of Fab 19 daily for 3 days. These observations suggest that human monoclonal Fabs grown in Escherichia coli may prove useful in the treatment of serious RSV disease as well as diseases caused by other viruses where replication in vivo is limited primarily to the lumenal lining of the respiratory tract.

Coexistent Superscan and Lincoln Sign on Bone Scintigraphy.

PubMed

Kulkarni, Mukta; Soni, Atul; Shetkar, Shubhangi; Amer, Momin; Mulavekar, Amruta; Joshi, Prathamesh

2017-08-01

A 70-year-old man underwent Tc-methylene diphosphonate scintigraphy for staging of adenocarcinoma prostate. Scintigraphy revealed diffuse increased tracer uptake in skeletal system along with faint renal visualization, a pattern compatible with metastatic superscan. The scintigraphy also revealed increased radiotracer uptake in the body of the mandible-Lincoln sign or black beard sign. Radiological imaging revealed sclerotic lesions throughout the skeleton including the mandible, confirming widespread skeletal metastases. Lincoln sign is previously described in monostotic Paget disease of the mandible and in contiguous spread of oral malignancy. We describe this pattern in distant metastatic involvement from carcinoma prostate with coexistent superscan pattern.

Immunoglobulin G (IgG) Fab glycosylation analysis using a new mass spectrometric high-throughput profiling method reveals pregnancy-associated changes.

PubMed

Bondt, Albert; Rombouts, Yoann; Selman, Maurice H J; Hensbergen, Paul J; Reiding, Karli R; Hazes, Johanna M W; Dolhain, Radboud J E M; Wuhrer, Manfred

2014-11-01

The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼ 20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Immunoglobulin G (IgG) Fab Glycosylation Analysis Using a New Mass Spectrometric High-throughput Profiling Method Reveals Pregnancy-associated Changes*

PubMed Central

Bondt, Albert; Rombouts, Yoann; Selman, Maurice H. J.; Hensbergen, Paul J.; Reiding, Karli R.; Hazes, Johanna M. W.; Dolhain, Radboud J. E. M.; Wuhrer, Manfred

2014-01-01

The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding. PMID:25004930

Acute adverse events associated with the administration of Crotalidae polyvalent immune Fab antivenom within the North American Snakebite Registry.

PubMed

Kleinschmidt, Kurt; Ruha, Anne-Michelle; Campleman, Sharan; Brent, Jeffrey; Wax, Paul

2018-04-24

Crotalidae Polyvalent Immune Fab (Fab Antivenom) is the primary Viperid antivenom used in the United States since 2000. Adverse event data associated with its use are limited. The purpose of this study is to describe the prevalence of acute adverse events associated with the use of Fab antivenom. The American College of Medical Toxicology's Toxicology Investigators Consortium maintains a prospective case registry of poisoned and envenomated patients managed by medical toxicologists at the bedside. This registry includes the North American Snakebite sub-registry. We performed a review of 438 cases entered into the Snakebite sub-registry. A total of 373 (85.2%) received at least one vial of Fab Antivenom. Forty percent were children. Adverse events occurred in 10 patients (2.7%) of whom six were adults. Rash was the most common adverse event. More severe adverse events (hypotension, bronchospasm, and/or angioedema) occurred in four (1.1%) patients. Prophylaxis was administered prior to Fab antivenom in 4.0%. Eight patients received various treatments for their adverse events. Neither the initial number of Fab antivenom vials, atopic history, nor prior envenomation correlated with the prevalence of adverse events. This prevalence of adverse events was lower than in previous studies and in a meta-analysis of 11 studies. The types of adverse events and treatments used are consistent with those in previous reports. There were no prior reports of prophylaxis use with which to compare. The prevalence of Fab antivenom adverse events in the North American Snakebite Registry was 2.7%.

Oriented Immobilization of Fab Fragments by Site-Specific Biotinylation at the Conserved Nucleotide Binding Site for Enhanced Antigen Detection.

PubMed

Mustafaoglu, Nur; Alves, Nathan J; Bilgicer, Basar

2015-09-08

Oriented immobilization of antibodies and antibody fragments has become increasingly important as a result of the efforts to reduce the size of diagnostic and sensor devices to miniaturized dimensions for improved accessibility to the end-user. Reduced dimensions of sensor devices necessitate the immobilized antibodies to conserve their antigen binding activity for proper operation. Fab fragments are becoming more commonly used in small-scaled diagnostic devices due to their small size and ease of manufacture. In this study, we used the previously described UV-NBS(Biotin) method to functionalize Fab fragments with IBA-EG11-Biotin linker utilizing UV energy to initiate a photo-cross-linking reaction between the nucleotide binding site (NBS) on the Fab fragment and IBA-Biotin molecule. Our results demonstrate that immobilization of biotinylated Fab fragments via UV-NBS(Biotin) method generated the highest level of immobilized Fab on surfaces when compared to other typical immobilization methods while preserving antigen binding activity. UV-NBS(Biotin) method provided 432-fold, 114-fold, and 29-fold improved antigen detection sensitivity than physical adsorption, NHS-Biotin, and ε-NH3(+), methods, respectively. Additionally, the limit of detection (LOD) for PSA utilizing Fab fragments immobilized via UV-NBS(Biotin) method was significantly lower than that of the other immobilization methods, with an LOD of 0.4 pM PSA. In summary, site-specific biotinylation of Fab fragments without structural damage or loss in antigen binding activity provides a wide range of application potential for UV-NBS immobilization technique across numerous diagnostic devices and nanotechnologies.

Strategy optimization for mask rule check in wafer fab

NASA Astrophysics Data System (ADS)

Yang, Chuen Huei; Lin, Shaina; Lin, Roger; Wang, Alice; Lee, Rachel; Deng, Erwin

2015-07-01

Photolithography process is getting more and more sophisticated for wafer production following Moore's law. Therefore, for wafer fab, consolidated and close cooperation with mask house is a key to achieve silicon wafer success. However, generally speaking, it is not easy to preserve such partnership because many engineering efforts and frequent communication are indispensable. The inattentive connection is obvious in mask rule check (MRC). Mask houses will do their own MRC at job deck stage, but the checking is only for identification of mask process limitation including writing, etching, inspection, metrology, etc. No further checking in terms of wafer process concerned mask data errors will be implemented after data files of whole mask are composed in mask house. There are still many potential data errors even post-OPC verification has been done for main circuits. What mentioned here are the kinds of errors which will only occur as main circuits combined with frame and dummy patterns to form whole reticle. Therefore, strategy optimization is on-going in UMC to evaluate MRC especially for wafer fab concerned errors. The prerequisite is that no impact on mask delivery cycle time even adding this extra checking. A full-mask checking based on job deck in gds or oasis format is necessary in order to secure acceptable run time. Form of the summarized error report generated by this checking is also crucial because user friendly interface will shorten engineers' judgment time to release mask for writing. This paper will survey the key factors of MRC in wafer fab.

Functional Dissection of the Blocking and Bypass Activities of the Fab-8 Boundary in the Drosophila Bithorax Complex.

PubMed

Kyrchanova, Olga; Mogila, Vladic; Wolle, Daniel; Deshpande, Girish; Parshikov, Alexander; Cléard, Fabienne; Karch, Francois; Schedl, Paul; Georgiev, Pavel

2016-07-01

Functionally autonomous regulatory domains direct the parasegment-specific expression of the Drosophila Bithorax complex (BX-C) homeotic genes. Autonomy is conferred by boundary/insulator elements that separate each regulatory domain from its neighbors. For six of the nine parasegment (PS) regulatory domains in the complex, at least one boundary is located between the domain and its target homeotic gene. Consequently, BX-C boundaries must not only block adventitious interactions between neighboring regulatory domains, but also be permissive (bypass) for regulatory interactions between the domains and their gene targets. To elucidate how the BX-C boundaries combine these two contradictory activities, we have used a boundary replacement strategy. We show that a 337 bp fragment spanning the Fab-8 boundary nuclease hypersensitive site and lacking all but 83 bp of the 625 bp Fab-8 PTS (promoter targeting sequence) fully rescues a Fab-7 deletion. It blocks crosstalk between the iab-6 and iab-7 regulatory domains, and has bypass activity that enables the two downstream domains, iab-5 and iab-6, to regulate Abdominal-B (Abd-B) transcription in spite of two intervening boundary elements. Fab-8 has two dCTCF sites and we show that they are necessary both for blocking and bypass activity. However, CTCF sites on their own are not sufficient for bypass. While multimerized dCTCF (or Su(Hw)) sites have blocking activity, they fail to support bypass. Moreover, this bypass defect is not rescued by the full length PTS. Finally, we show that orientation is critical for the proper functioning the Fab-8 replacement. Though the inverted Fab-8 boundary still blocks crosstalk, it disrupts the topology of the Abd-B regulatory domains and does not support bypass. Importantly, altering the orientation of the Fab-8 dCTCF sites is not sufficient to disrupt bypass, indicating that orientation dependence is conferred by other factors.

Functional Dissection of the Blocking and Bypass Activities of the Fab-8 Boundary in the Drosophila Bithorax Complex

PubMed Central

Wolle, Daniel; Deshpande, Girish; Parshikov, Alexander; Cléard, Fabienne; Karch, Francois; Schedl, Paul; Georgiev, Pavel

2016-01-01

Functionally autonomous regulatory domains direct the parasegment-specific expression of the Drosophila Bithorax complex (BX-C) homeotic genes. Autonomy is conferred by boundary/insulator elements that separate each regulatory domain from its neighbors. For six of the nine parasegment (PS) regulatory domains in the complex, at least one boundary is located between the domain and its target homeotic gene. Consequently, BX-C boundaries must not only block adventitious interactions between neighboring regulatory domains, but also be permissive (bypass) for regulatory interactions between the domains and their gene targets. To elucidate how the BX-C boundaries combine these two contradictory activities, we have used a boundary replacement strategy. We show that a 337 bp fragment spanning the Fab-8 boundary nuclease hypersensitive site and lacking all but 83 bp of the 625 bp Fab-8 PTS (promoter targeting sequence) fully rescues a Fab-7 deletion. It blocks crosstalk between the iab-6 and iab-7 regulatory domains, and has bypass activity that enables the two downstream domains, iab-5 and iab-6, to regulate Abdominal-B (Abd-B) transcription in spite of two intervening boundary elements. Fab-8 has two dCTCF sites and we show that they are necessary both for blocking and bypass activity. However, CTCF sites on their own are not sufficient for bypass. While multimerized dCTCF (or Su(Hw)) sites have blocking activity, they fail to support bypass. Moreover, this bypass defect is not rescued by the full length PTS. Finally, we show that orientation is critical for the proper functioning the Fab-8 replacement. Though the inverted Fab-8 boundary still blocks crosstalk, it disrupts the topology of the Abd-B regulatory domains and does not support bypass. Importantly, altering the orientation of the Fab-8 dCTCF sites is not sufficient to disrupt bypass, indicating that orientation dependence is conferred by other factors. PMID:27428541

Targeting Mast Cells and Basophils with Anti-FcεRIα Fab-Conjugated Celastrol-Loaded Micelles Suppresses Allergic Inflammation.

PubMed

Peng, Xia; Wang, Juan; Li, Xianyang; Lin, Lihui; Xie, Guogang; Cui, Zelin; Li, Jia; Wang, Yuping; Li, Li

2015-12-01

Mast cells and basophils are effector cells in the pathophysiology of allergic diseases. Targeted elimination of these cells may be a promising strategy for the treatment of allergic disorders. Our present study aims at targeted delivery of anti-FcεRIα Fab-conjugated celastrol-loaded micelles toward FcεRIα receptors expressed on mast cells and basophils to have enhanced anti-allergic effect. To achieve this aim, we prepared celastrol-loaded (PEO-block-PPO-block-PEO, Pluronic) polymeric nanomicelles using thin-film hydration method. The anti-FcεRIα Fab Fragment was then conjugated to carboxyl groups on drug-loaded micelles via EDC amidation reaction. The anti-FcεRIα Fab-conjugated celastrol-loaded micelles revealed uniform particle size (93.43 ± 12.93 nm) with high loading percentage (21.2 ± 1.5% w/w). The image of micelles showed oval and rod like. The anti-FcεRIα Fab-conjugated micelles demonstrated enhanced cellular uptake and cytotoxity toward target KU812 cells than non-conjugated micelles in vitro. Furthermore, diffusion of the drug into the cells allowed an efficient induction of cell apoptosis. In mouse model of allergic asthma, treatment with anti-FcεRIα Fab-conjugated micelles increased lung accumulation of micelles, and significantly reduced OVA-sIgE, histamine and Th2 cytokines (IL-4, IL-5, TNF-α) levels, eosinophils infiltration and mucus production. In addition, in mouse model of passive cutaneous anaphylaxis, anti-FcεRIα Fab-conjugated celastrol-loaded micelles treatment significantly decreased extravasated evan's in the ear. These results indicate that anti-FcεRIα Fab-conjugated celastrol-loaded micelles can target and selectively kill mast cells and basophils which express FcεRIα, and may be efficient reagents for the treatment of allergic disorders and mast cell related diseases.

A rapid fluorometric assay for the S-malonyltransacylase FabD and other sulfhydryl utilizing enzymes.

PubMed

Marcella, Aaron M; Barb, Adam W

The development of biorenewable chemicals will support green chemistry initiatives and supplement the catalog of starting materials available to the chemical industry. Bacterial fatty acid biosynthesis is being pursued as a source of protein catalysts to synthesize novel reduced carbon molecules in fermentation systems. The availability of methods to measure enzyme catalysis for native and engineered enzymes from this pathway remains a bottleneck because a simple quantitative enzyme assay for numerous enzymes does not exist. Here we present two variations of a fluorescence assay that is readily extendable to high-throughput screening and is appropriate for thiol consuming and generating enzymes including the Escherichia coli enzymes malonyl-coenzyme A transacylase (FabD) and keto-acylsynthase III (FabH). We measured K M values of 60 ± 20 µM (acetyl-CoA) and 20 ± 10 µM (malonyl-ACP) and a k cat of 7.4-9.0 s -1 with FabH. Assays of FabD included a precipitation step to remove the thiol-containing substrate holo-ACP from the reaction product coenzyme-A to estimate reaction rates. Analysis of initial velocity measurements revealed K M values of 60 ± 20 µM (malonyl-CoA) and 40 ± 10 µM (holo-ACP) and a k cat of 2100-2600 s -1 for the FabD enzyme. Our data show similar results when compared to existing radioactive and continuous coupled assays in terms of sensitivity while providing the benefit of simplicity, scalability and repeatability. Fluorescence detection also eliminates the need for radioactive substrates traditionally used to assay these enzymes.

Quantitative glycan profiling of normal human plasma derived immunoglobulin and its fragments Fab and Fc.

PubMed

Anumula, Kalyan Rao

2012-08-31

Typical clinical grade human IgG (intravenous immunoglobulin, IVIG), used for carbohydrate analysis, is derived from thousands of healthy donors. Quantitative high-resolution glycan profiles of IgG and its Fc-Fab fragments are presented here. Glycan profiles were established following digestions with Fc specific endoglycosidase S and generic PNGase F under denaturing and non-denaturing (native) conditions. The native PNGase F glycan profile of IgG was similar (but not identical) to that of Endo S. Endo S profiles did not contain the glycans with bisecting GlcNAc. PNGase F glycan profiles were the same for Fc fragments that were isolated from pepsin and Ide S protease digests. Both isolated Fab fragments and the previously deglycosylated IVIG (native conditions) yielded the same glycan profile. Glycan profiles were established using high resolution HPLC with 2-aminobenzoic acid (2AA) labeling. An accurate determination of sialylation levels can be made by this method. Carbohydrate content in Fc and Fab was determined using an internal standard and corrected for both protein and glycan recoveries. Fab portion contained about 14% of the total carbohydrate which translates to 2.3 sugar chains per mol in IVIG where 2 chains are located in the CH2 domain of the Fc. Fc glycans consisted of neutral (N) 84.5%; mono-sialylated (S1) 15% and di-sialylated (S2) 0.5%. In contrast, Fab contained N, 21%; S1, 43% and S2, 36%. The distribution of bisecting N-acetylglucosamine and fucose was found to be very different in various glycans (N, S1 and S2) found in Fab and Fc. Total IgG glycan profile (Fab plus Fc) contained N, 78.5%; S1, 17% and S2, 4.5%. Percent distribution of glycans G0, G1 and G2 (with 0, 1 and 2 two galactoses) was 26, 49 and 25 respectively within the 78% of the neutral glycans. Glycan profiles were nearly the same for various clinical grade IVIG preparations from various manufacturers. A fast HPLC profiling method was developed for the separation and quantitation

IgG4 autoantibodies against muscle-specific kinase undergo Fab-arm exchange in myasthenia gravis patients.

PubMed

Koneczny, Inga; Stevens, Jo A A; De Rosa, Anna; Huda, Saif; Huijbers, Maartje G; Saxena, Abhishek; Maestri, Michelangelo; Lazaridis, Konstantinos; Zisimopoulou, Paraskevi; Tzartos, Socrates; Verschuuren, Jan; van der Maarel, Silvère M; van Damme, Philip; De Baets, Marc H; Molenaar, Peter C; Vincent, Angela; Ricciardi, Roberta; Martinez-Martinez, Pilar; Losen, Mario

2017-02-01

Autoimmunity mediated by IgG4 subclass autoantibodies is an expanding field of research. Due to their structural characteristics a key feature of IgG4 antibodies is the ability to exchange Fab-arms with other, unrelated, IgG4 molecules, making the IgG4 molecule potentially monovalent for the specific antigen. However, whether those disease-associated antigen-specific IgG4 are mono- or divalent for their antigens is unknown. Myasthenia gravis (MG) with antibodies to muscle specific kinase (MuSK-MG) is a well-recognized disease in which the predominant pathogenic IgG4 antibody binds to extracellular epitopes on MuSK at the neuromuscular junction; this inhibits a pathway that clusters the acetylcholine (neurotransmitter) receptors and leads to failure of neuromuscular transmission. In vitro Fab-arm exchange-inducing conditions were applied to MuSK antibodies in sera, purified IgG4 and IgG1-3 sub-fractions. Solid-phase cross-linking assays were established to determine the extent of pre-existing and inducible Fab-arm exchange. Functional effects of the resulting populations of IgG4 antibodies were determined by measuring inhibition of agrin-induced AChR clustering in C2C12 cells. To confirm the results, κ/κ, λ/λ and hybrid κ/λ IgG4s were isolated and tested for MuSK antibodies. At least fifty percent of patients had IgG4, but not IgG1-3, MuSK antibodies that could undergo Fab-arm exchange in vitro under reducing conditions. Also MuSK antibodies were found in vivo that were divalent (monospecific for MuSK). Fab-arm exchange with normal human IgG4 did not prevent the inhibitory effect of serum derived MuSK antibodies on AChR clustering in C2C12 mouse myotubes. The results suggest that a considerable proportion of MuSK IgG4 could already be Fab-arm exchanged in vivo. This was confirmed by isolating endogenous IgG4 MuSK antibodies containing both κ and λ light chains, i.e. hybrid IgG4 molecules. These new findings demonstrate that Fab-arm exchanged antibodies

Evidence-based, multidisciplinary approach to the development of a crotalidae polyvalent antivenin (CroFab) protocol at a university hospital.

PubMed

Weant, Kyle A; Johnson, Peter N; Bowers, Rebecca C; Armitstead, John A

2010-03-01

Several thousand people are bitten annually by venomous snakes in the US. While the development of ovine Crotalidae polyvalent immune Fab antivenin (FabAV) for Crotalinae snakebite envenomations has greatly changed the way this clinical presentation is treated, multiple issues complicate its use. From patient assessment and evaluation, to medication preparation and administration, to the management of adverse drug reactions, the use of this antidote carries with it multiple points of possible medication variances. The inappropriate use of this agent can result in adverse patient consequences and a significant financial burden for both the hospital and the patient. To describe an evidence-based, multidisciplinary approach that was taken to ensure optimal, safe, and cost-effective treatment of patients with FabAV. Following an analysis of the available literature, a multidisciplinary committee was formed to construct a protocol for use of FabAV. This group included clinical pharmacists, pharmacy administrators, emergency medicine physicians who specialized in wilderness medicine and pharmacy residents. A multidisciplinary FabAV usage protocol was constructed and implemented to ensure appropriate patient evaluation, FabAV use and preparation, monitoring, and follow-up. This protocol was based on the available literature and the expert opinion of the committee. Through the use of a 24-hour in-house pharmacy resident on-call system, clinical pharmacy services were provided to ensure a multidisciplinary approach to the care of these patients emergently. Although limited, initial data show that this approach is effective and may result in substantial cost savings. Initial results from implementation of a protocol for use of FabAV have limited inappropriate use, reduced medication wastage, and decreased costs.

Native MS and ECD Characterization of a Fab-Antigen Complex May Facilitate Crystallization for X-ray Diffraction

NASA Astrophysics Data System (ADS)

Zhang, Ying; Cui, Weidong; Wecksler, Aaron T.; Zhang, Hao; Molina, Patricia; Deperalta, Galahad; Gross, Michael L.

2016-07-01

Native mass spectrometry (MS) and top-down electron-capture dissociation (ECD) combine as a powerful approach for characterizing large proteins and protein assemblies. Here, we report their use to study an antibody Fab (Fab-1)-VEGF complex in its near-native state. Native ESI with analysis by FTICR mass spectrometry confirms that VEGF is a dimer in solution and that its complex with Fab-1 has a binding stoichiometry of 2:2. Applying combinations of collisionally activated dissociation (CAD), ECD, and infrared multiphoton dissociation (IRMPD) allows identification of flexible regions of the complex, potentially serving as a guide for crystallization and X-ray diffraction analysis.

Relative stabilities of IgG1 and IgG4 Fab domains: Influence of the light–heavy interchain disulfide bond architecture

PubMed Central

Heads, James T; Adams, Ralph; D'Hooghe, Lena E; Page, Matt J T; Humphreys, David P; Popplewell, Andrew G; Lawson, Alastair D; Henry, Alistair J

2012-01-01

The stability of therapeutic antibodies is a prime pharmaceutical concern. In this work we examined thermal stability differences between human IgG1 and IgG4 Fab domains containing the same variable regions using the thermofluor assay. It was found that the IgG1 Fab domain is up to 11°C more stable than the IgG4 Fab domain containing the same variable region. We investigated the cause of this difference with the aim of developing a molecule with the enhanced stability of the IgG1 Fab and the biological properties of an IgG4 Fc. We found that replacing the seven residues, which differ between IgG1 CH1 and IgG4 CH1 domains, while retaining the native IgG1 light-heavy interchain disulfide (L–H) bond, did not affect thermal stability. Introducing the IgG1 type L–H interchain disulfide bond (DSB) into the IgG4 Fab resulted in an increase in thermal stability to levels observed in the IgG1 Fab with the same variable region. Conversely, replacement of the IgG1 L–H interchain DSB with the IgG4 type L–H interchain DSB reduced the thermal stability. We utilized the increased stability of the IgG1 Fab and designed a hybrid antibody with an IgG1 CH1 linked to an IgG4 Fc via an IgG1 hinge. This construct has the expected biophysical properties of both the IgG4 Fc and IgG1 Fab domains and may therefore be a pharmaceutically relevant format. PMID:22761163

Production and Characterization of F(Ab')2 Fragments Obtained by Enzymatic Digestion from Murine Anti-MRSA PBP2a Monoclonal Antibodies.

PubMed

de Araujo, Anna Erika Vieira; de Souza, Natalia Plinio; de Sousa, Alvaro Paiva Braga; Lara, Flavio Alves; Senna, Jose Procopio Moreno

2018-05-01

Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a worldwide health problem. In a previous study, a murine monoclonal antibody (mMAB), capable of binding to PBP2a within MRSA strains, was generated. F(ab') 2 antibody fragments are widely described in the literature as immunochemical tools and reagents for diagnostics and therapeutics, particularly because of their low immunogenicity and rapid pharmacokinetics. In this study, F(ab') 2 fragments from mMAB were generated by enzymatic digestion, using pepsin. They were purified by affinity chromatography using protein A and concentrated by a MWCO 50 kDa filtration unit. The results indicate that it is possible to obtain F(ab') 2 fragments by pepsin digestion. ELISA, western blotting, and fluorescence microscopy data demonstrated that F(ab') 2 affinity for PBP2a is not lost even after the enzymatic digestion process. As expected, in the pharmacokinetics tests, F(ab') 2 presented a faster elimination (between 12 and 18 h) compared to IgG. These F(ab') 2 fragments could be used in future immunodiagnostic applications, including in vitro or in situ radiolabeling and in the treatment of infections caused by this important pathogen.

20 CFR 30.312 - What will the FAB do if the claimant objects to the recommended decision but does not request a...

Code of Federal Regulations, 2010 CFR

2010-04-01

... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false What will the FAB do if the claimant objects....312 What will the FAB do if the claimant objects to the recommended decision but does not request a... period of time allotted in § 30.310 but does not request a hearing, the FAB will consider any objections...

20 CFR 30.317 - Can the FAB request a further response from the claimant or return a claim to the district office?

Code of Federal Regulations, 2010 CFR

2010-04-01

... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false Can the FAB request a further response from....317 Can the FAB request a further response from the claimant or return a claim to the district office? At any time before the issuance of its final decision, the FAB may request that the claimant submit...

dSAP18 and dHDAC1 contribute to the functional regulation of the Drosophila Fab-7 element.

PubMed

Canudas, Silvia; Pérez, Silvia; Fanti, Laura; Pimpinelli, Sergio; Singh, Navjot; Hanes, Steven D; Azorín, Fernando; Espinás, M Lluïsa

2005-01-01

It was described earlier that the Drosophila GAGA factor [Trithorax-like (Trl)] interacts with dSAP18, which, in mammals, was reported to be a component of the Sin3-HDAC co-repressor complex. GAGA-dSAP18 interaction was proposed to contribute to the functional regulation of the bithorax complex (BX-C). Here, we show that mutant alleles of Trl, dsap18 and drpd3/hdac1 enhance A6-to-A5 transformation indicating a contribution to the regulation of Abd-B expression at A6. In A6, expression of Abd-B is driven by the iab-6 enhancer, which is insulated from iab-7 by the Fab-7 element. Here, we report that GAGA, dSAP18 and dRPD3/HDAC1 co-localize to ectopic Fab-7 sites in polytene chromosomes and that mutant Trl, dsap18 and drpd3/hdac1 alleles affect Fab-7-dependent silencing. Consistent with these findings, chromatin immunoprecipitation analysis shows that, in Drosophila embryos, the endogenous Fab-7 element is hypoacetylated at histones H3 and H4. These results indicate a contribution of GAGA, dSAP18 and dRPD3/HDAC1 to the regulation of Fab-7 function.

Incidental Warthin Tumor on Pertechnetate Scintigraphy.

PubMed

Kulkarni, Mukta; Shetkar, Shubhangi; Joshi, Prathamesh; Kasaliwal, Sanket; Chaudhari, Shrikant

2016-09-01

A 30-year-old woman underwent Tc-pertechnetate scintigraphy for evaluation of thyrotoxicosis. The scintigraphy revealed hypervascular thyroid gland with markedly increased trapping function in both the lobes suggesting diagnosis of Graves disease. Incidentally, a hypervascular and pertechnetate avid focus was seen along the lateral margin of the right parotid gland. Pertechnetate avidity and site of uptake suggested possibility of Warthin tumor. Clinical examination and ultrasonography revealed a well-defined lesion in the superficial lobe of the right parotid gland favoring diagnosis of benign lesion. Postsurgery specimen confirmed diagnosis of Warthin tumor.

Elderly patients with suspected chronic digoxin toxicity: A comparison of clinical characteristics of patients receiving and not receiving digoxin-Fab.

PubMed

Arbabian, Hooman; Lee, Hwee Min; Graudins, Andis

2018-04-01

The aim of the present study was to compare clinical features of patients with elevated serum digoxin concentrations who were treated with digoxin-Fab with those where the immunotherapy was not given by a tertiary hospital toxicology service. This was a retrospective series of patients with supratherapeutic serum digoxin concentrations referred to the toxicology service from August 2013 to October 2015. Data collected included demographics, presenting complaint, digoxin dose, other medications taken, serum digoxin, potassium and creatinine concentration on presentation and initial and post-digoxin-Fab heart rate. There were 47 referrals. Digoxin-Fab was administered in 21 cases. It was given more commonly when the heart rate was <51/min or serum potassium was >5.0 mmol/L. Patients receiving digoxin-Fab were more likely to be on maintenance therapy with beta-blockers or calcium channel blockers (95% vs 61%; OR 13.1; 95% CI 1.5-113) and/or potassium-sparing medications (95% vs 54%; OR 17.1; 95% CI 2.0-147). They had elevated serum creatinine (76% vs 42%; OR 8.2; 95% CI 1.9-34), higher serum potassium (median: 5.1 mmol/L vs 4.2 mmol/L, P = 0.02), higher serum digoxin concentration (median: 3.5 nmol/L vs 2.3 nmol/L, P = 0.02) and pretreatment heart rate <51/min (66% vs 31%; OR 4.5; 95% CI 1.3-15). There were no patients with ventricular arrhythmias or hypotension. Median heart rate increased by 10/min 1 and 4 h after digoxin-Fab. However, individual heart rate response to digoxin-Fab was variable. Digoxin-Fab was more commonly administered when heart rate was <51/min. It had a small effect on increasing heart rate; however, individual response to digoxin-Fab was variable as patients were using other negative chronotropic medications. In symptomatic bradycardic patients on multiple heart failure medications, positive chronotropic and potassium-lowering therapies should be considered in concert with digoxin-Fab. © 2018 Australasian College for

Assessment of stenosis severity: Correlation of angiography, T1-201 scintigraphy, and intracoronary pressure gradients

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bateman, T.; Raymond, M.; Czer, L.

1984-01-01

To clarify the relationship between angiographic and hemodynamic stenosis severity and the appearance during stress-redistribution myocardial T1-201 scintigraphy (Ex-T1) of a visual (V) or quantitative (Q) perfusion defect (PD) or washout (WO) abnormality, 24 pts with CAD underwent intracoronary pressure gradient study at bypass surgery (CABG). All had pre-CABG Ex-T1 without interval deterioration. The mean diastolic pressure gradient (MDG) measured at reproducible hyperemic flow rates was determined for 34 stenoses (13 LAD, 7 LCX, 14 RCA) and compared with the results of Ex-T1 in subtended myocardial regions (LAD=anterior; LCX=posterolateral; RCA=inferior). Fourteen stenoses (50-99% diameter narrowing) were unassociated with VPD despitemore » maximal exercise: MDG was 9 +- 5mmHg, with MDG/mean aortic diastolic pressure (ADP) ratio of 0.12 +- 0.07. QPD and QWO analysis detected 8 of these. Thirteen stenoses (90-100% severity) led to reversible VPD: MDG was 36 +- 11 mm Hg, MDG/ADP ratio was 0.52 +- 0.17, and Q analysis was abnormal in 12/13. Seven stenoses (90-100% severity) subtended infarcted myocardium: MDG was 42 +- 21 mm Hg, MDG/ADP ratio was 0.52 +- 0.18, and V and Q analyses were abnormal in all. From this study, the authors derive the following conclusion: 1) Ex-T1 correlates better with hemodynamic severity of stenoses than does angiography; 2) V abnormalities identify stenoses of major angiographic and hemodynamic severity, while Q analysis detects some (57% in this study) stenoses of lesser severity; and 3) stenoses causing reversible Ex-T1 abnormalities present similar hemodynamic impediments to those causing myocardial infarcts.« less

Cholestatic Liver Disease after Rituximab and Adalimumab and the Possible Role of Cross-Reacting Antibodies to Fab 2 Fragments

PubMed Central

Koetter, Ina; Schwab, Matthias; Fritz, Peter; Kimmel, Martin; Alscher, M. Dominik; Braun, Niko

2013-01-01

Background Millions of patients are treated with therapeutic monoclonal antibodies (Tmabs) for miscellaneous diseases. We investigated sera from six patients who received immune globulin, from one patient with refractory anti-neutrophil-cytoplasmic antibody (ANCA)-associated granulomatosis with polyangiitis (GPA) who developed two episodes of acute cholestatic liver disease, one after treatment with rituximab and a second after adalimumab and a healthy control group. Methods Three sera from the patient and six sera from patients who received immune globulin were analyzed for antibodies to rituximab and adalimumab by ELISA. Additionally, sera from the patients and from nine healthy blood donors were coated with the Fab fragment of an unrelated humanized monoclonal antibody, with human Fc proteins as well as a mouse IgG globulin. Results Viral serology for hepatitis A, B, C and autoantibodies specific for autoimmune liver disorders were negative. In all three sera from the patient antibodies to rituximab could be detected, but also antibodies to adalimumab were present even at time points when the patient had not yet received adalimumab, indicating cross reactivity between both substances. Testing against an unrelated human Fab fragment revealed positive results, indicating that the patient had antibodies against human Fab fragments in general. The Fc proteins were negative, and patients’ sera did also not react with mouse IgG globulins. Remarkably, 2 out of 5 patients which were treated with immune globulin had antibodies against human Fab fragments in general whereas in none of the samples from healthy controls antibodies to Fab fragment could be detected. Conclusion This is the first study demonstrating cholestatic liver disease induced by two different Tmabs. Cross - reacting antibodies to Fab2 fragments in general are probably involved. Further studies must show if these Fab2 antibodies in general are related with drug-induced side effects and accelerated drug

Efficient production of antibody Fab fragment by transient gene expression in insect cells.

PubMed

Mori, Keita; Hamada, Hirotsugu; Ogawa, Takafumi; Ohmuro-Matsuyama, Yuki; Katsuda, Tomohisa; Yamaji, Hideki

2017-08-01

Transient gene expression allows a rapid production of diverse recombinant proteins in early-stage preclinical and clinical developments of biologics. Insect cells have proven to be an excellent platform for the production of functional recombinant proteins. In the present study, the production of an antibody Fab fragment by transient gene expression in lepidopteran insect cells was investigated. The DNA fragments encoding heavy-chain (Hc; Fd fragment) and light-chain (Lc) genes of an Fab fragment were individually cloned into the plasmid vector pIHAneo, which contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression. Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were co-transfected with the resultant plasmid vectors using linear polyethyleneimine. When the transfection efficiency was evaluated, a plasmid vector encoding an enhanced green fluorescent protein (EGFP) gene was also co-transfected. Transfection and culture conditions were optimized based on both the flow cytometry of the EGFP expression in transfected cells and the yield of the secreted Fab fragments determined by enzyme-linked immunosorbent assay (ELISA). Under optimal conditions, a yield of approximately 120 mg/L of Fab fragments was achieved in 5 days in a shake-flask culture. Transient gene expression in insect cells may offer a promising approach to the high-throughput production of recombinant proteins. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

A study on EUV reticle surface molecular contamination under different storage conditions in a HVM foundry fab

NASA Astrophysics Data System (ADS)

Singh, SherJang; Yatzor, Brett; Taylor, Ron; Wood, Obert; Mangat, Pawitter

2017-03-01

The prospect of EUVL (Extreme Ultraviolet Lithography) insertion into HVM (High Volume Manufacturing) has never been this promising. As technology is prepared for "lab to fab" transition, it becomes important to comprehend challenges associated with integrating EUVL infrastructure within existing high volume chip fabrication processes in a foundry fab. The existing 193nm optical lithography process flow for reticle handling and storage in a fab atmosphere is well established and in-fab reticle contamination concerns are mitigated with the reticle pellicle. However EUVL reticle pellicle is still under development and if available, may only provide protection against particles but not molecular contamination. HVM fab atmosphere is known to be contaminated with trace amounts of AMC's (Atmospheric Molecular Contamination). If such contaminants are organic in nature and get absorbed on the reticle surface, EUV photon cause photo-dissociation resulting into carbon generation which is known to reduce multilayer reflectivity and also degrades exposure uniformity. Chemical diffusion and aggregation of other ions is also reported under the e-beam exposure of a EUV reticle which is known to cause haze issues in optical lithography. Therefore it becomes paramount to mitigate absorbed molecular contaminant concerns on EUVL reticle surface. In this paper, we have studied types of molecular contaminants that are absorbed on an EUVL reticle surface under HVM fab storage and handling conditions. Effect of storage conditions (gas purged vs atmospheric) in different storage pods (Dual pods, Reticle Clamshells) is evaluated. Absorption analysis is done both on ruthenium capping layer as well as TaBN absorber. Ru surface chemistry change as a result of storage is also studied. The efficacy of different reticle cleaning processes to remove absorbed contaminant is evaluated as well.

Thyroid scintigraphy in veterinary medicine.

PubMed

Daniel, Gregory B; Neelis, Dana A

2014-01-01

Thyroid scintigraphy is performed in cats and dogs and has been used to a limited degree in other species such as the horse. Thyroid scintigraphy is most commonly used to aid in the diagnosis and treatment management of feline hyperthyroidism but is also used in the evaluation of canine hypothyroidism and canine thyroid carcinoma. This article reviews the normal scintigraphic appearance of the thyroid in the cat, the dog, and the horse and the principles of interpretation of abnormal scan results in the cat and the dog. Radioiodine is the treatment of choice for feline hyperthyroidism, and the principles of its use in the cat are reviewed. Copyright © 2014 Elsevier Inc. All rights reserved.

Comparison of childhood myelodysplastic syndrome, AML FAB M6 or M7, CCG 2891: report from the Children's Oncology Group.

PubMed

Barnard, Dorothy R; Alonzo, Todd A; Gerbing, Robert B; Lange, Beverly; Woods, William G

2007-07-01

Myelodysplastic syndromes (MDS), acute erythroleukemia (FAB M6), and acute megakaryocytic leukemia (FAB M7) have overlapping features. Children without Down syndrome or acute promyelocytic leukemia who were newly diagnosed with primary myelodysplastic syndrome or acute myeloid leukemia (AML) M6 or M7 were compared to children with de novo AML M0-M5. All children were entered on the Children's Cancer Group therapeutic research study CCG 2891. The presentation and outcomes of the 132 children diagnosed with MDS (60 children), AML FAB M6 (19 children), or AML FAB M7 (53 children) were similar. Children with AML FAB M7 were diagnosed at a significantly younger age (P = 0.001). Children with MDS, M6, or M7 had significantly lower white blood cell (WBC) counts (P = 0.001), lower peripheral blast counts (P < 0.001), and an increased frequency of -7/7q- (P = 0.003) at presentation. All three groups had significantly inferior overall survival (OS) (P < 0.001) and event free survival (P < 0.001) compared with the 748 children diagnosed with AML FAB M0-M5 when assessed from entry on study. This poor survival was largely attributable to induction death and failure. However, when assessed from successful completion of induction therapy, the 5-year OS (P = 0.090)(49.1 vs. 56.9%) and disease-free survival (DFS) (P = 0.113)(38.0 vs. 46.3%) therapy were not significantly different from other children with AML. Childhood AML FAB M6 and AML M7 resemble MDS in presentation, poor induction success rates, and outcomes.

Linkage of Recognition and Replication Functions by Assembling Combinatorial Antibody Fab Libraries Along Phage Surfaces

NASA Astrophysics Data System (ADS)

Kang, Angray S.; Barbas, Carlos F.; Janda, Kim D.; Benkovic, Stephen J.; Lerner, Richard A.

1991-05-01

We describe a method based on a phagemid vector with helper phage rescue for the construction and rapid analysis of combinatorial antibody Fab libraries. This approach should allow the generation and selection of many monoclonal antibodies. Antibody genes are expressed in concert with phage morphogenesis, thereby allowing incorporation of functional Fab molecules along the surface of filamentous phage. The power of the method depends upon the linkage of recognition and replication functions and is not limited to antibody molecules.

Feasibility study of the Fab fragment of a monoclonal antibody against tissue factor as a diagnostic tool.

PubMed

Tsumura, Ryo; Sato, Ryuta; Furuya, Fumiaki; Koga, Yoshikatsu; Yamamoto, Yoshiyuki; Fujiwara, Yuki; Yasunaga, Masahiro; Matsumura, Yasuhiro

2015-12-01

Tissue factor (TF) is expressed strongly in various types of cancer, especially cancers that are often refractory to treatment, such as pancreatic cancer. In this study, we compared the differences in the biophysical and pharmacological properties of whole IgG and the Fab fragment of anti-human TF monoclonal antibody (1849 antibodies), in order to determine their suitability for application in the diagnosis and treatment of cancers. In the biophysical examination, we investigated the characteristics of 1849-whole IgG and 1849-Fab by SPR sensing and confocal fluorescence microscopy analysis using recombinant human TF antigen and TF-overexpressing human pancreatic cancer cell line, BxPC3, respectively. After conjugation with Alexa-Flour-647, in vivo imaging was conducted in mice bearing BxPC3 xenograft tumors. Furthermore, the distribution of the conjugates in tumors and major organs was evaluated by ex vivo study. The in vitro experiments showed that 1849 antibodies had high affinity against TF antigen. In addition, 1849-Fab showed a faster dissociation rate from the antigen than 1849-whole IgG. In mice, 1849-Fab-Alexa-Flour-647 showed rapid renal clearance and faster tumor accumulation, achieving a high contrast signal over nearby normal tissues in the early phase and enhanced tumor penetration after administration. On the other hand, 1849-whole IgG-Alexa-Flour-647 showed slow clearance from the blood and sustained high tumor accumulation. These results suggest that 1849-Fab may be a useful tool for pancreatic cancer diagnosis.

Opening the Arms: The FAB Projects and Digital Resilience

ERIC Educational Resources Information Center

Longden, Alison; Monaghan, Tom; Mycroft, Lou; Kelly, Claire

2018-01-01

A chance observation in a staff training session led to a series of action research projects, which dug into why some educators are digitally resistant whilst others, however experienced, are not. The Education and Training Foundation-funded "FAB Projects" have so far spanned three years and three small-scale research investigations and…

Bringing optics to Fab Labs in Europe

NASA Astrophysics Data System (ADS)

Adam, Aurèle; Zuidwijk, Thim; Urbach, Paul

2017-08-01

The Optics Group of Delft University of Technology plays a major role in teaching optics to bachelor and master students. In addition, the group has a long record of introducing, demonstrating and teaching optics to quite diverse groups of people from outside of the university. We will describe some of these activities and focus on a recently started project funded by the European Commission called Phablabs 4.0, which aims to bring photonics to European Fab labs.

[Molecular dynamics of immune complex of photoadduct-containing DNA with Fab-Anti-DNA antibody fragment].

PubMed

Akberova, N I; Zhmurov, A A; Nevzorova, T A; Litvinov, R I

2016-01-01

Antibodies to DNA play an important role in the pathogenesis of autoimmune diseases. The elucidation of structural mechanisms of both the antigen recognition and the interaction of anti-DNA antibodies with DNA will help to understand the role of DNA-containing immune complexes in various pathologies and can provide a basis for new treatment modalities. Moreover, the DNA-antibody complex is an analog of specific intracellular DNA-protein interactions. In this work, we used in silico molecular dynamic simulations of bimolecular complexes of the dsDNA segment containing the Fab fragment of an anti-DNA antibody to obtain the detailed thermodynamic and structural characteristics of dynamic intermolecular interactions. Using computationally modified crystal structure of the Fab-DNA complex (PDB ID: 3VW3), we studied the equilibrium molecular dynamics of the 64M-5 antibody Fab fragment associated with the dsDNA fragment containing the thymine dimer, the product of DNA photodamage. Amino acid residues that constitute paratopes and the complementary nucleotide epitopes for the Fab-DNA construct were identified. Stacking and electrostatic interactions were found to play the main role in mediating the most specific antibody-dsDNA contacts, while hydrogen bonds were less significant. These findings may shed light on the formation and properties of pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus associated with skin photosensitivity and DNA photodamage.

Biophysical characterization and structure of the Fab fragment from the NIST reference antibody, RM 8671.

PubMed

Karageorgos, Ioannis; Gallagher, Elyssia S; Galvin, Connor; Gallagher, D Travis; Hudgens, Jeffrey W

2017-11-01

Monoclonal antibody pharmaceuticals are the fastest-growing class of therapeutics, with a wide range of clinical applications. To assure their safety, these protein drugs must demonstrate highly consistent purity and stability. Key to these objectives is higher order structure measurements validated by calibration to reference materials. We describe preparation, characterization, and crystal structure of the Fab fragment prepared from the NIST Reference Antibody RM 8671 (NISTmAb). NISTmAb is a humanized IgG1κ antibody, produced in murine cell culture and purified by standard biopharmaceutical production methods, developed at the National Institute of Standards and Technology (NIST) to serve as a reference material. The Fab fragment was derived from NISTmAb through papain cleavage followed by protein A based purification. The purified Fab fragment was characterized by SDS-PAGE, capillary gel electrophoresis, multi-angle light scattering, size exclusion chromatography, mass spectrometry, and x-ray crystallography. The crystal structure at 0.2 nm resolution includes four independent Fab molecules with complete light chains and heavy chains through Cys 223, enabling assessment of conformational variability and providing a well-characterized reference structure for research and engineering applications. This nonproprietary, publically available reference material of known higher-order structure can support metrology in biopharmaceutical applications, and it is a suitable platform for validation of molecular modeling studies. Published by Elsevier Ltd.

Increase in electrocardiographic R-waves after revascularization in patients with acute myocardial infarction.

PubMed

Isobe, Satoshi; Takada, Yasuo; Ando, Akitada; Ohshima, Satoru; Yamada, Kiyoyasu; Nanasato, Mamoru; Unno, Kazumasa; Ogawa, Takuo; Kondo, Takahisa; Izawa, Hideo; Inden, Yasuya; Hirai, Makoto; Murohara, Toyoaki

2006-11-01

The physiological mechanism of the increase in the electrocardiographic (ECG) R-wave voltage after revascularization in patients with acute myocardial infarction (MI) needs to be elucidated. One hundred and thirty-eight MI patients (83: anterior MI, 45: inferior MI, 10: lateral MI) underwent ECG and echocardiography in both the acute and subacute phases after emergency revascularization, as well as a resting thallium-201/iodine-123 15-p-iodophenyl-3-(R,S)-methyl pentadecanoic acid myocardial scintigraphy in the acute phase. The total sum of the R-wave voltage (SigmaR) was calculated over multiple leads on ECG for each infarcted lesion. Scintigraphic defect on each tracer was expressed as the percentage (%) defect of the total left ventricular (LV) myocardium. The % defect-discordance on both images in the acute phase and the % increase in SigmaR and the absolute increase in LV ejection fraction from the acute to the subacute phase (DeltaEF) were also calculated. The SigmaR in the subacute phase was significantly greater than that in the acute phase (p<0.0001). The % increase in SigmaR significantly correlated with the DeltaEF (r=0.57, p<0.0001). The % increase in SigmaR also correlated with the % defect-discordance (r=0.68, p<0.0001). The increase in the ECG R-wave voltage reflects not only the improvement in myocardial perfusion but also the presence of salvaged myocardium after revascularization in acute MI patients.

Characterization of a recombinant humanized anti-cocaine monoclonal antibody and its Fab fragment.

PubMed

Kirley, Terence L; Norman, Andrew B

2015-01-01

Variations of post-translational modifications are important for stability and in vivo behavior of therapeutic antibodies. A recombinant humanized anti-cocaine monoclonal antibody (h2E2) was characterized for heterogeneity of N-linked glycosylation and disulfide bonds. In addition, charge heterogeneity, which is partially due to the presence or absence of C-terminal lysine on the heavy chains, was examined. For cocaine overdose therapy, Fab fragments may be therapeutic, and thus, a simplified method of generation, purification, and characterization of the Fab fragment generated by Endoproteinase Lys-C digestion was devised. Both the intact h2E2 antibody and purified Fab fragments were analyzed for their affinities for cocaine and 2 of its metabolites, benzoylecgonine and cocaethylene, by fluorescence quenching of intrinsic antibody tyrosine and tryptophan fluorescence resulting from binding of these drugs. Binding constants obtained from fluorescence quenching measurements are in agreement with recently published radioligand and ELISA binding assays. The dissociation constants determined for the h2E2 monoclonal and its Fab fragment are approximately 1, 5, and 20 nM for cocaethylene, cocaine, and benzoylecgonine, respectively. Tryptophan fluorescence quenching (emission at 330 nm) was measured after either excitation of tyrosine and tryptophan (280 nm) or selective excitation of tryptophan alone (295 nm). More accurate binding constants are obtained using tryptophan selective excitation at 295 nm, likely due to interfering absorption of cocaine and metabolites at 280 nm. These quenching results are consistent with multiple tryptophan and tyrosine residues in or near the predicted binding location of cocaine in a previously published 3-D model of this antibody's variable region.

A novel antibody-drug conjugate anti-CD19(Fab)-LDM in the treatment of B-cell non-Hodgkin lymphoma xenografts with enhanced anticancer activity.

PubMed

Jiang, Linlin; Yang, Ming; Zhang, Xiaoyun; Bao, Shiqi; Ma, Li; Fan, Dongmei; Zhou, Yuan; Xiong, Dongsheng; Zhen, Yongsu

2016-01-01

Rituximab is widely used in clinical setting for the treatment of B malignant lymphoma and has achieved remarkable success. However, in most patients, the disease ultimately relapses and become resistant to rituximab. To overcome the limitation, there is still a need to find novel strategy for improving therapeutic efficacy. To construct genetically engineered antibody anti-CD19(Fab)-LDM, and verify the anticancer activity targeted toward B-lymphoma. The anticancer activity of anti-CD19(Fab)-LDM in vitro and in vivo was examined. In vitro, the binding activity and internalization of anti-CD19(Fab)-LDP were measured. Using comet assay and apoptosis, the cytotoxicity of energized fusion proteins was observed. From in vivo experiments, targeting of therapeutic effect and anticancer efficacy bythe fusion protein was verified. Data showed that anti-CD19(Fab)-LDM does not only binding the cell surface but is also internalized into the cell. The energized fusion proteins anti-CD19(Fab)-LDM can induce DNA damage. Furthermore, significant in vivo therapeutic efficacy was observed. The present study demonstrated that the genetically engineered antibody anti-CD19(Fab)-LDM exhibited enhanced cytotoxicity compared to LDM alone. One of the most powerful advantages of anti-CD19(Fab)-LDM, however, is that it can be internalized within the cells and carry out cytotoxic effects. Therefore, anti-CD19(Fab)-LDM may be as a useful targeted therapy for B-cell lymphoma.

Generation and selection of naïve Fab library for parasitic antigen: Anti-BmSXP antibodies for lymphatic filariasis.

PubMed

Omar, Noorsharmimi; Hamidon, Nurul Hamizah; Yunus, Muhammad Hafiznur; Noordin, Rahmah; Choong, Yee Siew; Lim, Theam Soon

2018-05-01

Phage display has been applied successfully as a tool for the generation of monoclonal antibodies (mAbs). Naive antibody libraries are unique as they are able to overcome several limitations associated with conventional mAb generation methods like the hybridoma technology. Here, we performed an in vitro selection and generation of Fab antibodies against Brugia malayi SXP protein (BmSXP), a recombinant antigen for the detection of lymphatic filariasis. We developed a naïve multi ethnic Fab antibody library with an estimated diversity of 2.99 × 10 9 . The antibody library was used to screen for mAbs against BmSXP recombinant antigen. Soluble monoclonal Fab antibodies against BmSXP were successfully isolated from the naïve library. The Fab antibodies obtained were expressed and analyzed to show its binding capability. The diversity obtained from a pool of donors from various ethnic groups allowed for a diverse antibody library to be generated. The mAbs obtained were also functional in soluble form, which makes it useful for further downstream applications. We believe that the Fab mAbs are valuable for further studies and could also contribute to improvements in the diagnosis of filariasis. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

Synthetic Fab Fragments that Bind the HIV-1 gp41 Heptad Repeat Regions

PubMed Central

Liu, Yanyun; Regula, Lauren K.; Stewart, Alex; Lai, Jonathan R.

2011-01-01

Recent work has demonstrated that antibody phage display libraries containing restricted diversity in the complementarity determining regions (CDRs) can be used to target a wide variety of antigens with high affinity and specificity. In the most extreme case, antibodies whose combining sites are comprised of only two residues – tyrosine and serine – have been identified against several protein antigens. [F. A. Fellouse, B. Li, D. M. Compaan, A. A. Peden, S. G. Hymowitz, and S. S. Sidhu, J. Mol. Biol., 348 (2005) 1153–1162.] Here, we report the isolation and characterization of antigen-binding fragments (Fabs) from such “minimalist” diversity synthetic antibody libraries that bind the heptad repeat regions of human immunodeficiency virus type 1 (HIV-1) gp41. We show that these Fabs are highly specific for the HIV-1 epitope and comparable in affinity to a single chain variable fragment (scFv) derived from a natural antibody repertoire that targets the same region. Since the heptad repeat regions of HIV-1 gp41 are required for viral entry, these Fabs have potential for use in therapeutic, research, or diagnostic applications. PMID:21925149

EphB4-targeted imaging with antibody h131, h131-F(ab′)2 and h131-Fab

PubMed Central

Li, Dan; Liu, Shuanglong; Liu, Ren; Zhou, Yue; Park, Ryan; Naga, Kranthi; Krasnoperov, Valery; Gill, Parkash S.; Li, Zibo; Shan, Hong; Conti, Peter S.

2013-01-01

Accumulating evidence suggests that overexpression of the tyrosine kinase receptor EphB4, a mediator of vascular development, is a novel target for tumor diagnosis, prognosis and therapy. Noninvasive imaging of EphB4 expression could therefore be valuable for evaluating disease course and therapeutic efficacy at the earliest stages of anti-EphB4 treatment. In this study, we systematically investigated the use of anti-EphB4 antibody h131 (150 kD) and its fragments (h131-F(ab′)2, 110 kD; h131-Fab, 50 kD) for near-infrared fluorescence (NIRF) imaging of EphB4 expression in vivo. h131-F(ab′)2 and h131-Fab were produced through pepsin and papain digestion of h131 respectively, whose purity was confirmed by FPLC and SDS-PAGE. After conjugation with Cy5.5, in vivo characteristics of h131, h131-F(ab′)2 and h131-Fab were evaluated in EphB4-positive HT29 tumor model. Although h131-Cy5.5 demonstrated highest tumor uptake among these probes, its optimal tumor uptake level was obtained at 2 d post injection (p.i.). For h131-Fab-Cy5.5, maximum tumor uptake was achieved at 4 h p.i.. However, no significant difference was observed between h131-Fab-Cy5.5 and hIgG-Fab-Cy5.5, indicating the tumor accumulation was mainly caused by passive targeting. In contrast, h131-F(ab′)2-Cy5.5 demonstrated prominent tumor uptake at 6 h p.i. The target specificity was confirmed by hIgG-F(ab′)2-Cy5.5 control and immunofluorescent staining. Collectively, h131-F(ab′)2 exhibited prominent and specific tumor uptake at early time points, which suggests it is a promising agent for EphB4-targeted imaging. PMID:24147882

Extracorporeal life support and digoxin-specific Fab fragments for successful management of Taxus baccata intoxication with low output and ventricular arrhythmia.

PubMed

Farag, Mina; Badowski, Dominika; Koschny, Ronald; Skopp, Gisela; Brcic, Andreas; Szabo, Gabor B

2017-12-01

Yew plants are evergreen shrubs which are widely spread throughout the northern hemisphere. Taxane alkaloid derivatives, mainly taxine B, represent the main toxins of Taxus baccata and are highly cardiotoxic. Due to the lack of randomized clinical trials, case reports on accidental or suicidal yew intoxications build the only source of knowledge of clinical treatment options. We report the case of a suicidal yew ingestion admitted to our hospital under prolonged cardiopulmonary resuscitation due to pulseless electrical activity. Extra-corporeal life support (ECLS) was established to maintain adequate organ perfusion. Repeated administration of digoxin-specific Fab antibody fragments, which cross-react with taxine, was associated with an immediate conversion from asystole to broad-complex bradycardia and a gradual normalization of the electrocardiogram (ECG). This was paralleled by a recovery of the cardiac function and weaning from the ECLS. The taxine metabolite 3,5-dimethoxyphenol could be detected by mass spectrometry before but not after the first Fab-fragment treatment. In contrast, the total amount of taxine (including the neutralized, Fab fragment-bound fraction) was increased after each Fab fragment administration, suggesting an accumulation of neutralized, since antibody-bound taxine in the blood by anti-digoxin Fab fragments. In conclusion, the successful clinical course of this case suggests a benefit of an early anti-digoxin Fab-fragment administration for the treatment of yew intoxication. Copyright © 2017 Elsevier Inc. All rights reserved.

Fab Four self-interaction in quantum regime

NASA Astrophysics Data System (ADS)

Arbuzov, A. B.; Latosh, B. N.

2017-10-01

Quantum behavior of the John Lagrangian from the Fab Four class of covariant Galileons is studied. We consider one-loop corrections to the John interaction due to cubic scalar field interaction. Counter terms are calculated, one appears because of massless scalar field theory infrared issues, another one lies in the George class, and the rest of them can be reduced to the initial Lagrangian up to surface terms. The role of quantum corrections in the context of cosmological applications is discussed.

Incidence of immediate hypersensitivity reaction and serum sickness following administration of Crotalidae polyvalent immune Fab antivenom: a meta-analysis.

PubMed

Schaeffer, Tammi H; Khatri, Vaishali; Reifler, Liza M; Lavonas, Eric J

2012-02-01

  Crotalidae polyvalent immune Fab (ovine) (FabAV) is commonly used in the treatment of symptomatic North American crotaline snake envenomation. When approved by the U.S. Food and Drug Administration in 2000, the incidences of immediate hypersensitivity reactions and serum sickness were reported as 0.14 and 0.18, respectively. The objective of this meta-analysis was to evaluate the incidence of immediate hypersensitivity reactions and serum sickness reported in studies of patients treated with FabAV therapy after North American crotaline envenomation.   The authors searched PubMed, Ovid MEDLINE, and EMBASE from January 1, 1997, to September 20, 2010, for English-language medical literature and cross-referenced bibliographies of reviewed articles. The published abstracts of the major toxicology conferences were also searched. All prospective and retrospective cohort studies with patients receiving FabAV therapy for North American crotaline envenomations were eligible for data abstraction. Two content experts reviewed full-text articles and extracted relevant study design and outcome data. Proportions of immediate hypersensitivity and serum sickness for each study were analyzed in a random-effects model to produce an overall estimate of immediate hypersensitivity and serum sickness incidence associated with FabAV administration.   The literature search revealed 11 unique studies of patients who received FabAV that contained information on immediate hypersensitivity reactions and serum sickness. The meta-analysis produced a combined estimate of the incidence of immediate hypersensitivity of 0.08 (95% confidence interval [CI] = 0.05 to 0.11) and a combined estimate of the incidence of serum sickness of 0.13 (95% CI = 0.07 to 0.21).   In this systematic literature review and meta-analysis, the combined estimates of the incidence of immediate hypersensitivity reactions and serum sickness from FabAV in the treatment of symptomatic North American crotaline

Antimyosin imaging in acute transmural myocardial infarctions: results of a multicenter clinical trial.

PubMed

Johnson, L L; Seldin, D W; Becker, L C; LaFrance, N D; Liberman, H A; James, C; Mattis, J A; Dean, R T; Brown, J; Reiter, A

1989-01-01

Murine monoclonal antimyosin antibody has been shown experimentally to bind selectively to irreversibly damaged myocytes. To evaluate the safety and efficacy of monoclonal antimyosin for identifying acute transmural infarction, 50 patients with acute Q wave myocardial infarction were entered into a phase I/II multicenter trial involving three clinical sites. Indium-111 antimyosin was prepared from an instant kit formulation containing 0.5 mg of diethylene triamine pentaacetic acid (DTPA)-coupled Fab fragment (R11D10) and 1.2 to 2.4 mCi of indium-111. Average labeling efficiency was 92%. Antimyosin was injected 27 +/- 16 h after the onset of chest pain. Planar or tomographic imaging was performed 27 +/- 9 h after injection in all patients, and repeat imaging was done 24 h later in 39 patients. Of the 50 patients entered, 46 showed myocardial uptake of antimyosin (sensitivity 92%). Thirty-one of 39 planar scans performed at 24 h were diagnostic; 8 showed persistent blood pool activity that cleared by 48 h. Focal myocardial uptake of antimyosin corresponded to electrocardiographic infarct localization. No patient had an adverse reaction to antimyosin. In addition, 125 serum samples, including 21 collected greater than 42 days after injection, were tested for human antimouse antibodies, and all samples were assessed as having undetectable titers. Intensity of antimyosin uptake was correlated with infarct location and the presence or absence of collateral vessels. There was a significant correlation between faint uptake and inferoposterior infarct location. In 21 patients who had coronary angiography close to the time of antimyosin injection, there was a significant correlation between faint tracer uptake and closed infarct-related vessel with absent collateral flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-colchicine Fab fragments prevent lethal colchicine toxicity in a porcine model: a pharmacokinetic and clinical study.

PubMed

Eddleston, Michael; Fabresse, Nicolas; Thompson, Adrian; Al Abdulla, Ibrahim; Gregson, Rachael; King, Tim; Astier, Alain; Baud, Frederic J; Clutton, R Eddie; Alvarez, Jean-Claude

2018-08-01

Colchicine poisoning is commonly lethal. Colchicine-specific Fab fragments increase rat urinary colchicine clearance and have been associated with a good outcome in one patient. We aimed to develop a porcine model of colchicine toxicity to study the pharmacokinetics and efficacy of ovine Fab. A Göttingen minipig critical care model was established and serial blood samples taken for colchicine and Fab pharmacokinetics, clinical chemistry, and haematology. Animals were euthanised when the mean arterial pressure fell below 45 mmHg without response to vasopressor, or at study completion. Initial studies indicated that oral dosing produced variable pharmacokinetics and time-to-euthanasia. By contrast, intravenous infusion of 0.25 mg/kg colchicine over 1 h produced reproducible pharmacokinetics (AUC 0-20 343 [SD = 21] µg/L/h), acute multi-organ injury, and cardiotoxicity requiring euthanasia a mean of 22.5 (SD = 3.2) h after dosing. A full-neutralising equimolar Fab dose given 6 h after the infusion (50% first hour, 50% next 6 h [to reduce renal-loss of unbound Fab]) produced a 7.35-fold increase in plasma colchicine (AUC 0-20 2,522 [SD = 14] µg/L/h), and removed all free plasma colchicine, but did not prevent toxicity (euthanasia at 29.1 [SD = 3.4] h). Earlier administration over 1 h of the full-neutralising dose, 1 or 3 h after the colchicine, produced a 12.9-fold (AUC 0-20 4,433 [SD = 607] µg/L/h) and 6.0-fold (AUC 0-20 2,047 [SD = 51] µg/L/h) increase in plasma colchicine, respectively, absence of free plasma colchicine until 20 h, and survival to study end without marked cardiotoxicity. Colchicine-specific Fab given early, in equimolar dose, bound colchicine, eliciting its movement into the blood, and preventing severe toxicity. Clinical studies are now needed to determine how soon this antidote must be given to work in human poisoning.

99 mTc-MIBI washout as a complementary factor in the evaluation of idiopathic dilated cardiomyopathy (IDCM) using myocardial perfusion imaging.

PubMed

Shiroodi, Mohammad Kazem; Shafiei, Babak; Baharfard, Nastaran; Gheidari, Mohammad Esmail; Nazari, Babak; Pirayesh, Elaheh; Kiasat, Ali; Hoseinzadeh, Samaneh; Hashemi, Abolghassem; Akbarzadeh, Mohammad Ali; Javadi, Hamid; Nabipour, Iraj; Assadi, Majid

2012-01-01

Rapid technetium-99 m methoxyisobutylisonitrile (99 mTc-MIBI) washout has been shown to occur in impaired myocardia. This study is based on the hypothesis that scintigraphy can be applied to calculate the myocardial 99 mTc-MIBI washout rate (WR) to diagnose and evaluate heart failure severity and other left ventricular functional parameters specifically in idiopathic dilated cardiomyopathy (IDCM) patients. Patients with IDCMP (n = 17; 52.65 ± 11.47 years) and normal subjects (n = 6; 49.67 ± 10.15 years) were intravenously administered 99 mTc-hexakis-2-methoxyisobutylisonitrile (99 mTc-MIBI). Next, early and delayed planar data were acquired (at 3.5-h intervals), and electrocardiogram (ECG)-gated myocardial perfusion single photon emission computed tomography (SPECT) was performed. The 99 mTc-MIBI WR was calculated using early and delayed planar images. Left ventricular functional parameters were also analyzed using quantitative gated SPECT (QGS) data. In target group, myocardial WRs (29.13 ± 6.68%) were significantly higher than those of control subjects (14.17 ± 3.31%; P < 0.001). The 99 mTc-MIBI WR increased with the increasing severity of the NYHA functional class (23.16 ± 1.72% for class I, 30.25 ± 0.95% for class II, 32.60 ± 6.73% for class III, and 37.50 ± 7.77% for class IV; P = 0.02). The WR was positively correlated with the end-diastolic volume (EDV) index (r (2) = 0.216; β = 0.464; P = 0.02 [ml/m(2)], the end-systolic volume (ESV) index (r (2) = 0.234; β = 0.484; P = 0.01 [ml/m(2)]), the summed motion score (SMS) (r (2) = 0.544; β = 0.738; P = 0.00), and the summed thickening score (STS) (r (2) = 0.656; β = 0.810; P = 0.00); it was negatively correlated with the left ventricular ejection fraction (LVEF) (r (2) = 0.679; β = -0.824; P = 0.00). It can be concluded that 99 mTc-MIBI scintigraphy might be a valuable molecular imaging tool for the diagnosis and evaluation of myocardial damage or dysfunction severity.

Myocardial Bridge

MedlinePlus

... Center > Myocardial Bridge Menu Topics Topics FAQs Myocardial Bridge En español Your heart is made of muscle, ... surface of the heart. What is a myocardial bridge? A myocardial bridge is a band of heart ...

Aerial image measurement technique for automated reticle defect disposition (ARDD) in wafer fabs

NASA Astrophysics Data System (ADS)

Zibold, Axel M.; Schmid, Rainer M.; Stegemann, B.; Scheruebl, Thomas; Harnisch, Wolfgang; Kobiyama, Yuji

2004-08-01

The Aerial Image Measurement System (AIMS)* for 193 nm lithography emulation has been brought into operation successfully worldwide. A second generation system comprising 193 nm AIMS capability, mini-environment and SMIF, the AIMS fab 193 plus is currently introduced into the market. By adjustment of numerical aperture (NA), illumination type and partial illumination coherence to match the conditions in 193 nm steppers or scanners, it can emulate the exposure tool for any type of reticles like binary, OPC and PSM down to the 65 nm node. The system allows a rapid prediction of wafer printability of defects or defect repairs, and critical features, like dense patterns or contacts on the masks without the need to perform expensive image qualification consisting of test wafer exposures followed by SEM measurements. Therefore, AIMS is a mask quality verification standard for high-end photo masks and established in mask shops worldwide. The progress on the AIMS technology described in this paper will highlight that besides mask shops there will be a very beneficial use of the AIMS in the wafer fab and we propose an Automated Reticle Defect Disposition (ARDD) process. With smaller nodes, where design rules are 65 nm or less, it is expected that smaller defects on reticles will occur in increasing numbers in the wafer fab. These smaller mask defects will matter more and more and become a serious yield limiting factor. With increasing mask prices and increasing number of defects and severability on reticles it will become cost beneficial to perform defect disposition on the reticles in wafer production. Currently ongoing studies demonstrate AIMS benefits for wafer fab applications. An outlook will be given for extension of 193 nm aerial imaging down to the 45 nm node based on emulation of immersion scanners.

Factors associated with difficulty achieving initial control with crotalidae polyvalent immune fab antivenom in snakebite patients.

PubMed

Yin, Shan; Kokko, Jamie; Lavonas, Eric; Mlynarchek, Sara; Bogdan, Greg; Schaeffer, Tammi

2011-01-01

The prescribing information for Crotalidae Fab antivenom (FabAV) instructs clinicians to administer FabAV until initial control of the envenomation syndrome is achieved. Risk factors for difficulty achieving initial control are not known. The study aim was to identify factors present before administration of antivenom associated with difficulty achieving initial control. The authors conducted a retrospective study of all patients presenting to any one of 17 centers and receiving FabAV from 2002 to 2004. Demographic and historical information, as well as data about nine specific venom effects, were collected prior to the administration of antivenom. An expert panel used standard criteria to determine if initial control was achieved. The patient group that had difficulty achieving initial control was compared to the group that achieved initial control, and adjusted odds ratios were calculated using stepwise logistic regression. A total of 247 patients were included in the final analysis. The majority of patients were envenomated on the upper extremity and were young males. A total of 203 patients (82.2%) achieved initial control. In univariate analysis, thrombocytopenia, bleeding, neurologic effects, and a severe bite were significantly associated with difficulty achieving initial control. After logistic regression, the presence of neurologic effects and thrombocytopenia remained significantly associated with difficulty achieving initial control. When both factors were present, the patient was 13.8 times more likely to have difficulty achieving initial control. A number of factors were present before the administration of FabAV that were independently associated with difficulty achieving initial control of the envenomation syndrome. Predicting which patients will have difficulty achieving initial control has important ramifications for patient disposition and may provide insight into the mechanisms for lack of antivenom efficacy. © 2010 by the Society for Academic

PEGylation prolongs the pulmonary retention of an anti-IL-17A Fab' antibody fragment after pulmonary delivery in three different species.

PubMed

Freches, Danielle; Patil, Harshad P; Machado Franco, Maria; Uyttenhove, Catherine; Heywood, Sam; Vanbever, Rita

2017-04-15

The PEGylation of antibody fragments has been shown to greatly prolong their residence time in the lungs in mice. The purpose of this research was to confirm the effect of PEGylation in higher animal species, that is, the rat and the rabbit. An anti-IL-17A Fab' antibody fragment was conjugated to a two-armed 40kDa polyethylene glycol (PEG) via site-selective thiol PEGylation. PEGylation did not significantly alter the binding activity of the Fab' fragment but it largely enhanced its inhibitory potency. PEGylation increased the residence time of the Fab' in the lungs of mice, rats and rabbits. Following intratracheal administration, the unconjugated Fab' was cleared from the lungs within 24h while large quantities of the PEGylated Fab' remained present up to 48h. No significant differences in clearance were noted between the three animal species although there was a tendency of longer residence time in higher species. PEGylation represents a promising approach to sustain the presence of antibody fragments in the lungs and to enhance their therapeutic efficacy in respiratory diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

Myocardial Rupture in Acute Myocardial Infarction: Mechanistic Explanation Based on the Ventricular Myocardial Band Hypothesis.

PubMed

Vargas-Barron, Jesús; Antunez-Montes, Omar-Yassef; Roldán, Francisco-Javier; Aranda-Frausto, Alberto; González-Pacheco, Hector; Romero-Cardenas, Ángel; Zabalgoitia, Miguel

2015-01-01

Torrent-Guasp explains the structure of the ventricular myocardium by means of a helical muscular band. Our primary purpose was to demonstrate the utility of echocardiography in human and porcine hearts in identifying the segments of the myocardial band. The second purpose was to evaluate the relation of the topographic distribution of the myocardial band with some post-myocardial infarction ruptures. Five porcine and one human heart without cardiopathy were dissected and the ventricular myocardial segments were color-coded for illustration and reconstruction purposes. These segments were then correlated to the conventional echocardiographic images. Afterwards in three cases with post-myocardial infarction rupture, a correlation of the topographic location of the rupture with the distribution of the ventricular band was made. The human ventricular band does not show any differences from the porcine band, which confirms the similarities of the four segments; these segments could be identified by echocardiography. In three cases with myocardial rupture, a correlation of the intra-myocardial dissection with the distribution of the ventricular band was observed. Echocardiography is helpful in identifying the myocardial band segments as well as the correlation with the topographic distribution of some myocardial post-infarction ruptures.

A dual host vector for Fab phage display and expression of native IgG in mammalian cells.

PubMed

Tesar, Devin; Hötzel, Isidro

2013-10-01

A significant bottleneck in antibody discovery by phage display is the transfer of immunoglobulin variable regions from phage clones to vectors that express immunoglobulin G (IgG) in mammalian cells for screening. Here, we describe a novel phagemid vector for Fab phage display that allows expression of native IgG in mammalian cells without sub-cloning. The vector uses an optimized mammalian signal sequence that drives robust expression of Fab fragments fused to an M13 phage coat protein in Escherichia coli and IgG expression in mammalian cells. To allow the expression of Fab fragments fused to a phage coat protein in E.coli and full-length IgG in mammalian cells from the same vector without sub-cloning, the sequence encoding the phage coat protein was embedded in an optimized synthetic intron within the immunoglobulin heavy chain gene. This intron is removed from transcripts in mammalian cells by RNA splicing. Using this vector, we constructed a synthetic Fab phage display library with diversity in the heavy chain only and selected for clones binding different antigens. Co-transfection of mammalian cells with DNA from individual phage clones and a plasmid expressing the invariant light chain resulted in the expression of native IgG that was used to assay affinity, ligand blocking activity and specificity.

Successful application of the dual-vector system II in creating a reliable phage-displayed combinatorial Fab library.

PubMed

Song, Suk-yoon; Hur, Byung-ung; Lee, Kyung-woo; Choi, Hyo-jung; Kim, Sung-soo; Kang, Goo; Cha, Sang-hoon

2009-03-31

The dual-vector system-II (DVS-II), which allows efficient display of Fab antibodies on phage, has been reported previously, but its practical applicability in a phage-displayed antibody library has not been verified. To resolve this issue, we created two small combinatorial human Fab antibody libraries using the DVS-II, and isolation of target-specific antibodies was attempted. Biopanning of one antibody library, termed DVFAB-1L library, which has a 1.3 x 10(7) combinatorial antibody complexity, against fluorescein-BSA resulted in successful isolation of human Fab clones specific for the antigen despite the presence of only a single light chain in the library. By using the unique feature of the DVS-II, an antibody library of a larger size, named DVFAB-131L, which has a 1.5 x 10(9) combinatorial antibody complexity, was also generated in a rapid manner by combining 1.3 x 10(7) heavy chains and 131 light chains and more diverse anti-fluorescein-BSA Fab antibody clones were successfully obtained. Our results demonstrate that the DVS-II can be applied readily in creating phage-displayed antibody libraries with much less effort, and target-specific antibody clones can be isolated reliably via light chain promiscuity of antibody molecule.

Pharmacological efficacy of anti-IL-1β scFv, Fab and full-length antibodies in treatment of rheumatoid arthritis.

PubMed

Qi, Jianying; Ye, Xianlong; Ren, Guiping; Kan, Fangming; Zhang, Yu; Guo, Mo; Zhang, Zhiyi; Li, Deshan

2014-02-01

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease that mainly causes the synovial joint inflammation and cartilage destruction. Interleukin-1β (IL-1β) is an important proinflammatory cytokine involved in the pathogenesis of RA. In this study, we constructed and expressed anti-IL-1β-full-length antibody in CHO-K1-SV, anti-IL-1β-Fab and anti-IL-1β-scFv in Rosetta. We compared the therapeutic efficacy of three anti-IL-1β antibodies for CIA mice. Mice with CIA were subcutaneously injected with humanized anti-IL-1β-scFv, anti-IL-1β-Fab or anti-IL-1β-full-length antibody. The effects of treatment were determined by arthritis severity score, autoreactive humoral, cellular immune responses, histological lesion and cytokines production. Compared with anti-IL-1β-scFv treatments, anti-IL-1β-Fab and anti-IL-1β-full-length antibody therapy resulted in more significant effect in alleviating the severity of arthritis by preventing bone damage and cartilage destruction, reducing humoral and cellular immune responses, and down-regulating the expression of IL-1β, IL-6, IL-2, IFN-γ, TNF-α and MMP-3 in inflammatory tissue. The therapeutic effects of anti-IL-1β-Fab and anti-IL-1β-full-length antibodies on CIA mice had no significant difference. However, production of anti-IL-1β-full-length antibody in eukaryotic system is, in general, time-consuming and more expensive than that of anti-IL-1β-Fab in prokaryotic systems. In conclusion, as a small molecule antibody, anti-IL-1β-Fab is an ideal candidate for RA therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

Severe adverse drug reaction following Crotalidae Polyvalent Immune Fab (Ovine) administration for copperhead snakebite.

PubMed

Lepak, Maryjoy R; Bochenek, Samantha H; Bush, Sean P

2015-01-01

To present the case of a severe anaphylactic/anaphylactoid reaction to Crotalidae Polyvalent Immune Fab (Ovine) in a patient bitten by a copperhead snake. A 68-year-old man presented with progressive envenomation after receiving a copperhead snakebite on each hand. Crotalinae Fab antivenom was administered. While the initial and only dose was partially infusing, the patient developed an adverse drug reaction (ADR) of urticaria and hypotension, which resolved with cessation of the infusion, recurred with resumption of the infusion, and ultimately was completed with supportive care. An additional episode of hypotension, urticaria, and angioedema occurred shortly after antivenom therapy completion. Epinephrine was administered, resolving the reaction with complete patient recovery. The event received a Naranjo score of 10, indicating a definite ADR. Treating copperhead snakebites with antivenom is a matter of debate. Concern over adverse events and cost induce some physicians to manage copperhead bites without antivenom because they are generally milder in severity. As demonstrated in this case, severe ADR can occur with Crotalinae Fab antivenom, and its efficacy for copperhead envenoming needs to be better established via placebo-controlled, randomized trials. © The Author(s) 2014.

Conjugation of 10 kDa Linear PEG onto Trastuzumab Fab' Is Sufficient to Significantly Enhance Lymphatic Exposure while Preserving in Vitro Biological Activity.

PubMed

Chan, Linda J; Ascher, David B; Yadav, Rajbharan; Bulitta, Jürgen B; Williams, Charlotte C; Porter, Christopher J H; Landersdorfer, Cornelia B; Kaminskas, Lisa M

2016-04-04

The lymphatic system is a major conduit by which many diseases spread and proliferate. There is therefore increasing interest in promoting better lymphatic drug targeting. Further, antibody fragments such as Fabs have several advantages over full length monoclonal antibodies but are subject to rapid plasma clearance, which can limit the lymphatic exposure and activity of Fabs against lymph-resident diseases. This study therefore explored ideal PEGylation strategies to maximize biological activity and lymphatic exposure using trastuzumab Fab' as a model. Specifically, the Fab' was conjugated with single linear 10 or 40 kDa PEG chains at the hinge region. PEGylation led to a 3-4-fold reduction in binding affinity to HER2, but antiproliferative activity against HER2-expressing BT474 cells was preserved. Lymphatic pharmacokinetics were then examined in thoracic lymph duct cannulated rats after intravenous and subcutaneous dosing at 2 mg/kg, and the data were evaluated via population pharmacokinetic modeling. The Fab' displayed limited lymphatic exposure, but conjugation of 10 kDa PEG improved exposure by approximately 11- and 5-fold after intravenous (15% dose collected in thoracic lymph over 30 h) and subcutaneous (9%) administration, respectively. Increasing the molecular weight of the PEG to 40 kDa, however, had no significant impact on lymphatic exposure after intravenous (14%) administration and only doubled lymphatic exposure after subcutaneous administration (18%) when compared to 10 kDa PEG-Fab'. The data therefore suggests that minimal PEGylation has the potential to enhance the exposure and activity of Fab's against lymph-resident diseases, while no significant benefit is achieved with very large PEGs.

Pharmacological stress myocardial perfusion scintigraphy: use of a modified adenosine protocol in patients with asthma.

PubMed

Sundram, Francis; Notghi, Alp; Smith, Neil B

2009-03-01

Stress radionuclide myocardial perfusion scintigraphy (MPS) using adenosine pharmacological vasodilatation is the preferred method in many centres because of its convenience, safety and speed. It can, however, cause bronchospasm and hence its use is avoided in patients with known or suspected bronchospasm. Owing to service pressures, we use technologist-led adenosine stressing for patients referred for MPS studies. We use a modified adenosine infusion protocol under medical supervision for patients with asthma to prevent and minimize adenosine-induced bronchospasm. In this study, we audited our use of this modified protocol in asthmatic patients and compared the side-effect profile with the standard adenosine protocol used in nonasthmatic patients. We audited 50 consecutive patients with asthma attending our department for stress MPS. All patients were taking regular inhalers+/-oral steroids. Patients who had exacerbation of asthma requiring hospital admission during the preceding 6 months were excluded. Before commencing the infusion, two inhaled puffs of salbutamol were administered. A modified adenosine infusion protocol was used, starting initially at a rate of 70 microg/kg/min and increasing to the standard 140 microg/kg/min within 1 min and then maintained for a further 5 min. Technetium-99m tetrofosmin was injected at 3 min. Blood pressure (BP), pulse rate (PR), oxygen saturation and ECG were monitored before, during and at the end of the infusion. All side effects were recorded. Fifty-eight consecutive patients without asthma were included as controls and received the standard 140 microg/kg/min infusion over 6 min. One hundred and eight patients, 50 with asthma and 58 without asthma, were entered into the study. The test was stopped early in two patients (4%) with asthma and 11 patients (19%) without asthma (chi=5.679; P=0.017). Proportionally, more nonasthmatics developed shortness of breath (SOB) (47 of 58, 81% without asthma vs. 35 of 50, 70% with asthma

Genomic Landscape of Intrahost Variation in Group A Streptococcus: Repeated and Abundant Mutational Inactivation of the fabT Gene Encoding a Regulator of Fatty Acid Synthesis

PubMed Central

Eraso, Jesus M.; Olsen, Randall J.; Beres, Stephen B.; Kachroo, Priyanka; Porter, Adeline R.; Nasser, Waleed; Bernard, Paul E.; DeLeo, Frank R.

2016-01-01

To obtain new information about Streptococcus pyogenes intrahost genetic variation during invasive infection, we sequenced the genomes of 2,954 serotype M1 strains recovered from a nonhuman primate experimental model of necrotizing fasciitis. A total of 644 strains (21.8%) acquired polymorphisms relative to the input parental strain. The fabT gene, encoding a transcriptional regulator of fatty acid biosynthesis genes, contained 54.5% of these changes. The great majority of polymorphisms were predicted to deleteriously alter FabT function. Transcriptome-sequencing (RNA-seq) analysis of a wild-type strain and an isogenic fabT deletion mutant strain found that between 3.7 and 28.5% of the S. pyogenes transcripts were differentially expressed, depending on the growth temperature (35°C or 40°C) and growth phase (mid-exponential or stationary phase). Genes implicated in fatty acid synthesis and lipid metabolism were significantly upregulated in the fabT deletion mutant strain. FabT also directly or indirectly regulated central carbon metabolism genes, including pyruvate hub enzymes and fermentation pathways and virulence genes. Deletion of fabT decreased virulence in a nonhuman primate model of necrotizing fasciitis. In addition, the fabT deletion strain had significantly decreased survival in human whole blood and during phagocytic interaction with polymorphonuclear leukocytes ex vivo. We conclude that FabT mutant progeny arise during infection, constitute a metabolically distinct subpopulation, and are less virulent in the experimental models used here. PMID:27600505

Sortase-catalyzed in vitro functionalization of a HER2-specific recombinant Fab for tumor targeting of the plant cytotoxin gelonin

PubMed Central

Kornberger, Petra; Skerra, Arne

2014-01-01

We report on the preparation of a new type of immunotoxin via in vitro ligation of the αHer2 antigen binding fragment (Fab) of the clinically-validated antibody trastuzumab to the plant toxin gelonin, employing catalysis by the bacterial enzyme sortase A (SrtA). The αHer2 Fab was fused with the extended SrtA recognition motif LPET↓GLEH6 at the C-terminus of its heavy chain, thereby preventing interference with antigen binding, while the toxin was equipped with a Gly2 sequence at its N-terminus, distant to the catalytically active site in the C-terminal region. Site-specific in vitro transpeptidation led to a novel antibody-toxin conjugate wherein gelonin had effectively replaced the Fc region of a conventional (monomerized) immunoglobulin. After optimization of reaction conditions and incubation time, the resulting Fab-Gelonin ligation product was purified to homogeneity in a two-step procedure by means of Strep-Tactin affinity chromatography—utilizing the Strep-tag II appended to gelonin—and size exclusion chromatography. Binding activity of the immunotoxin for the Her2 ectodomain was indistinguishable from the unligated Fab as measured by real-time surface plasmon resonance spectroscopy. Specific cytotoxic potency of Fab-Gelonin was demonstrated against two Her2-positive cell lines, resulting in EC50 values of ~1 nM or lower, indicating a 1000-fold enhanced cell-killing activity compared with gelonin itself. Thus, our strategy provides a convenient route to the modular construction of functional immunotoxins from Fabs of established tumor-specific antibodies with gelonin or related proteotoxins, also avoiding the elevated biosafety levels that would be mandatory for the direct biotechnological preparation of corresponding fusion proteins. PMID:24492291

Guidelines for radioiodinated MIBG scintigraphy in children.

PubMed

Olivier, Pierre; Colarinha, Paula; Fettich, Jure; Fischer, Sibylle; Frökier, Jörgen; Giammarile, Francesco; Gordon, Isky; Hahn, Klaus; Kabasakal, Levent; Mann, Mike; Mitjavila, Mercedes; Piepsz, Amy; Porn, Ute; Sixt, Rune; van Velzen, Jeannette

2003-05-01

These guidelines on the use of radioiodinated (99m)Tc-MIBG scintigraphy in children, which summarise the views of the Paediatric Committee of the European Association of Nuclear Medicine, provide a framework which may prove helpful to nuclear medicine teams in daily practice. They have been influenced by the conclusions of the "Consensus Guidelines for MIBG Scintigraphy" (Paris, November 6, 1997) of the European Neuroblastoma Group and by those of the Oncological Committee of the French Society of Nuclear Medicine. The guidelines should be taken in the context of "good practice" and any local/national rules which apply to nuclear medicine examinations.

Successful use of digoxin-specific immune Fab in the treatment of severe Nerium oleander toxicosis in a dog.

PubMed

Pao-Franco, Amaris; Hammond, Tara N; Weatherton, Linda K; DeClementi, Camille; Forney, Scott D

2017-09-01

To describe a case in which digoxin-specific immune Fab was used successfully in a dog with severe oleander toxicosis secondary to ingesting plant material. A 6-year-old intact female Rhodesian Ridgeback mixed breed dog was presented for severe oleander toxicosis and was refractory to all antiarrhythmic therapies and supportive care. Digoxin-specific immune Fab was successful in treating this dog. The dog recovered but suffered ischemic injuries, the long-term effects of which are unknown. This report describes the successful use of digoxin-specific immune Fab in the treatment of oleander toxicosis in a dog, which has not previously been published in veterinary literature. Oleander poisoning can be associated with permanent cardiac arrhythmias due to the ischemic damage. © Veterinary Emergency and Critical Care Society 2017.

Structural and biophysical characterization of an epitope-specific engineered Fab fragment and complexation with membrane proteins: implications for co-crystallization.

PubMed

Johnson, Jennifer L; Entzminger, Kevin C; Hyun, Jeongmin; Kalyoncu, Sibel; Heaner, David P; Morales, Ivan A; Sheppard, Aly; Gumbart, James C; Maynard, Jennifer A; Lieberman, Raquel L

2015-04-01

Crystallization chaperones are attracting increasing interest as a route to crystal growth and structure elucidation of difficult targets such as membrane proteins. While strategies to date have typically employed protein-specific chaperones, a peptide-specific chaperone to crystallize multiple cognate peptide epitope-containing client proteins is envisioned. This would eliminate the target-specific chaperone-production step and streamline the co-crystallization process. Previously, protein engineering and directed evolution were used to generate a single-chain variable (scFv) antibody fragment with affinity for the peptide sequence EYMPME (scFv/EE). This report details the conversion of scFv/EE to an anti-EE Fab format (Fab/EE) followed by its biophysical characterization. The addition of constant chains increased the overall stability and had a negligible impact on the antigen affinity. The 2.0 Å resolution crystal structure of Fab/EE reveals contacts with larger surface areas than those of scFv/EE. Surface plasmon resonance, an enzyme-linked immunosorbent assay, and size-exclusion chromatography were used to assess Fab/EE binding to EE-tagged soluble and membrane test proteins: namely, the β-barrel outer membrane protein intimin and α-helical A2a G protein-coupled receptor (A2aR). Molecular-dynamics simulation of the intimin constructs with and without Fab/EE provides insight into the energetic complexities of the co-crystallization approach.

Label-free Fab and Fc affinity/avidity profiling of the antibody complex half-life for polyclonal and monoclonal efficacy screening.

PubMed

Read, Thomas; Olkhov, Rouslan V; Williamson, E Diane; Shaw, Andrew M

2015-09-01

A unified approach to affinity screening for Fab and Fc interactions of an antibody for its antigen and FcγR receptor has been developed. An antigen array is used for the Fab affinity and cross-reactivity screening and protein A/G proxy is the FcγR receptor. The affinities are derived using a simple 1:1 binding model with a consistent error analysis. The association and dissociation kinetics are measured over optimised times for accurate determination. The Fab/Fc affinities are derived for ten antibodies: mAb-actin (mouse), pAb-BSA (sheep), pAb-collagen V (rabbit), pAb-CRP (goat), mAb-F1 (mouse), mAbs (mouse) 7.3, 12.3, 29.3, 36.3 and 46.3 raised against LcrV in Yersinia pestis. The rate of the dissociation of antigen-antibody complexes relates directly to their immunological function as does the Fc-FcγR complex and a new half-life plot has been defined with a Fab/Fc half-life range of 17-470 min. The upper half-life value points to surface avidity. Two antibodies that are protective as an immunotherapy define a Fab half-life >250 min and an Fc half-life >50 min as characteristics of ideal interactions which can form the basis of an antibody screen for immunotherapy.

Studies of nontarget-mediated distribution of human full-length IgG1 antibody and its FAb fragment in cardiovascular and metabolic-related tissues.

PubMed

Davidsson, Pia; Söderling, Ann-Sofi; Svensson, Lena; Ahnmark, Andrea; Flodin, Christine; Wanag, Ewa; Screpanti-Sundqvist, Valentina; Gennemark, Peter

2015-05-01

Tissue distribution and pharmacokinetics (PK) of full-length nontargeted antibody and its antigen-binding fragment (FAb) were evaluated for a range of tissues primarily of interest for cardiovascular and metabolic diseases. Mice were intravenously injected with a dose of 10 mg/kg of either human IgG1or its FAb fragment; perfused tissues were collected at a range of time points over 3 weeks for the human IgG1 antibody and 1 week for the human FAb antibody. Tissues were homogenized and antibody concentrations were measured by specific immunoassays on the Gyros system. Exposure in terms of maximum concentration (Cmax ) and area under the curve was assessed for all nine tissues. Tissue exposure of full-length antibody relative to plasma exposure was found to be between 1% and 10%, except for brain (0.2%). Relative concentrations of FAb antibody were the same, except for kidney tissue, where the antibody concentration was found to be ten times higher than in plasma. However, the absolute tissue uptake of full-length IgG was significantly higher than the absolute tissue uptake of the FAb antibody. This study provides a reference PK state for full-length whole and FAb antibodies in tissues related to cardiovascular and metabolic diseases that do not include antigen or antibody binding. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Detection of deep venous thrombophlebitis by gallium 67 scintigraphy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller, J.H.

1981-07-01

Deep venous thrombophlebitis may escape clinical detection. Three cases are reported in which whole-body gallium 67 scintigraphy was used to detect unsuspected deep venous thrombophlebitis related to indwelling catheters in three children who were being evaluated for fevers of unknown origin. Two of these children had septicemia from Candida organisms secondary to these venous lines. Gallium 67 scintigraphy may be useful in the detection of complications of indwelling venous catheters.

Detection of deep venous thrombophlebitis by Gallium 67 scintigraphy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miller, J.H.

1981-07-01

Deep venous thrombophlebitis may escape clinical detection. Three cases are reported in which whole-body gallium 67 scintigraphy was used to detect unsuspected deep venous thrombophlebitis related to indwelling catheters in three children who were being evaluated for fevers of unknown origin. Two of these children had septicemia from Candida organisms secondary to these venous lines. Gallium 67 scintigraphy may be useful in the detection of complications of indwelling venous catheters.

Cytosolic expression of functional Fab fragments in Escherichia coli using a novel combination of dual SUMO expression cassette and EnBase® cultivation mode.

PubMed

Rezaie, F; Davami, F; Mansouri, K; Agha Amiri, S; Fazel, R; Mahdian, R; Davoudi, N; Enayati, S; Azizi, M; Khalaj, V

2017-05-08

The Escherichia coli expression system is highly effective in producing recombinant proteins. However, there are some limitations in this system, especially in obtaining correctly folded forms of some complex proteins such as Fab fragments. To improve the solubility and folding quality of Fab fragments, we have examined the effect of simultaneous application of a SUMO fusion tag, EnBase ® cultivation mode and a redox mutant strain in the E. coli expression system. A bicistronic gene construct was designed to express an antivascular endothelial growth factor (VEGF) Fab fragment as a model system. The construct contained a dual SUMO fusion gene fragment to encode SUMO-tagged heavy and light chains. While the expression of the construct in batch cultures of BL21 or SHuffle ® transformants produced insoluble and unfolded products, the induction of the transformants in EnBase ® medium resulted in soluble and correctly folded Fab fragment, reaching as high as 19% of the total protein in shuffle strain. The functional assays indicated that the biological activity of the target Fab is similar to the commercial anti-VEGF, Lucentis ® . This study demonstrated that the combination of SUMO fusion technology, EnBase ® cultivation system and recruiting a redox mutant of E. coli can efficiently enhance the solubility and productivity of recombinant Fab fragments. The presented strategy provides not only a novel method to produce soluble and active form of an anti-VEGF Fab but also may use in the efficient production of other antibody fragments. © 2017 The Society for Applied Microbiology.

Evaluation of hepatic function with (99m)Tc-galactosylated serum albumin scintigraphy in patients with malaria: comparison with (99m)Tc-colloid scintigraphy and liver ultrasonography.

PubMed

Lee, Sang-Woo; Lee, Jaetae; Lee, Deog-Young; Chun, Kyung-Ah; Ahn, Byeong-Cheol; Kang, Young-Mo; Lee, Kyubo

2007-02-01

Malarial parasites injected by the mosquito rapidly target hepatocytes, and hepatomegaly is commonly observed during the progress of the disease in malaria patients. To evaluate the degree of hepatic damage and functional status of hepatocytes in malaria patients, we performed liver scintigraphy using (99m)Tc-galactosylated serum albumin (GSA) prospectively and the findings were compared with those of (99m)Tc-colloid scintigraphy, ultrasonography and clinical results in the same subject. Eight malaria patients (all male, mean age 22 years) confirmed to be infected with Plasmodium vivax underwent (99m)Tc-GSA liver scintigraphy, followed by liver ultrasonography and (99m)Tc-colloid scintigraphy using phytate within 3 days. For hepatocyte scintigraphy, anterior images of cardiac blood-pool and liver were continuously acquired for 30 min after injection of 185 MBq (99m)Tc-GSA (3 mg). In addition to visual interpretation of the images, quantitative measurement of hepatic function was performed with several functional parameters, such as hepatic uptake index (LHL15), blood clearance index (HH15) and modified receptor index (LHL/HH) calculated from the radioactivity of the liver and heart. (99m)Tc-colloid images were assessed and graded visually. Severity of hepatic dysfunction or reticuloendothelial system activation was classified as normal, mild, moderate and severe on GSA or colloid images. Hepatomegaly was observed in five and splenomegaly in seven of the eight patients. Serum levels of transaminase and alkaline phosphatase were mildly elevated in two. Visual assessment of GSA scintigraphy revealed normal findings in all subjects, except for mild increases in size. The mean values of LHL15, HH15 and LHL/HH were 0.928+/-0.014, 0.537+/-0.031 and 1.732+/-0.106, respectively. They were graded as normal in five, and near-normal to mild dysfunction in three subjects. In contrast, (99m)Tc-colloid scintigraphy revealed abnormal findings in all of the subjects, and graded as

Chimeric rabbit/human Fab antibodies against the hepatitis Be-antigen and their potential applications in assays, characterization, and therapy.

PubMed

Zhuang, Xiaolei; Watts, Norman R; Palmer, Ira W; Kaufman, Joshua D; Dearborn, Altaira D; Trenbeath, Joni L; Eren, Elif; Steven, Alasdair C; Rader, Christoph; Wingfield, Paul T

2017-10-06

Hepatitis B virus (HBV) infection afflicts millions worldwide, causing cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a soluble variant of the viral capsid protein. HBeAg is not required for viral replication but is implicated in establishing immune tolerance and chronic infection. The structure of recombinant e-antigen (rHBeAg) was recently determined, yet to date, the exact nature and quantitation of HBeAg still remain uncertain. Here, to further characterize HBeAg, we used phage display to produce a panel of chimeric rabbit/human monoclonal antibody fragments (both Fab and scFv) against rHBeAg. Several of the Fab/scFv, expressed in Escherichia coli , had unprecedentedly high binding affinities ( K d ∼10 -12 m) and high specificity. We used Fab/scFv in the context of an enzyme-linked immunosorbent assay (ELISA) for HBeAg quantification, which we compared with commercially available kits and verified with seroconversion panels, the WHO HBeAg standard, rHBeAg, and patient plasma samples. We found that the specificity and sensitivity are superior to those of existing commercial assays. To identify potential fine differences between rHBeAg and HBeAg, we used these Fabs in microscale immunoaffinity chromatography to purify HBeAg from individual patient plasmas. Western blotting and MS results indicated that rHBeAg and HBeAg are essentially structurally identical, although HBeAg from different patients exhibits minor carboxyl-terminal heterogeneity. We discuss several potential applications for the humanized Fab/scFv.

A comparison of 111In- or 64Cu-DOTA-trastuzumab Fab fragments for imaging subcutaneous HER2-positive tumor xenografts in athymic mice using microSPECT/CT or microPET/CT.

PubMed

Chan, Conrad; Scollard, Deborah A; McLarty, Kristin; Smith, Serena; Reilly, Raymond M

2011-08-17

Our objective was to compare 111In- or 64Cu-DOTA-trastuzumab Fab fragments for imaging small or large s.c. tumor xenografts in athymic mice that display a wide range of human epidermal growth factor receptor-2 (HER2) expression using microSPECT/CT or microPET/CT. Trastuzumab Fab were labeled with 111In or 64Cu by conjugation to 1,4,7,10-tetraazacyclododecane N, N', N'', N'''-tetraacetic acid (DOTA). The purity of 111In- and 64Cu-DOTA-trastuzumab Fab was measured by SDS-PAGE and HPLC. HER2 binding affinity was determined in saturation radioligand binding assays using SKBR-3 cells (1.3 × 106 HER2/cell). MicroSPECT/CT and microPET/CT were performed in athymic mice bearing s.c. BT-20 and MDA-MB-231 xenografts with low (0.5 to 1.6 × 105 receptors/cell), MDA-MB-361 tumors with intermediate (5.1 × 105 receptors/cell) or SKOV-3 xenografts with high HER2 expression (1.2 × 106 receptors/cell) at 24 h p.i. of 70 MBq (10 μg) of 111In-DOTA-trastuzumab Fab or 22 MBq (10 μg) of 64Cu-DOTA-trastuzumab Fab or irrelevant 111In- or 64Cu-DOTA-rituximab Fab. Tumor and normal tissue uptake were quantified in biodistribution studies. 111In- and 64Cu-DOTA-trastuzumab were > 98% radiochemically pure and bound HER2 with high affinity (Kd = 20.4 ± 2.5 nM and 40.8 ± 3.5 nM, respectively). MDA-MB-361 and SKOV-3 tumors were most clearly imaged using 111In- and 64Cu-DOTA-trastuzumab Fab. Significantly higher tumor/blood (T/B) ratios were found for 111In-DOTA-trastuzumab Fab than 111In-DOTA-rituximab Fab for BT-20, MDA-MB-231 and MDA-MB-361 xenografts, and there was a direct association between T/B ratios and HER2 expression. In contrast, tumor uptake of 64Cu-DOTA-trastuzumab Fab was significantly higher than 64Cu-DOTA-rituximab Fab in MDA-MB-361 tumors but no direct association with HER2 expression was found. Both 111In- and 64Cu-DOTA-trastuzumab Fab imaged small (5 to 10 mm) or larger (10 to 15 mm) MDA-MB-361 tumors. Higher blood, liver, and spleen radioactivity were observed for 64Cu

A comparison of 111In- or 64Cu-DOTA-trastuzumab Fab fragments for imaging subcutaneous HER2-positive tumor xenografts in athymic mice using microSPECT/CT or microPET/CT

PubMed Central

2011-01-01

Background Our objective was to compare 111In- or 64Cu-DOTA-trastuzumab Fab fragments for imaging small or large s.c. tumor xenografts in athymic mice that display a wide range of human epidermal growth factor receptor-2 (HER2) expression using microSPECT/CT or microPET/CT. Methods Trastuzumab Fab were labeled with 111In or 64Cu by conjugation to 1,4,7,10-tetraazacyclododecane N, N', N'', N'''-tetraacetic acid (DOTA). The purity of 111In- and 64Cu-DOTA-trastuzumab Fab was measured by SDS-PAGE and HPLC. HER2 binding affinity was determined in saturation radioligand binding assays using SKBR-3 cells (1.3 × 106 HER2/cell). MicroSPECT/CT and microPET/CT were performed in athymic mice bearing s.c. BT-20 and MDA-MB-231 xenografts with low (0.5 to 1.6 × 105 receptors/cell), MDA-MB-361 tumors with intermediate (5.1 × 105 receptors/cell) or SKOV-3 xenografts with high HER2 expression (1.2 × 106 receptors/cell) at 24 h p.i. of 70 MBq (10 μg) of 111In-DOTA-trastuzumab Fab or 22 MBq (10 μg) of 64Cu-DOTA-trastuzumab Fab or irrelevant 111In- or 64Cu-DOTA-rituximab Fab. Tumor and normal tissue uptake were quantified in biodistribution studies. Results 111In- and 64Cu-DOTA-trastuzumab were > 98% radiochemically pure and bound HER2 with high affinity (Kd = 20.4 ± 2.5 nM and 40.8 ± 3.5 nM, respectively). MDA-MB-361 and SKOV-3 tumors were most clearly imaged using 111In- and 64Cu-DOTA-trastuzumab Fab. Significantly higher tumor/blood (T/B) ratios were found for 111In-DOTA-trastuzumab Fab than 111In-DOTA-rituximab Fab for BT-20, MDA-MB-231 and MDA-MB-361 xenografts, and there was a direct association between T/B ratios and HER2 expression. In contrast, tumor uptake of 64Cu-DOTA-trastuzumab Fab was significantly higher than 64Cu-DOTA-rituximab Fab in MDA-MB-361 tumors but no direct association with HER2 expression was found. Both 111In- and 64Cu-DOTA-trastuzumab Fab imaged small (5 to 10 mm) or larger (10 to 15 mm) MDA-MB-361 tumors. Higher blood, liver, and spleen

Rational optimization of drug-target residence time: Insights from inhibitor binding to the S. aureus FabI enzyme-product complex

PubMed Central

Chang, Andrew; Schiebel, Johannes; Yu, Weixuan; Bommineni, Gopal R.; Pan, Pan; Baxter, Michael V.; Khanna, Avinash; Sotriffer, Christoph A.; Kisker, Caroline; Tonge, Peter J.

2013-01-01

Drug-target kinetics has recently emerged as an especially important facet of the drug discovery process. In particular, prolonged drug-target residence times may confer enhanced efficacy and selectivity in the open in vivo system. However, the lack of accurate kinetic and structural data for series of congeneric compounds hinders the rational design of inhibitors with decreased off-rates. Therefore, we chose the Staphylococcus aureus enoyl-ACP reductase (saFabI) - an important target for the development of new anti-staphylococcal drugs - as a model system to rationalize and optimize the drug-target residence time on a structural basis. Using our new, efficient and widely applicable mechanistically informed kinetic approach, we obtained a full characterization of saFabI inhibition by a series of 20 diphenyl ethers complemented by a collection of 9 saFabI-inhibitor crystal structures. We identified a strong correlation between the affinities of the investigated saFabI diphenyl ether inhibitors and their corresponding residence times, which can be rationalized on a structural basis. Due to its favorable interactions with the enzyme, the residence time of our most potent compound exceeds 10 hours. In addition, we found that affinity and residence time in this system can be significantly enhanced by modifications predictable by a careful consideration of catalysis. Our study provides a blueprint for investigating and prolonging drug-target kinetics and may aid in the rational design of long-residence-time inhibitors targeting the essential saFabI enzyme. PMID:23697754

Structure of Mycobacterium tuberculosis mtFabD, a malonyl-CoA:acyl carrier protein transacylase (MCAT)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ghadbane, Hemza; Brown, Alistair K.; Kremer, Laurent

2007-10-01

Binding of Ni{sup 2+} ions to the uncleaved affinity tag facilitated de novo phasing of the crystal structure of M. tuberculosis mtFabD to 3.0 Å resolution. Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosismore » mtFabD, the mycobacterial MCAT, has been determined to 3.0 Å resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni{sup 2+} ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.« less

Nurturing Creativity and Innovation through FabKids: A Case Study

ERIC Educational Resources Information Center

Beyers, Ronald Noel

2010-01-01

This paper will report on a case study that was conducted involving Grade 10 learners who were exposed to a high-tech rapid-prototyping environment of a Fabrication Laboratory as part of a FabKids experience. This project must be viewed in the context of a global shortage of key skills placing a higher priority on the initiation and development of…

Family of Advanced Beyond Line-of-Sight Terminals (FAB-T)

DTIC Science & Technology

2015-12-01

Architecture (DoD IEA), excepting tactical and non- operational (OP) communications 3) Compliant with GIG Technical Guidance ( GTG ) to include...Information Technology (IT) standards identified in the Standards FAB-T December 2015 SAR March 21, 2016 15:24:15 UNCLASSIFIED 24 Technical Guidance ( GTG ...Availability Anti-spoofing Module (SAASM), Spectrum and Joint Tactical Radio System (JTRS) requirements. Compliant GTG to include IT standards

Identification of a new binding site in E. coli FabH using Molecular dynamics simulations: validation by computational alanine mutagenesis and docking studies.

PubMed

Ramamoorthy, Divya; Turos, Edward; Guida, Wayne C

2013-05-24

FabH (Fatty acid biosynthesis, enzyme H, also referred to as β-ketoacyl-ACP-synthase III) is a key condensing enzyme in the type II fatty acid synthesis (FAS) system. The FAS pathway in bacteria is essential for growth and survival and vastly differs from the human FAS pathway. Enzymes involved in this pathway have arisen as promising biomolecular targets for discovery of new antibacterial drugs. However, currently there are no clinical drugs that selectively target FabH, and known inhibitors of FabH all act within the active site. FabH exerts its catalytic function as a dimer, which could potentially be exploited in developing new strategies for inhibitor design. The aim of this study was to elucidate structural details of the dimer interface region by means of computational modeling, including molecular dynamics (MD) simulations, in order to derive information for the structure-based design of new FabH inhibitors. The dimer interface region was analyzed by MD simulations, trajectory snapshots were collected for further analyses, and docking studies were performed with potential small molecule disruptors. Alanine mutation and docking studies strongly suggest that the dimer interface could be a potential target for anti-infection drug discovery.

The role of bone scintigraphy in detecting child abuse.

PubMed

Conway, J J; Collins, M; Tanz, R R; Radkowski, M A; Anandappa, E; Hernandez, R; Freeman, E L

1993-10-01

This review of diagnostic imaging in cases of suspected child abuse characterizes the significant differences between bone scintigraphy and x-ray evaluation, describes the advantages and disadvantages of each modality, postulates on the specific mechanisms of injury that produce the characteristic scintigraphic findings, and emphasizes the influences that scintigraphic studies have on the medical, social, and legal aspects of child abuse. The major advantages of bone scintigraphy are its increased sensitivity (25% to 50%) in detecting evidence of soft tissue as well as bone trauma in child abuse. Furthermore, it is postulated that the specific mechanisms of inflicting the trauma relate to the patient's size and are characterized by bone scintigraphy. During fits of anger or frustration, the perpetrator of child abuse grasps the small infant or child by the thorax during the shaking activity. This produces characteristic rib injuries. The older and heavier child is more likely to be grabbed by the extremities, which produces periosteal injuries manifested as characteristic abnormal localizations in the diaphyses of the extremities. The roentgenograms of these injuries are frequently normal. The importance of bone scintigraphy is its complementary nature in defining and characterizing the extent and severity of trauma from child abuse. Such findings have direct bearing on the medical, social, and legal outcomes for the abused child. The quality of scintigraphic imaging is important, requiring the use of magnification techniques in the infant. The interpretation of the scintigraphic images depends on an understanding of the mechanisms by which the radionuclide localizes in bone. The same traumatic incident can lead to decreased, normal, or increased localization at the trauma site. Radionuclide scintigraphy is a complementary rather than competitive imaging modality to X-ray evaluation in the diagnosis and management of physical child abuse.

Role of scintigraphy in urinary tract infection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Conway, J.J.

1988-10-01

There is controversy regarding the role of radiological imaging for urinary tract infection (UTI). The gold standard has been the intravenous pyelogram (IVP). Yet, the IVP has a very limited value with only about 25% of children with pyelonephritis demonstrating abnormalities. Ultrasound (US) has recently been advocated as a replacement for the poorly sensitive and poorly specific IVP. However, comparative studies between US and IVP indicate only an equivalent sensitivity and specificity. Cortical scintigraphy with Technetium-99m glucoheptonate (99mTc GH) or 99mTc dimercaptosuccinic acid (99mTc DMSA) has also been advocated as a means of differentiating parenchymal (pyelonephritis) from nonparenchymal (lower UTI)more » involvement in UTI. The clinical presentation may be misleading especially in the infant and child in whom an elevated temperature, flank pain, shaking chills, or an elevated sedimentation rate are often lacking. The clinician attempts to localize the site of infection for it has a direct bearing upon the therapy. A collecting system infection can often be eradicated with a single oral dose of an appropriate antibiotic, whereas renal parenchymal involvement requires IV therapy for an extended interval. Cortical scintigraphy can localize the site of infection with a high degree of accuracy. Recent studies report a sensitivity of 86% and specificity of 81% of pyelonephritis. This is in contrast to the IVP with a sensitivity of only 24% and US with a sensitivity of only 42%. The scintigraphic appearance of parenchymal infection of the kidney is a spectrum of minimal to gross defects reflecting the degree of histologic involvement that spans from a mild infection to frank abscess. Cortical scintigraphy can be used to monitor the evolution of scarring following infection. Cortical scintigraphy with 99mTc DMSA or 99mTc GH is the method of choice for the initial evaluation of UTI. 37 references.« less

Optimization of a protocol for myocardial perfusion scintigraphy by using an anthropomorphic phantom.

PubMed

Ramos, Susie Medeiros Oliveira; Glavam, Adriana Pereira; Kubo, Tadeu Takao Almodovar; de Sá, Lidia Vasconcellos

2014-01-01

To develop a study aiming at optimizing myocardial perfusion imaging. Imaging of an anthropomorphic thorax phantom with a GE SPECT Ventri gamma camera, with varied activities and acquisition times, in order to evaluate the influence of these parameters on the quality of the reconstructed medical images. The (99m)Tc-sestamibi radiotracer was utilized, and then the images were clinically evaluated on the basis of data such as summed stress score, and on the technical image quality and perfusion. The software ImageJ was utilized in the data quantification. The results demonstrated that for the standard acquisition time utilized in the procedure (15 seconds per angle), the injected activity could be reduced by 33.34%. Additionally, even if the standard scan time is reduced by 53.34% (7 seconds per angle), the standard injected activity could still be reduced by 16.67%, without impairing the image quality and the diagnostic reliability. The described method and respective results provide a basis for the development of a clinical trial of patients in an optimized protocol.

Optimization of a protocol for myocardial perfusion scintigraphy by using an anthropomorphic phantom*

PubMed Central

Ramos, Susie Medeiros Oliveira; Glavam, Adriana Pereira; Kubo, Tadeu Takao Almodovar; de Sá, Lidia Vasconcellos

2014-01-01

Objective To develop a study aiming at optimizing myocardial perfusion imaging. Materials and Methods Imaging of an anthropomorphic thorax phantom with a GE SPECT Ventri gamma camera, with varied activities and acquisition times, in order to evaluate the influence of these parameters on the quality of the reconstructed medical images. The 99mTc-sestamibi radiotracer was utilized, and then the images were clinically evaluated on the basis of data such as summed stress score, and on the technical image quality and perfusion. The software ImageJ was utilized in the data quantification. Results The results demonstrated that for the standard acquisition time utilized in the procedure (15 seconds per angle), the injected activity could be reduced by 33.34%. Additionally, even if the standard scan time is reduced by 53.34% (7 seconds per angle), the standard injected activity could still be reduced by 16.67%, without impairing the image quality and the diagnostic reliability. Conclusion The described method and respective results provide a basis for the development of a clinical trial of patients in an optimized protocol. PMID:25741088

The use of pre-test and post-test probability values as criteria before selecting patients to undergo coronary angiography in patients who have ischemic findings on myocardial perfusion scintigraphy.

PubMed

Karahan Şen, Nazlı Pınar; Bekiş, Recep; Ceylan, Ali; Derebek, Erkan

2016-07-01

Myocardial perfusion scintigraphy (MPS) is a diagnostic test which is frequently used in the diagnosis of coronary heart disease (CHD). MPS is generally interpreted as ischemia present or absent; however, it has a power in predicting the disease, similar to other diagnostic tests. In this study, we aimed to assist in directing the high-risk patients to undergo coronary angiography (CA) primarily by evaluating patients without prior CHD history with pre-test and post-test probabilities. The study was designed as a retrospective study. Between January 2008 and July 2011, 139 patients with positive MPS results and followed by CA recently (<6 months) were evaluated from patient files. Patients' pre-test probabilities based on the Diamond and Forrester method and the likelihood ratios that were obtained from the literature were used to calculate the patients' post exercise and post-MPS probabilities. Patients were evaluated in risk groups as low, intermediate, and high, and an ROC curve analysis was performed for the post-MPS probabilities. Coronary artery stenosis (CAS) was determined in 59 patients (42.4%). A significant difference was determined between the risk groups according to CAS, both for the pre-test and post-test probabilities (p<0.001, p=0.024). The ROC analysis provided a cut-off value of 80.4% for post- MPS probability in predicting CAS with 67.9% sensitivity and 77.8% specificity. When the post-MPS probability is ≥80% in patients who have reversible perfusion defects on MPS, we suggest interpreting the MPS as "high probability positive" to improve the selection of true-positive patients to undergo CA, and these patients should be primarily recommended CA.

Generation of human Fab antibody libraries: PCR amplification and assembly of light- and heavy-chain coding sequences.

PubMed

Andris-Widhopf, Jennifer; Steinberger, Peter; Fuller, Roberta; Rader, Christoph; Barbas, Carlos F

2011-09-01

The development of therapeutic antibodies for use in the treatment of human diseases has long been a goal for many researchers in the antibody field. One way to obtain these antibodies is through phage-display libraries constructed from human lymphocytes. This protocol describes the construction of human Fab (fragment antigen binding) antibody libraries. In this method, the individual rearranged heavy- and light-chain variable regions are amplified separately and are linked through a series of overlap polymerase chain reaction (PCR) steps to give the final Fab products that are used for cloning.

Crotalidae polyvalent immune Fab antivenom limits the decrease in perfusion pressure of the anterior leg compartment in a porcine crotaline envenomation model.

PubMed

Tanen, David A; Danish, David C; Clark, Richard F

2003-03-01

Crotalidae Polyvalent Immune Fab (CroFab; FabAV) antivenom prevents a decrease in perfusion pressures in intramuscular crotaline envenomation compared with normal saline solution. We used a randomized, blinded, controlled acute animal preparation. Twenty anesthetized and instrumented swine were injected intramuscularly with 6 mg/kg Crotalus atrox venom into the anterior tibialis muscle of each hind limb (time 0). One hour after envenomation (time 1 hour), animals were randomized to receive 8 vials of reconstituted FabAV or an equal volume of normal saline solution (control) through a central venous line. The main outcome variable was the area under the perfusion-time curve (AUC) of the anterior compartment of the hind limb measured from time 1 hour to time 8 hours. Perfusion pressure was defined as mean arterial pressure-compartment pressure. Additionally, physiologic variables, including pulse rate, prothrombin time, fibrinogen level, platelet count, hemoglobin level, and hematocrit level, were monitored. Venom injection resulted in a decrease in average perfusion pressures from 54.1 mm Hg (time 0) to 31.7 mm Hg (time 1 hour). Comparison of AUC between groups from time 1 hour (time of treatment) to the completion of the study at time 8 hours revealed a 57% greater AUC in animals that received FabAV (mean+/-SD: 211.1+/-67.9 versus 134.5+/-55.8 mm Hg/h; P =.036; 95% confidence interval for difference 5.9 to 147.3). Comparison of the time curves for the mean prothrombin time from time 1 hour to the completion of the study by means of repeated-measures analysis of variance revealed a significant increase in the control group (P =.02). No significant difference was detected in the time curves for the means of mean arterial pressure, compartment pressure, pulse rate, hemoglobin level, hematocrit level, fibrinogen level, or platelet count over the course of the study. FabAV was found to significantly increase survival time when compared with the effect of the normal

[Gastrointestinal bleeding from capillary hemangioma of the ileum detected by 99mTc-HSAD scintigraphy].

PubMed

Asano, Y; Ishii, K; Sagiuchi, T; Aoki, Y; Yanaihara, H; Hayakawa, K

2001-05-01

It has been well-known that technetium-99m-human serum albumin-diethylenetriaminepenta-acetic acid (99mTc-HSAD) scintigraphy is useful for diagnosis of the localization of the gastrointestinal arterial or venous bleeding. In this report, we describe a case of venous bleeding from capillary hemangioma of the ileum end detected by 99mTc-HSAD scintigraphy. This patient was a 9-year-old girl with severe anemia. Gastrointestinal bleeding was suspected from her clinical course and laboratory tests. Immediately after melena occurred, 99mTc-HSAD scintigraphy showed the extravasation of RI suggesting gastrointestinal bleeding in the ileum end. Abdominal angiography immediately after 99mTc-HSAD scintigraphy, however, could not show the extravasation of contrast agent. Because the condition of the patient became worse, laparotomy was performed on the basis of 99mTc-HSAD scintigraphy findings. At surgery, venous bleeding from capillary hemangioma in the ileum end was observed. It was suggested that 99mTc-HSAD scintigraphy was very useful for identifying the gastrointestinal venous bleeding.

Somatostatin receptor scintigraphy in patients with cat-scratch disease.

PubMed

Krause, R; Piswanger-Soelkner, C; Lipp, R W; Daxböck, F; Schnedl, W J; Hoier, S; Reisinger, E C

2006-01-01

Somatostatin receptor scintigraphy images various neoplastic, granulomatous, and auto-immune diseases. Cat-scratch disease in an infectious granulomatous disease usually affecting the lymphnodes. It is not known whether cat-scratch disease provides positive somatostatin receptor scintigrams. Twelve patients with lymphadenitis and suspected cat-scratch disease were investigated by immunofluorescence antibody testing and somatostatin receptor scintigraphy. Suppurated lymphnodes were extracted or drained and Bartonella henselae specific PCR was then performed. Eleven of 12 patients showed IgG antibodies against B. henselae. SRS showed positive scintigraphic results in 6 of 11 patients with CSD. B. henselae DNA was detected in tissue of lymphnodes from 4 of 5 patients with lymphnode extraction or lymphnode drainage. SRS demonstrated positive scintigrams in all patients with a positive PCR. In one patient with suspected CSD SRS was negative as well as antibody testing. Somatostatin receptor scintigraphy correlated with positive Bartonella henselae specific PCR tests and positive Bartonella henselae specific antibody tests in patients with CSD.

AIDS-related Kaposi sarcoma: findings on thallium-201 scintigraphy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, V.W.; Rosen, M.P.; Baum, A.

1988-12-01

No simple, noninvasive method is available for evaluating extracutaneous Kaposi sarcoma in AIDS patients or for following the tumor's response to treatment. We report our preliminary experience with thallium-201 scintigraphy in nine AIDS patients with proved Kaposi sarcoma. Eight of the nine had abnormal uptake of the radionuclide in skin, lymph nodes, oral cavity, vagina, and lungs. Only four of the nine had cutaneous Kaposi sarcoma at the time of scanning. All cutaneous and mucosal lesions were thallium avid. Two of the six patients with thallium-avid nodes underwent nodal biopsy. Both biopsies confirmed the diagnosis of Kaposi sarcoma. Cutaneous Kaposimore » sarcoma developed later in one of these patients, showing the efficacy of thallium scintigraphy for the early detection of extracutaneous lesions. These preliminary results show thallium avidity in Kaposi sarcoma involving the skin and various extracutaneous sites (lymph nodes, lung, mucosa, and vagina). Thallium scintigraphy is a potentially useful procedure for detecting extracutaneous Kaposi sarcoma in AIDS patients.« less

Nuclear cardiograph and scintigraphy

NASA Technical Reports Server (NTRS)

Mclaughlin, P.

1975-01-01

Extensive advances in the technology of detectors, data analysis systems, and tracers used have resulted in greatly expanded applications of radioisotopes to the assessment of cardiac function and disease. The development of nuclear cardiology has proceeded along four lines: (1) radionuclide angiography, (2) myocardial perfusion imaging, (3) intracoronary microsphere imaging, and (4) regional myocardial blood flow determination using inert gases.

Antistaphylococcal activities of CG400549, a new bacterial enoyl-acyl carrier protein reductase (FabI) inhibitor.

PubMed

Park, Hee Soo; Yoon, Yu Min; Jung, Sung Ji; Kim, Cheol Min; Kim, Jeong Mi; Kwak, Jin-Hwan

2007-09-01

This study was performed to analyse in vitro and in vivo activities of CG400549, a new FabI inhibitor, against clinical isolates of staphylococci. The mode of action of CG400549 and resistance mechanism of Staphylococcus aureus against CG400549 were also investigated by genetic approaches. In vitro activity of CG400549 was evaluated by the 2-fold agar sdilution method as described by the CLSI, and compared with those of oxacillin, erythromycin, ciprofloxacin, sparfloxacin, moxifloxacin, gemifloxacin, vancomycin, linezolid and quinupristin-dalfopristin. In vivo activity of CG400549 was determined against systemic infections in mice. Time-kill curves of CG400549 were analysed at concentrations of 1 x , 2 x and 4 x MIC against S. aureus strains. CG400549 had the lowest MICs among the test compounds against 238 clinical isolates of S. aureus (MIC90, 0.25 mg/L) and 51 clinical isolates of coagulase-negative staphylococci (MIC90, 1 mg/L). The activity of CG400549 was irrespective of whether the strains were methicillin-susceptible or -resistant. Furthermore, CG400549 was effective by oral or subcutaneous administration against systemic infections in mice. In a time-kill study, CG400549 at concentrations of 1 x MIC, 2 x MIC and 4 x MIC had a bacteriostatic activity during 24 h. A FabI-overexpressing S. aureus strain gave rise to an increase in the MIC of CG400549 compared with the parental strain, while the susceptibilities of the FabI-overexpressing S. aureus strain to the other antibacterial agents such as oxacillin, erythromycin and ciprofloxacin were not affected. This result showed that the mode of action of CG400549 was via inhibition of FabI, which is involved in biosynthesis of fatty acids in bacteria. Study of the resistance mechanism of S. aureus showed that CG400549-resistant mutants of S. aureus had an alteration in FabI at Phe-204 to Leu. CG400549 had potent in vitro and in vivo activity against staphylococci, including methicillin-, ciprofloxacin- and

Fab-based inhibitors reveal ubiquitin independent functions for HIV Vif neutralization of APOBEC3 restriction factors

PubMed Central

Smith, Amber M.; Hultquist, Judd F.; Caretta Cartozo, Nathalie; Campbell, Melody G.; Burton, Lily; La Greca, Florencia; McGregor, Michael J.; Ta, Hai M.; Bartholomeeusen, Koen; Peterlin, B. Matija; Krogan, Nevan J.; Sevillano, Natalia

2018-01-01

The lentiviral protein Viral Infectivity Factor (Vif) counteracts the antiviral effects of host APOBEC3 (A3) proteins and contributes to persistent HIV infection. Vif targets A3 restriction factors for ubiquitination and proteasomal degradation by recruiting them to a multi-protein ubiquitin E3 ligase complex. Here, we describe a degradation-independent mechanism of Vif-mediated antagonism that was revealed through detailed structure-function studies of antibody antigen-binding fragments (Fabs) to the Vif complex. Two Fabs were found to inhibit Vif-mediated A3 neutralization through distinct mechanisms: shielding A3 from ubiquitin transfer and blocking Vif E3 assembly. Combined biochemical, cell biological and structural studies reveal that disruption of Vif E3 assembly inhibited A3 ubiquitination but was not sufficient to restore its packaging into viral particles and antiviral activity. These observations establish that Vif can neutralize A3 family members in a degradation-independent manner. Additionally, this work highlights the potential of Fabs as functional probes, and illuminates how Vif uses a multi-pronged approach involving both degradation dependent and independent mechanisms to suppress A3 innate immunity. PMID:29304101

Quantitative bone and 67Ga scintigraphy in the differentiation of necrotizing external otitis from severe external otitis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Uri, N.; Gips, S.; Front, A.

1991-06-01

Qualitative technetium Tc 99m bone scintigraphy using phosphate compounds and gallium 67 scintigraphy were described as a helpful means in diagnosing necrotizing external otitis (NEO). They were, however, claimed to be nonspecific. Quantitative Tc 99m methylene diphosphonate bone scintigraphy and gallium 67 scintigraphy were performed in eight patients with NEO and in 20 patients with severe external otitis, in order to prove usefulness of scintigraphy in the diagnosis of NEO. Ratios of lesion to nonlesion for bone scintigraphy were 1.67 {plus minus} 0.16 in patients with NEO and 1.08 {plus minus} 0.09 in patients with severe external otitis, and formore » gallium 67 scintigraphy they were 1.35 {plus minus} 0.24 in NEO patients and 1.05 {plus minus} 0.03 in patients with severe external otitis. There was no difference in uptake between diabetic patients with severe external otitis and nondiabetic patients. The scintigraphic studies were also evaluated using a qualitative scoring method (scores 0 to +4), according to the intensity of the radiopharmaceutical uptake. This method was found to be inferior in the diagnosis of NEO compared with the quantitative method. We conclude that lesion-to-nonlesion ratios greater than 1.5 and 1.3 on bone and gallium 67 scintigraphy, respectively, are indicative of NEO. Quantitative bone scintigraphy, which is quicker to perform, may be used as a single imaging modality for the diagnosis of NEO.« less

(99m)Tc-HYNIC-TOC scintigraphy in evaluation of active Graves' ophthalmopathy (GO).

PubMed

Sun, Hua; Jiang, Xu-Feng; Wang, Shu; Chen, Hao-Yan; Sun, Jiao; Li, Pei-Yong; Ning, Guang; Zhao, Yong-Ju

2007-06-01

A promising radiopharmaceutical (99m)Tc-HYNIC-TOC ((99m)Tc-HYNIC-Octreotide) can be applied for somatostatin receptor scintigraphy with the potential to replace Indium-111 labeled somatostatin analogus. Here we evaluate whether orbital (99m)Tc-HYNIC-TOC scintigraphy can be used as a Graves' ophthalmopathy (GO) activity parameter to predict the retrobulbar irradiation response. Orbital (99m)Tc-HYNIC-TOC scintigraphy was performed on 14 consecutive patients demonstrating moderated to severe Graves' ophthalmopathy. The patients were treated with retrobulbar irradiation following the octreoscan and the response to this therapy was assessed at 3 months after the start of treatment. The orbital (99m)Tc-HYNIC-TOC uptake was calculated to assess the effects of treatment. Among the 14 GO patients, eight (57.1%) responded to retrobulbar radiotherapy; six (42.9%) showed no change. We compared the eight responders and six non-responders in terms of orbital (99m)Tc-HYNIC-TOC uptake, using the orbital/occipital ratio. On the 4-h (99m)Tc-HYNIC-TOC scintigraphy, responders had a higher orbital/occipital uptake ratio than the no-responders (P = 0.001). A significant correlation was found between the orbital/occipital ratio and the clinical activity score (CAS) (P = 0.034). The Receiving-Operator-Characteristic curve showed the best threshold for discriminating active and inactive disease was 1.40 (sensitivity, 100%; specificity, 83.3%). In the responders group, all these eight patients had positive scintigraphy. While there were five patients who had negative scintigraphy in the non-responders group. Orbital (99m)Tc-HYNIC-TOC scintigraphy can be a useful method for the estimation of disease activity and prediction the response to subsequent radiotherapy in GO patient. And the patients with positive octreoscan were more likely to respond to irradiation.

Impact of Fab Lab Tulsa on Student Self-Efficacy toward STEM Education

ERIC Educational Resources Information Center

Dubriwny, Nicholas; Pritchett, Nathan; Hardesty, Michelle; Hellman, Chan M.

2016-01-01

Student self-confidence is important to any attempt to increase interest and achievement in Science, Technology, Engineering, and Math (STEM) education. This study presents a longitudinal examination of Fab Lab Tulsa's impact on attitude and self-efficacy toward STEM education among middle-school aged students. Paired samples t-test showed a…

Systematic review of cost-effectiveness of myocardial perfusion scintigraphy in patients with ischaemic heart disease: A report from the cardiovascular committee of the European Association of Nuclear Medicine. Endorsed by the European Association of Cardiovascular Imaging.

PubMed

Trägårdh, Elin; Tan, Siok Swan; Bucerius, Jan; Gimelli, Alessia; Gaemperli, Oliver; Lindner, Oliver; Agostini, Denis; Übleis, Christopher; Sciagrà, Roberto; Slart, Riemer H; Underwood, S Richard; Hyafil, Fabien; Hacker, Marcus; Verberne, Hein J

2017-05-01

Coronary artery disease (CAD) is a major cause of death and disability. Several diagnostic tests, such as myocardial perfusion scintigraphy (MPS), are accurate for the detection of CAD, as well as having prognostic value for the prediction of cardiovascular events. Nevertheless, the diagnostic and prognostic value of these tests should be cost-effective and should lead to improved clinical outcome. We have reviewed the literature on the cost-effectiveness of MPS in different circumstances: (i) the diagnosis and management of CAD; (ii) comparison with exercise electrocardiography (ECG) and other imaging tests; (iii) as gatekeeper to invasive coronary angiography (ICA), (iv) the impact of appropriate use criteria; (v) acute chest pain, and (vi) screening of asymptomatic patients with type-2 diabetes. In total 57 reports were included. Although most non-invasive imaging tests are cost-effective compared with alternatives, the data conflict on which non-invasive strategy is the most cost-effective. Different definitions of cost-effectiveness further confound the subject. Computer simulations of clinical diagnosis and management are influenced by the assumptions made. For instance, diagnostic accuracy is often defined against an anatomical standard that is wrongly assumed to be perfect. Conflicting data arise most commonly from these incorrect or differing assumptions. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.

On-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry using Fab´antibody fragments for the analysis of serum transthyretin.

PubMed

Pont, Laura; Benavente, Fernando; Barbosa, José; Sanz-Nebot, Victoria

2017-08-01

This paper describes an on-line immunoaffinity solid-phase extraction capillary electrophoresis mass spectrometry (IA-SPE-CE-MS) method using an immunoaffinity sorbent with Fab' antibody fragments (Fab'-IA) for the analysis of serum transthyretin (TTR), a homotetrameric protein (M r ~56,000) involved in different types of amyloidosis. The IA sorbent was prepared by covalent attachment of Fab' fragments obtained from a polyclonal IgG antibody against TTR to succinimidyl silica particles. The Fab'-IA-SPE-CE-MS methodology was first established analyzing TTR standard solutions. Under optimized conditions, repeatability and reproducibility were acceptable, the method was linear between 1 and 25µgmL -1 , limits of detection (LODs) were around 0.5µgmL -1 (50-fold lower than by CE-MS, ~25µgmL -1 ) and different TTR conformations were observed (folded and unfolded). The applicability of the developed method to screen for familial amyloidotic polyneuropathy type I (FAP-I), which is the most common hereditary systemic amyloidosis, was evaluated analyzing serum samples from healthy controls and FAP-I patients. For the analysis of sera, the most abundant proteins were precipitated with 5% (v/v) of phenol before Fab'-IA-SPE-CE-MS. The current method enhanced our previous results for the analysis of TTR using intact antibodies immobilized on magnetic beads. It allowed a slight improvement on LODs (2-fold), the detection of proteoforms found at lower concentrations and the preparation of microcartridges with extended durability. Copyright © 2017. Published by Elsevier B.V.

Extending the half-life of a fab fragment through generation of a humanized anti-human serum albumin Fv domain: An investigation into the correlation between affinity and serum half-life.

PubMed

Adams, Ralph; Griffin, Laura; Compson, Joanne E; Jairaj, Mark; Baker, Terry; Ceska, Tom; West, Shauna; Zaccheo, Oliver; Davé, Emma; Lawson, Alastair Dg; Humphreys, David P; Heywood, Sam

2016-10-01

We generated an anti-albumin antibody, CA645, to link its Fv domain to an antigen-binding fragment (Fab), thereby extending the serum half-life of the Fab. CA645 was demonstrated to bind human, cynomolgus, and mouse serum albumin with similar affinity (1-7 nM), and to bind human serum albumin (HSA) when it is in complex with common known ligands. Importantly for half-life extension, CA645 binds HSA with similar affinity within the physiologically relevant range of pH 5.0 - pH 7.4, and does not have a deleterious effect on the binding of HSA to neonatal Fc receptor (FcRn). A crystal structure of humanized CA645 Fab in complex with HSA was solved and showed that CA645 Fab binds to domain II of HSA. Superimposition with the crystal structure of FcRn bound to HSA confirmed that CA645 does not block HSA binding to FcRn. In mice, the serum half-life of humanized CA645 Fab is 84.2 h. This is a significant extension in comparison with < 1 h for a non-HSA binding CA645 Fab variant. The Fab-HSA structure was used to design a series of mutants with reduced affinity to investigate the correlation between the affinity for albumin and serum half-life. Reduction in the affinity for MSA by 144-fold from 2.2 nM to 316 nM had no effect on serum half-life. Strikingly, despite a reduction in affinity to 62 µM, an extension in serum half-life of 26.4 h was still obtained. CA645 Fab and the CA645 Fab-HSA complex have been deposited in the Protein Data Bank (PDB) with accession codes, 5FUZ and 5FUO, respectively.

Hybrid Vision-Fusion system for whole-body scintigraphy.

PubMed

Barjaktarović, Marko; Janković, Milica M; Jeremić, Marija; Matović, Milovan

2018-05-01

Radioiodine therapy in the treatment of differentiated thyroid carcinoma (DTC) is used in clinical practice for the ablation of thyroid residues and/or destruction of tumour tissue. Whole-body scintigraphy for visualization of the spatial 131I distribution performed by a gamma camera (GC) is a standard procedure in DTC patients after application of radioiodine therapy. A common problem is the precise topographic localization of regions where radioiodine is accumulated even in SPECT imaging. SPECT/CT can provide precise topographic localization of regions where radioiodine is accumulated, but it is often unavailable, especially in developing countries because of the high price of the equipment. In this paper, we present a Vision-Fusion system as an affordable solution for 1) acquiring an optical whole-body image during routine whole-body scintigraphy and 2) fusing gamma and optical images (also available for the auto-contour mode of GC). The estimated prediction error for image registration is 1.84 mm. The validity of fusing was tested by performing simultaneous optical and scintigraphy image acquisition of the bar phantom. The fusion result shows that the fusing process has a slight influence and is lower than the spatial resolution of GC (mean value ± standard deviation: 1.24 ± 0.22 mm). The Vision-Fusion system was used for radioiodine post-therapeutic treatment, and 17 patients were followed (11 women and 6 men, with an average age of 48.18 ± 13.27 years). Visual inspection showed no misregistration. Based on our first clinical experience, we noticed that the Vision-Fusion system could be very useful for improving the diagnostic possibility of whole-body scintigraphy after radioiodine therapy. Additionally, the proposed Vision-Fusion software can be used as an upgrade for any GC to improve localizations of thyroid/tumour tissue. Copyright © 2018 Elsevier Ltd. All rights reserved.

The use of pre-test and post-test probability values as criteria before selecting patients to undergo coronary angiography in patients who have ischemic findings on myocardial perfusion scintigraphy

PubMed Central

Şen, Nazlı Pınar Karahan; Bekiş, Recep; Ceylan, Ali; Derebek, Erkan

2016-01-01

Objective: Myocardial perfusion scintigraphy (MPS) is a diagnostic test which is frequently used in the diagnosis of coronary heart disease (CHD). MPS is generally interpreted as ischemia present or absent; however, it has a power in predicting the disease, similar to other diagnostic tests. In this study, we aimed to assist in directing the high-risk patients to undergo coronary angiography (CA) primarily by evaluating patients without prior CHD history with pre-test and post-test probabilities. Methods: The study was designed as a retrospective study. Between January 2008 and July 2011, 139 patients with positive MPS results and followed by CA recently (<6 months) were evaluated from patient files. Patients’ pre-test probabilities based on the Diamond and Forrester method and the likelihood ratios that were obtained from the literature were used to calculate the patients’ post-exercise and post-MPS probabilities. Patients were evaluated in risk groups as low, intermediate, and high, and an ROC curve analysis was performed for the post-MPS probabilities. Results: Coronary artery stenosis (CAS) was determined in 59 patients (42.4%). A significant difference was determined between the risk groups according to CAS, both for the pre-test and post-test probabilities (p<0.001, p=0.024). The ROC analysis provided a cut-off value of 80.4% for post-MPS probability in predicting CAS with 67.9% sensitivity and 77.8% specificity. Conclusion: When the post-MPS probability is ≥80% in patients who have reversible perfusion defects on MPS, we suggest interpreting the MPS as “high probability positive” to improve the selection of true-positive patients to undergo CA, and these patients should be primarily recommended CA. PMID:27004704

Functional and proteomic comparison of different techniques to produce equine anti-tetanus immunoglobulin F(ab')2 fragments.

PubMed

Zhang, Xue-Jun; Li, Hai-Ling; Deng, Da-Yi; Ji, Chong; Yao, Xiao-Dong; Liu, Jia-Xin

2018-05-29

Tetanus is still a major cause of human deaths in several developing countries. In particular, the neonatal form remains a significant public health problem. According to the World Health Organization, administration of tetanus toxoid is recommended for neonatal tetanus patients. Furthermore, tetanus antitoxin or anti-tetanus immunoglobulin (Ig) are used for mild case or intensive care. This paper discusses a novel purification technique for improving equine anti-tetanus Ig production. First, equine plasma dealt with two steps salting out with ammonium sulfate; second, ultrafiltration concentration liquid purified by one successive protein G based affinity chromatography steps; finally, the purified F(ab')2 fragments was characterized using biochemical and proteomic methods and shown to be pure and homogeneous. Compared with the original technique product, specific activity increased by 80% (about 90,000 IU/g) and recovery of F(ab')2 is approximately equal 75%. Furthermore, Proteomic profiling of total technique process is demonstrated by nano-HPLC-MS and bioinformatics analysis. New technique to produce equine anti-tetanus immunoglobulin F(ab')2 fragments from crude plasma in high quality and yield. And it also could be used for industrial amplification. Copyright © 2018 Elsevier B.V. All rights reserved.

Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria*

PubMed Central

Yao, Jiangwei; Bruhn, David F.; Frank, Matthew W.; Lee, Richard E.; Rock, Charles O.

2016-01-01

Neisseria is a Gram-negative pathogen with phospholipids composed of straight chain saturated and monounsaturated fatty acids, the ability to incorporate exogenous fatty acids, and lipopolysaccharides that are not essential. The FabI inhibitor, AFN-1252, was deployed as a chemical biology tool to determine whether Neisseria can bypass the inhibition of fatty acid synthesis by incorporating exogenous fatty acids. Neisseria encodes a functional FabI that was potently inhibited by AFN-1252. AFN-1252 caused a dose-dependent inhibition of fatty acid synthesis in growing Neisseria, a delayed inhibition of growth phenotype, and minimal inhibition of DNA, RNA, and protein synthesis, showing that its mode of action is through inhibiting fatty acid synthesis. Isotopic fatty acid labeling experiments showed that Neisseria encodes the ability to incorporate exogenous fatty acids into its phospholipids by an acyl-acyl carrier protein-dependent pathway. However, AFN-1252 remained an effective antibacterial when Neisseria were supplemented with exogenous fatty acids. These results demonstrate that extracellular fatty acids are activated by an acyl-acyl carrier protein synthetase (AasN) and validate type II fatty acid synthesis (FabI) as a therapeutic target against Neisseria. PMID:26567338

Stable expression and purification of a functional processed Fab' fragment from a single nascent polypeptide in CHO cells expressing the mCAT-1 retroviral receptor.

PubMed

Camper, Nicolas; Byrne, Teresa; Burden, Roberta E; Lowry, Jenny; Gray, Breena; Johnston, James A; Migaud, Marie E; Olwill, Shane A; Buick, Richard J; Scott, Christopher J

2011-09-30

Monoclonal antibodies and derivative formats such as Fab' fragments are used in a broad range of therapeutic, diagnostic and research applications. New systems and methodologies that can improve the production of these proteins are consequently of much interest. Here we present a novel approach for the rapid production of processed Fab' fragments in a CHO cell line that has been engineered to express the mouse cationic amino acid transporter receptor 1 (mCAT-1). This facilitated the introduction of the target antibody gene through retroviral transfection, rapidly producing stable expression. Using this system, we designed a single retroviral vector construct for the expression of a target Fab' fragment as a single polypeptide with a furin cleavage site and a FMDV 2A self-cleaving peptide introduced to bridge the light and truncated heavy chain regions. The introduction of these cleavage motifs ensured equimolar expression and processing of the heavy and light domains as exemplified by the production of an active chimeric Fab' fragment against the Fas receptor, routinely expressed in 1-2mg/L yield in spinner-flask cell cultures. These results demonstrate that this method could have application in the facile production of bioactive Fab' fragments. Copyright © 2011 Elsevier B.V. All rights reserved.

Effect of a hydrophilic and a hydrophobic statin on cardiac salvage after ST-elevated acute myocardial infarction - a pilot study.

PubMed

Chitose, Tadasuke; Sugiyama, Seigo; Sakamoto, Kenji; Shimomura, Hideki; Yamashita, Takuro; Hokamaki, Jun; Tsunoda, Ryusuke; Shiraishi, Shinya; Yamashita, Yasuyuki; Ogawa, Hisao

2014-11-01

Early statin therapy after acute coronary syndrome reduces atherothrombotic vascular events. This study aimed to compare the effects of hydrophilic and hydrophobic statins on myocardial salvage and left ventricular (LV) function in patients with ST-elevated myocardial infarction (STEMI). Seventy-five STEMI patients who had received emergency reperfusion therapy were enrolled and randomized into the hydrophilic statin group (rosuvastatin; 5 mg/day, n = 38) and hydrophobic statin group (atorvastatin; 10 mg/day, n = 37) for 6 months. LV ejection fraction (LVEF), and B-type natriuretic peptide (BNP) and co-enzyme Q10 (CoQ10) levels were measured at baseline and the end of treatment. The myocardial salvage index was assessed by single photon emission computed tomography with (123-)I-β-methyl-iodophenylpentadecanoic acid (ischemic area-at-risk at onset of STEMI: AAR) and (201-)thallium scintigraphy (area-at-infarction at 6 months: AAI) [myocardial salvage index = (AAR-AAI) × 100/AAR (%)]. Onset-to-balloon time and maximum creatine phosphokinase levels were comparable between the groups. After 6 months, rosuvastatin (-37.6% ± 17.2%) and atorvastatin (-32.4% ± 22.4%) equally reduced low-density lipoprotein-cholesterol (LDL-C) levels (p = 0.28). However, rosuvastatin (+3.1% ± 5.9%, p < 0.05), but not atorvastatin (+1.6% ± 5.7%, p = 0.15), improved LVEF. Rosuvastatin reduced BNP levels compared with atorvastatin (-53.3% ± 48.8% versus -13.8% ± 82.9%, p < 0.05). The myocardial salvage index was significantly higher in the rosuvastatin group than the atorvastatin group (78.6% ± 29.1% versus 52.5% ± 38.0%, p < 0.05). CoQ10/LDL-C levels at 6 months were increased in the rosuvastatin group (+23.5%, p < 0.01) and percent changes in CoQ10/LDL-C were correlated with the myocardial salvage index (r = 0.56, p < 0.01). Rosuvastatin shows better beneficial effects on myocardial salvage than atorvastatin in STEMI patients, including long-term cardiac function, associated with

Crystal structure of FabZ-ACP complex reveals a dynamic seesaw-like catalytic mechanism of dehydratase in fatty acid biosynthesis.

PubMed

Zhang, Lin; Xiao, Jianfeng; Xu, Jianrong; Fu, Tianran; Cao, Zhiwei; Zhu, Liang; Chen, Hong-Zhuan; Shen, Xu; Jiang, Hualiang; Zhang, Liang

2016-12-01

Fatty acid biosynthesis (FAS) is a vital process in cells. Fatty acids are essential for cell assembly and cellular metabolism. Abnormal FAS directly correlates with cell growth delay and human diseases, such as metabolic syndromes and various cancers. The FAS system utilizes an acyl carrier protein (ACP) as a transporter to stabilize and shuttle the growing fatty acid chain throughout enzymatic modules for stepwise catalysis. Studying the interactions between enzymatic modules and ACP is, therefore, critical for understanding the biological function of the FAS system. However, the information remains unclear due to the high flexibility of ACP and its weak interaction with enzymatic modules. We present here a 2.55 Å crystal structure of type II FAS dehydratase FabZ in complex with holo-ACP, which exhibits a highly symmetrical FabZ hexamer-ACP 3 stoichiometry with each ACP binding to a FabZ dimer subunit. Further structural analysis, together with biophysical and computational results, reveals a novel dynamic seesaw-like ACP binding and catalysis mechanism for the dehydratase module in the FAS system, which is regulated by a critical gatekeeper residue (Tyr100 in FabZ) that manipulates the movements of the β-sheet layer. These findings improve the general understanding of the dehydration process in the FAS system and will potentially facilitate drug and therapeutic design for diseases associated with abnormalities in FAS.

Prognostic significance of normal quantitative Tl-201 stress scintigraphy in relation to pretest likelihood of coronary artery disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Russo, D.J.; Russo, D.; Clements, J.

A previous study reported an excellent prognosis and low cardiac event rate in patients(pts) with chest pain and normal quantitative T1-201 scintigraphy(SC). Such result would not be unexpected if the population under study had a predominance of pts with low pre-T1-201 likelihood(L) of significant coronary artery disease (CAD). Hence, the authors undertook telephone follow-up in pts with chest pain syndrome and normal quantitative T1-201 exercise SC, and related outcome to pretest L of CAD. Pretest L was determined by serial L analysis on the basis of: symptoms, age, sex, and exercise ECG. All pts had T1-201 SC immediately post exercisemore » and 2 hrs later. After interpolative background correction, circumferential count and washout profiles were generated. All pts had unequivocally normal studies. Of a total of 96 pts studied in 1981-82, 20 pts were lost to follow-up. Of the remaining 76 pts, 45 were males and 31 females. The pretest L of CAD had an inverted Gaussian distribution: thirty-four pts(47%) had <33% L of CAD, 3 pts (5%) had 33-66% L of CAD, and 35 pts(48%) had >66% L of CAD. Mean follow-up was 22+- 3 months. No deaths occurred. Two pts(3%) (with pretest L of 54% and 94%) had myocardial infarctions, 8 and 22 months respectively after T1-201 stress SC. One pt underwent percutaneous transluminal coronary angioplasty (PTCA) 16 months after T1-201 stress SC. These findings confirm excellent prognostic significance of normal quantitative T1-201 stress scintigraphy.« less

Limitations and pitfalls of 99mTc-EDDA/HYNIC-TOC (Tektrotyd) scintigraphy.

PubMed

Garai, Ildikó; Barna, Sandor; Nagy, Gabor; Forgacs, Attila

2016-01-01

Tektrotyd kit was developed by Polatom company for 99mTc labeling to make an alternative tracer of somatostatin receptor scintigraphy available. Since 2005, 99mTc-EDDA/HYNIC-Tyr3-Octreotide has been used in clinical imaging and achieved high impact in management of patients with neuroendocrine tumors. Knowing the limitations and pitfalls is essential to provide ac-curate diagnosis. Therefore, the potential pitfalls associated with the use of 99mTc-EDDA/HYNIC-TOC are reviewed on the basis of own experience. Data were analyzed of 310 patients who underwent somatostatin receptor scintigraphy with 99mTc-Tektrotyd. Pitfalls during radiolabeling process or acquisition can worsen the sensitivity of SRS (somatostatin receptor scintigraphy). Recognizing physi-ological and clinical pitfalls, the diagnostic accuracy will improve.

Viable myocardium scintigraphy with intravenous nitroglycerine by computed tomography with Tc-99m (MIBI).

PubMed

Ramos Filho, José; Nascimento, Marcos Welber; Silva, Rafael Mariano Gislon da; Camargo, Thiago Negrini de; Almeida, Roberto Simões de; Lima, Eloá Jacinto

2008-09-01

The selection of patients with chronic coronary disease for recanalization is based on the detection of the affected myocardium that is potentially viable. To evaluate the potentially viable ischemic myocardium through single photon emission computed tomography (SPECT) with MIBI after a maximum tolerated dose of I.V. nitroglycerin. We prospectively investigated by SPECT with Tc-99m (MIBI), from April 2004 to November 2005, 40 patients (mean age: 62 +/- 8.9 yrs, 30 men) with coronary obstruction demonstrated angiographically; the myocardium scintigraphy was carried out at rest and after intravenous (I.V.) nitroglycerin, which was started at a dose of 1 microg/kg/min and increased every minute until the systolic blood pressure decreased by 20 mmHg. The decrease in the perfusion of the segments was classified as moderate or severe and compared after the nitroglycerin. The angiographic, hemodynamic and myocardial perfusion variables were analyzed. We analyzed 680 myocardial segments at rest: 538 with a homogenous distribution and 142 with hypoperfusion (54 with moderate and 88 with severe decrease). After the nitroglycerin, there was an increase in the perfusion in 19 (47.5%) of 40 patients and 55 of 142 segments became viable: 33 (61.1%) with moderate and 22 (25%) with severe decrease; both presented a significant increase in the radiotracer distribution (p < 0.001, Chi-square). One of the components with Tc-99m is Tc-99m 2-methoxy-isobutyl-isonitrile (MIBI), which, when used with an optimized dose of I.V. nitroglycerin, can increase the radiotracer uptake in areas with moderate and severe hypoperfusion. The results of the present study suggest the increase in the Tc-99m (MIBI) sensitivity by nitroglycerin for the detection of viable myocardium.

The Efficacy of Crotalidae Polyvalent Immune Fab (Ovine) Antivenom Versus Placebo Plus Optional Rescue Therapy on Recovery From Copperhead Snake Envenomation: A Randomized, Double-Blind, Placebo-Controlled, Clinical Trial.

PubMed

Gerardo, Charles J; Quackenbush, Eugenia; Lewis, Brandon; Rose, S Rutherfoord; Greene, Spencer; Toschlog, Eric A; Charlton, Nathan P; Mullins, Michael E; Schwartz, Richard; Denning, David; Sharma, Kapil; Kleinschmidt, Kurt; Bush, Sean P; Ryan, Samantha; Gasior, Maria; Anderson, Victoria E; Lavonas, Eric J

2017-08-01

Copperhead snake (Agkistrodon contortrix) envenomation causes limb injury resulting in pain and disability. It is not known whether antivenom administration improves limb function. We determine whether administration of antivenom improves recovery from limb injury in patients envenomated by copperhead snakes. From August 2013 through November 2015, we performed a multicenter, randomized, double-blind, placebo-controlled, clinical trial to evaluate the effect of ovine Crotalidae polyvalent immune Fab (ovine) (CroFab; FabAV) antivenom therapy on recovery of limb function in patients with copperhead snake envenomation at 14 days postenvenomation. The study setting was 18 emergency departments in regions of the United States where copperhead snakes are endemic. Consecutive patients aged 12 years or older with mild- to moderate-severity envenomation received either FabAV or placebo. The primary outcome was limb function 14 days after envenomation, measured by the Patient-Specific Functional Scale. Additional outcomes included the Patient-Specific Functional Scale at other points; the Disorders of the Arm, Shoulder, and Hand, Lower Extremity Functional Scale, and Patient's Global Impression of Change instruments; grip strength; walking speed; quality of life (Patient-Reported Outcomes Measurement Information System Physical Fucntion-10); pain; and analgesic use. Seventy-four patients received study drug (45 FabAV, 29 placebo). Mean age was 43 years (range 12 to 86 years). Fifty-three percent were men, 62% had lower extremity envenomation, and 88% had mild initial severity. The primary outcome, the least square mean Patient-Specific Functional Scale score at 14 days postenvenomation, was 8.6 for FabAV-treated subjects and 7.4 for placebo recipients (difference 1.2; 95% confidence interval 0.1 to 2.3; P=.04). Additional outcome assessments generally favored FabAV. More FabAV-treated subjects experienced treatment-emergent adverse events (56% versus 28%), but few were

Myocardial infarction caused by myocardial bridging in a male adolescent athlete.

PubMed

Zhu, Cheng-Gang; Liu, Jun; Liu, Wei-Dong; Xu, Yan-Lu; Wu, Na-Qiong; Guo, Yuan-Lin; Tang, Yi-Da; Jiang, Li-Xin; Li, Jian-Jun

2012-02-01

Myocardial bridging is a common congenital abnormality of a coronary artery, and is usually thought to be a benign anatomical variant. Although rare, previous studies have reported that patients with myocardial bridging may suffer from myocardial ischemia, myocardial infarction (MI), arrhythmias and even sudden death. Here we report the case of an 18-year-old adolescent athlete with myocardial bridging resulting in MI. Coronary angiography revealed 80% luminal narrowing by systolic compression in the proximal and mid segments of the left anterior descending coronary artery, which returned to normal during diastole. We considered that heavy sports might be a potential trigger for his MI attack. Therefore, special attention should be paid to this kind of athlete, especially if adolescent.

Metabolic Flux Between Unsaturated and Saturated Fatty Acids is Controlled by the FabA:FabB Ratio in the Fully Reconstituted Fatty Acid Biosynthetic Pathway of E. coli#

PubMed Central

Xiao, Xirui; Yu, Xingye; Khosla, Chaitan

2013-01-01

The entire fatty acid biosynthetic pathway from Escherichia coli, starting from the acetyl-CoA carboxylase, has been reconstituted in vitro from fourteen purified protein components. Radiotracer analysis verified stoichiometric conversion of acetyl-CoA and NAD(P)H into the free fatty acid product, allowing implementation of a facile spectrophotometric assay for kinetic analysis of this multi-enzyme system. At steady state, a maximum turnover rate of 0.5 s−1 was achieved. Under optimal turnover conditions, the predominant products were C16 and C18 saturated as well as monounsaturated fatty acids. The reconstituted system allowed us to quantitatively interrogate the factors that influence metabolic flux toward unsaturated versus saturated fatty acids. In particular, the concentrations of the dehydratase FabA and the β-ketoacyl synthase FabB were found to be crucial for controlling this property. By altering these variables, the percentage of unsaturated fatty acid produced could be adjusted between 10 and 50% without significantly affecting the maximum turnover rate of the pathway. Our reconstituted system provides a powerful tool to understand and engineer rate-limiting and regulatory steps in this complex and practically significant metabolic pathway. PMID:24147979

Somatostatin receptor scintigraphy in sarcoidosis: relation to selected clinical and laboratory markers.

PubMed

Piotrowski, Wojciech J; Bieńkiewicz, Małgorzata; Frieske, Izabella; Marczak, Jerzy; Antczak, Adam; Górski, Paweł; Kuśmierek, Jacek; Płachcińska, Anna

2012-01-01

Discriminating between active and inactive sarcoidosis may be problematic in everyday clinical practice. There are numerous biochemical markers used in the diagnosis and monitoring of sarcoidosis. Somatostatin receptor (SR) scintigraphy with the use of 99mTc-octreotide may be used to estimate disease activity. The aim of the paper was to assess the value of traditional biomarkers (serum angiotensin-converting enzyme [SACE], C-reactive protein, markers of calcium metabolism, bronchoalveolar lavage fluid [BALF] lymphocytes) and a novel biomarker, 8-isoprostane (8-IP) in exhaled breath condensate (EBC), in the assessment of sarcoidosis activity in relation to somatostatin receptor scintigraphy. The study included 32 patients with sarcoidosis. Scintigraphy was performed using somatostatin analogue, 99mTc-HYNIC-TOC; planar and SPECT/CT images were recorded. The study group was divided into a subgroup with positive radiotracer uptake (n = 20) and without a visible uptake (n = 12). 8-IP levels were measured in EBC by an immunoenzymatic assay. RESULTS We observed a significantly higher EBC 8-IP levels in the subgroup with positive uptake compared with those with negative uptake (19.1 ± 19.8 vs. 5.4 ± 3.5 pg/ml, P = 0.02). The levels of SACE and the percentage of BALF lymphocytes were also nonsignificantly elevated. In the group of patients with positive scintigraphy results, a positive correlation was observed between the uptake ratio and SACE (r = 0.44, P = 0.041). The results indicate low value of biochemical markers in the assessment of disease activity. SR scintigraphy may have practical usefulness in the monitoring of sarcoidosis.

Evaluation of the utility of 99m Tc-MDP bone scintigraphy versus MIBG scintigraphy and cross-sectional imaging for staging patients with neuroblastoma.

PubMed

Gauguet, Jean-Marc; Pace-Emerson, Tamara; Grant, Frederick D; Shusterman, Suzanne; DuBois, Steven G; Frazier, A Lindsay; Voss, Stephan D

2017-11-01

Accurate staging of neuroblastoma requires multiple imaging examinations. The purpose of this study was to determine the relative contribution of 99m Tc-methylene diphosphonate (MDP) bone scintigraphy (bone scan) versus metaiodobenzylguanidine scintigraphy (MIBG scan) for accurate staging of neuroblastoma. A medical record search by the identified patients with neuroblastoma from 1993 to 2012 who underwent both MIBG and bone scan for disease staging. Cross-sectional imaging was used to corroborate the scintigraphy results. Clinical records were used to correlate imaging findings with clinical staging and patient management. One hundred thirty-two patients underwent both MIBG and bone scan for diagnosis. All stage 1 (n = 12), 2 (n = 8), and 4S (n = 4) patients had a normal bone scan with no skeletal MIBG uptake. Six of 30 stage 3 patients had false (+) bone scans. In the 78 stage 4 patients, 58/78 (74%) were both skeletal MIBG(+)/bone scan (+). In 56 of the 58 cases, skeletal involvement detected with MIBG was equal to or greater than that detected by bone scan. Only 3/78 had (-) skeletal MIBG uptake and (+) bone scans; all 3 had other sites of metastatic disease. Five of 78 had (+) skeletal MIBG with a (-) bone scan, while 12/78 had no skeletal involvement by either MIBG or bone scan. In no case did a positive bone scan alone determine a stage 4 designation. In the staging of neuroblastoma, 99m Tc-MDP bone scintigraphy does not identify unique sites of disease that affect disease stage or clinical management, and in the majority of cases bone scans can be omitted from the routine neuroblastoma staging algorithm. © 2017 Wiley Periodicals, Inc.

PET imaging of osteosarcoma in dogs using a fluorine-18-labeled monoclonal antibody fab fragment

DOE Office of Scientific and Technical Information (OSTI.GOV)

Page, R.L.; Garg, P.K.; Gard, S.

Four dogs with histologically confirmed osteogenic sarcoma were studied with PET following intravenous injection of the {sup 18}F-labeled Fab fragment of TP-3, a monoclonal antibody specific for human and canine osteosarcomas. The antibody fragment was labeled using the N-succinimidyl (8-(4{prime}-({sup 18}F)fluorobenzyl)amino)suberate acylation agent. Blood clearance of activity was biphasic in all dogs but half-times were variable (T{sub 1/2{beta}} = 2-13 hr). Catabolism of labeled Fab was reflected by the decrease in protein-associated activity in serum from more than 90% at 1 min to 60%-80% at 4 hr. PET images demonstrated increased accumulation of {sup 18}F at the primary tumor sitemore » relative to normal contralateral bone in one dog as early as 15 min after injection. Biopsies obtained after euthanasia indicated higher uptake at the edges of the tumor as observed on the PET scans. Tumor uptake was 1-3 x 10{sup -3}% injected dose/g, a level similar to that reported for other Fab fragments in human tumors. In the three dogs with metastatic disease, early PET images reflected activity in the blood pool but later uptake was observed in suspected metastatic sites. These results, although preliminary, suggest that PET imaging of {sup 18}F-labeled antibody fragments is feasible and that dogs with spontaneous tumors could be a valuable model for preclinical research with radioimmunoconjugates. 34 refs., 6 figs., 2 tabs.« less

Discovery of a novel and potent class of F. tularensis enoyl-reductase (FabI) inhibitors by molecular shape and electrostatic matching

PubMed Central

Hevener, Kirk E.; Mehboob, Shahila; Su, Pin-Chih; Truong, Kent; Boci, Teuta; Deng, Jiangping; Ghassemi, Mahmood; Cook, James L.; Johnson, Michael E.

2011-01-01

Enoyl-acyl carrier protein (ACP) reductase, FabI, is a key enzyme in the bacterial fatty acid biosynthesis pathway (FAS II). FabI is an NADH-dependent oxidoreductase that acts to reduce enoyl-ACP substrates in a final step of the pathway. The absence of this enzyme in humans makes it an attractive target for the development of new antibacterial agents. FabI is known to be unresponsive to structure-based design efforts due to a high degree of induced fit and a mobile flexible loop encompassing the active site. Here we discuss the development, validation, and careful application of a ligand-based virtual screen used for the identification of novel inhibitors of the Francisella tularensis FabI target. In this study, four known classes of FabI inhibitors were used as templates for virtual screens that involved molecular shape and electrostatic matching. The program ROCS was used to search a high-throughput screening library for compounds that matched any of the four molecular shape queries. Matching compounds were further refined using the program EON, which compares and scores compounds by matching electrostatic properties. Using these techniques, 50 compounds were selected, ordered, and tested. The tested compounds possessed novel chemical scaffolds when compared to the input query compounds. Several hits with low micromolar activity were identified and follow-up scaffold-based searches resulted in the identification of a lead series with sub-micromolar enzyme inhibition, high ligand efficiency, and a novel scaffold. Additionally, one of the most active compounds showed promising whole-cell antibacterial activity against several Gram-positive and Gram-negative species, including the target pathogen. The results of a preliminary structure-activity relationship analysis are presented. PMID:22098466

Microbiota-Dependent Marker TMAO Is Elevated in Silent Ischemia but Is Not Associated With First-Time Myocardial Infarction in HIV Infection.

PubMed

Haissman, Judith M; Knudsen, Andreas; Hoel, Hedda; Kjær, Andreas; Kristoffersen, Ulrik S; Berge, Rolf K; Katzenstein, Terese L; Svardal, Asbjørn; Ueland, Thor; Aukrust, Pål; Lebech, Anne-Mette; Nielsen, Susanne D; Trøseid, Marius

2016-02-01

HIV infection is associated with increased risk of coronary heart disease beyond that explained by traditional risk factors, and altered gut microbiota has been proposed as a potential trigger. Trimethylamine-N-oxide (TMAO) is a proatherogenic substance formed in the liver from trimethylamine, exclusively generated by gut microbiota from dietary phosphatidylcholine. We aimed to investigate whether TMAO is associated with subclinical and clinical coronary heart disease in HIV infection. Two previously described cohorts were examined as follows: (1) cross-sectional cohort of HIV-infected persons and uninfected controls with known atherosclerotic plaque burden as assessed by myocardial perfusion scintigraphy, coronary artery calcium score, and intima-media thickness and (2) nested case-control study of HIV-infected persons with first-time myocardial infarction (MI) compared with HIV-infected persons without MI, assessed at 4 time points from before initiation of antiretroviral therapy (ART) to last sample before the case's MI (median: 51, range: 0-239 days). There was no difference in plasma TMAO when comparing HIV-infected persons and uninfected controls. TMAO was elevated in HIV-infected persons with myocardial perfusion defects but was not associated with coronary artery calcium score, intima media thickness, or Framingham risk score. In the nested case control study, plasma TMAO was not associated with first-time MI. However, TMAO increased after ART introduction and was associated with the use of protease inhibitors in both cohorts. TMAO was elevated in HIV-infected persons with myocardial perfusion defects, but was not associated with first-time MI. Our data question TMAO as a useful biomarker of cardiovascular risk in HIV infection, at least in ART-treated individuals.

The R117A variant of the Escherichia coli transacylase FabD synthesizes novel acyl-(acyl carrier proteins).

PubMed

Marcella, Aaron M; Barb, Adam W

2017-12-01

The commercial impact of fermentation systems producing novel and biorenewable chemicals will flourish with the expansion of enzymes engineered to synthesize new molecules. Though a small degree of natural variability exists in fatty acid biosynthesis, the molecular space accessible through enzyme engineering is fundamentally limitless. Prokaryotic fatty acid biosynthesis enzymes build carbon chains on a functionalized acyl carrier protein (ACP) that provides solubility, stability, and a scaffold for interactions with the synthetic enzymes. Here, we identify the malonyl-coenzyme A (CoA)/holo-ACP transacylase (FabD) from Escherichia coli as a platform enzyme for engineering to diversify microbial fatty acid biosynthesis. The FabD R117A variant produced novel ACP-based primer and extender units for fatty acid biosynthesis. Unlike the wild-type enzyme that is highly specific for malonyl-CoA to produce malonyl-ACP, the R117A variant synthesized acetyl-ACP, succinyl-ACP, isobutyryl-ACP, 2-butenoyl-ACP, and β-hydroxybutyryl-ACP among others from holo-ACP and the corresponding acyl-CoAs with specific activities from 3.7 to 120 nmol min -1  mg -1 . FabD R117A maintained K M values for holo-ACP (~ 40 μM) and displayed small changes in K M for acetoacetyl-CoA (110 ± 30 μM) and acetyl-CoA (200 ± 70 μM) when compared to malonyl-CoA (80 ± 30 μM). FabD R117A represents a novel catalyst that synthesizes a broad range of acyl-acyl-ACPs.

Anticomplementary activity of horse IgG and F(ab')2 antivenoms.

PubMed

Squaiella-Baptistão, Carla Cristina; Marcelino, José Roberto; Ribeiro da Cunha, Luiz Eduardo; Gutiérrez, José María; Tambourgi, Denise V

2014-03-01

Envenomation by poisonous animals is a neglected condition according to the World Health Organization (WHO). Antivenoms are included in the WHO Essential Medicines List. It has been assumed that immunoglobulin G (IgG) antivenoms could activate the complement system through Fc and induce early adverse reactions (EARs). However, data in the literature indicate that F(ab')2 fragments can also activate the complement system. Herein, we show that several batches of IgG and F(ab')2 antivenoms from the Butantan, Vital Brazil, and Clodomiro Picado Institutes activated the complement classical pathway and induced the production of C3a; however, only those antivenoms from Clodomiro Picado generated C5a. Different protein profiles (IgG heavy chain, protein contaminants, and aggregates) were observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses. Our results show that various antivenoms from different producers are able to activate the classical pathway of the complement system and generate anaphylatoxins, and these findings suggest that factors, such as composition, contaminant proteins, and aggregates, may influence the anticomplementary activity of antivenoms in vitro. Therefore, there is a need to further improve antivenom production methods to reduce their anticomplementary activity and potential to cause EARs.

Specific Conjugation of the Hinge Region for Homogeneous Preparation of Antibody Fragment-Drug Conjugate: A Case Study for Doxorubicin-PEG-anti-CD20 Fab' Synthesis.

PubMed

Zhou, Zhan; Zhang, Jing; Zhang, Yan; Ma, Guanghui; Su, Zhiguo

2016-01-20

Conventional preparation strategies for antibody-drug conjugates (ADCs) result in heterogeneous products with various molecular sizes and species. In this study, we developed a homogeneous preparation strategy by site-specific conjugation of the anticancer drug with an antibody fragment. The model drug doxorubicin (DOX) was coupled to the Fab' fragment of anti-CD20 IgG at its permissive sites through a heterotelechelic PEG linker, generating an antibody fragment-drug conjugate (AFDC). Anti-CD20 IgG was digested and reduced specifically with β-mercaptoethylamine to generate the Fab' fragment with two free mercapto groups in its hinge region. Meanwhile, DOX was conjugated with α-succinimidylsuccinate ω-maleimide polyethylene glycol (NHS-PEG-MAL) to form MAL-PEG-DOX, which was subsequently linked to the free mercapto containing Fab' fragment to form a Fab'-PEG-DOX conjugate. The dual site-specific bioconjugation was achieved through the combination of highly selective reduction of IgG and introduction of heterotelechelic PEG linker. The resulting AFDC provides an utterly homogeneous product, with a definite ratio of one fragment to two drugs. Laser confocal microscopy and cell ELISA revealed that the AFDC could accumulate in the antigen-positive Daudi tumor cell. In addition, the Fab'-PEG-DOX retained appreciable targeting ability and improved antitumor activity, demonstrating an excellent therapeutic effect on the lymphoma mice model for better cure rate and significantly reduced side effects.

A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties

PubMed Central

Tiller, Thomas; Schuster, Ingrid; Deppe, Dorothée; Siegers, Katja; Strohner, Ralf; Herrmann, Tanja; Berenguer, Marion; Poujol, Dominique; Stehle, Jennifer; Stark, Yvonne; Heßling, Martin; Daubert, Daniela; Felderer, Karin; Kaden, Stefan; Kölln, Johanna; Enzelberger, Markus; Urlinger, Stefanie

2013-01-01

This report describes the design, generation and testing of Ylanthia, a fully synthetic human Fab antibody library with 1.3E+11 clones. Ylanthia comprises 36 fixed immunoglobulin (Ig) variable heavy (VH)/variable light (VL) chain pairs, which cover a broad range of canonical complementarity-determining region (CDR) structures. The variable Ig heavy and Ig light (VH/VL) chain pairs were selected for biophysical characteristics favorable to manufacturing and development. The selection process included multiple parameters, e.g., assessment of protein expression yield, thermal stability and aggregation propensity in fragment antigen binding (Fab) and IgG1 formats, and relative Fab display rate on phage. The framework regions are fixed and the diversified CDRs were designed based on a systematic analysis of a large set of rearranged human antibody sequences. Care was taken to minimize the occurrence of potential posttranslational modification sites within the CDRs. Phage selection was performed against various antigens and unique antibodies with excellent biophysical properties were isolated. Our results confirm that quality can be built into an antibody library by prudent selection of unmodified, fully human VH/VL pairs as scaffolds. PMID:23571156

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hartmann, A.; Frenkel, J.; Hopf, R.

Amyloidosis is a systemic disease frequently involving the myocardium and leading to functional disturbances of the heart. Amyloidosis can mimic other cardiac diseases. A conclusive clinical diagnosis of cardiac involvement can only be made by a combination of different diagnostic methods. In 7 patients with myocardial amyloidosis we used a combined first-pass and static scintigraphy with technetium-99 m-pyrophosphate. There was only insignificant myocardial uptake of the tracer. The first-pass studies however revealed reduced systolic function in 4/7 patients and impaired diastolic function in 6/7 patients. Therefore, although cardiac amyloid could not be demonstrated in the static scintigraphy due to amyloidmore » fibril amount and composition, myocardial functional abnormalities were seen in the first-pass study.« less

Pancreas transplants: Evaluation using perfusion scintigraphy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuni, C.C.; du Cret, R.P.; Boudreau, R.J.

1989-07-01

To determine the value of scintigraphic perfusion studies in evaluating pancreas transplant patients, we reviewed 56 of these studies in 22 patients who had 27 transplants. Seventeen patients underwent two or more studies. The perfusion studies were performed with 20 mCi (740 MBq) of 99mTc-DTPA injected as a bolus followed by eight to 16 serial 2-sec images and a 500,000-count immediate static image. Images were evaluated for (1) the time and intensity of pancreatic peak radioactivity relative to the time and intensity of the iliac arterial peak; (2) relative pancreatic to iliac arterial intensity on the static image; and (3)more » size, homogeneity, and definition of the pancreas. Clinical diagnoses at the time of scintigraphy of normal function (n = 36), rejection (n = 13), pancreatitis (n = 6), or arterial thrombosis (n = 1) were based on insulin requirement, urine amylase, serum glucose, serum amylase, response to therapy, cultures, CT, MR, sonography, scintigraphy with 67Ga or 111In-WBCs, percutaneous drainage results, angiography, surgery, and pathologic examination of resected transplants. Three 99mTc-DTPA perfusion studies showed no pancreatic perfusion, four showed decreasing perfusion on serial studies, and five showed progressive loss of definition of the pancreas on serial studies. Of the three patients with no detectable perfusion, one had a normally functioning transplant, one had arterial thrombosis with transplant infarction, and one had severe rejection with minimal function. Decreasing perfusion was associated with rejection in three patients and pancreatitis in one. Decreasing definition was seen in four patients with rejection and one with pancreatitis. We conclude that perfusion scintigraphy is useful, primarily when performed serially, although nonspecific for evaluating pancreas transplants.« less

Matching the Decay Half-Life with the Biological Half-Life: ImmunoPET Imaging with 44Sc-Labeled Cetuximab Fab Fragment

PubMed Central

2015-01-01

Scandium-44 (t1/2 = 3.9 h) is a relatively new radioisotope of potential interest for use in clinical positron emission tomography (PET). Herein, we report, for the first time, the room-temperature radiolabeling of proteins with 44Sc for in vivo PET imaging. For this purpose, the Fab fragment of Cetuximab, a monoclonal antibody that binds with high affinity to epidermal growth factor receptor (EGFR), was generated and conjugated with N-[(R)-2-amino-3-(para-isothiocyanato-phenyl)propyl]-trans-(S,S)-cyclohexane-1,2-diamine-N,N,N′,N″,N″-pentaacetic acid (CHX-A″-DTPA). The high purity of Cetuximab-Fab was confirmed by SDS-PAGE and mass spectrometry. The potential of the bioconjugate for PET imaging of EGFR expression in human glioblastoma (U87MG) tumor-bearing mice was investigated after 44Sc labeling. PET imaging revealed rapid tumor uptake (maximum uptake of ∼12% ID/g at 4 h postinjection) of 44Sc–CHX-A″-DTPA–Cetuximab-Fab with excellent tumor-to-background ratio, which might allow for same day PET imaging in future clinical studies. Immunofluorescence staining was conducted to correlate tracer uptake in the tumor and normal tissues with EGFR expression. This successful strategy for immunoPET imaging of EGFR expression using 44Sc–CHX-A″-DTPA–Cetuximab-Fab can make clinically translatable advances to select the right population of patients for EGFR-targeted therapy and also to monitor the therapeutic efficacy of anti-EGFR treatments. PMID:25389697

Different Causes of Death in Patients with Myocardial Infarction Type 1, Type 2, and Myocardial Injury.

PubMed

Lambrecht, Sascha; Sarkisian, Laura; Saaby, Lotte; Poulsen, Tina S; Gerke, Oke; Hosbond, Susanne; Diederichsen, Axel C P; Thygesen, Kristian; Mickley, Hans

2018-05-01

Data outlining the mortality and the causes of death in patients with type 1 myocardial infarction, type 2 myocardial infarction, and those with myocardial injury are limited. During a 1-year period from January 2010 to January 2011, all hospitalized patients who had cardiac troponin I measured on clinical indication were prospectively studied. Patients with at least one cardiac troponin I value >30 ng/L underwent case ascertainment and individual evaluation by an experienced adjudication committee. Patients were classified as having type 1 myocardial infarction, type 2 myocardial infarction, or myocardial injury according to the criteria of the universal definition of myocardial infarction. Follow-up was ensured until December 31, 2014. Data on mortality and causes of death were obtained from the Danish Civil Registration System and the Danish Register of Causes of Death. Overall, 3762 consecutive patients were followed for a mean of 3.2 years (interquartile range 1.3-3.6 years). All-cause mortality differed significantly among categories: Type 1 myocardial infarction 31.7%, type 2 myocardial infarction 62.2%, myocardial injury 58.7%, and 22.2% in patients with nonelevated troponin values (log-rank test; P < .0001). In patients with type 1 myocardial infarction, 61.3% died from cardiovascular causes, vs 42.6% in patients with type 2 myocardial infarction (P = .015) and 41.2% in those with myocardial injury (P < .0001). The overall mortality and the causes of death did not differ substantially between patients with type 2 myocardial infarction and those with myocardial injury. Patients with type 2 myocardial infarction and myocardial injury exhibit a significantly higher long-term mortality compared with patients with type 1 myocardial infarction . However, most patients with type 1 myocardial infarction die from cardiovascular causes in contrast to patients with type 2 myocardial infarction and myocardial injury, in whom noncardiovascular causes of death

Diagnosis of pyogenic pelvic inflammatory diseases by 99mTc-HMPAO leucocyte scintigraphy.

PubMed

Rachinsky, I; Boguslavsky, L; Goldstein, D; Golan, H; Pak, I; Katz, M; Lantsberg, S

2000-12-01

Pelvic inflammatory disease (PID) is one of the major health problems of women of child-bearing age. Among the most serious complications of PID is the formation of a tubo-ovarian abscess (TOA). Early diagnosis of this condition may prevent serious surgical complications such as peritonitis and sepsis, which may be fatal. The purpose of this study was to investigate the efficacy of technetium-99m hexamethylpropylene amine oxime (HMPAO) leucocyte scintigraphy in the diagnosis of TOA. Twenty women with high clinical suspicion of TOA underwent 99mTc-HMPAO leucocyte scintigraphy. The labelling of leucocytes with 99mTc-HMPAO was performed according to a standard protocol. Scans were obtained at 1, 3 and 24 h following the injection of the labelled leucocytes. In eight cases the early and/or late scan was positive, in 11 cases it was negative, and in one case of ovarian cyst torsion, confirmed by laparoscopy, it showed slight uptake in the capsule of the cyst (false-positive). The sensitivity of 99mTc-HMPAO leucocyte scintigraphy was 100%, specificity 91.6%, positive predictive value 89%, negative predictive value 100% and overall accuracy 95%. It is concluded that leucocyte scintigraphy is a non-invasive, safe, physiological and accurate procedure for the diagnosis of TOA. The 24-h scan is crucial, since in some cases the abscess was not clearly visualized on the early scan. Leucocyte scintigraphy may reduce the need for CT, diagnostic laparoscopy and unnecessary invasive surgical procedures.

Identification of Warthin tumor: magnetic resonance imaging versus salivary scintigraphy with technetium-99m pertechnetate.

PubMed

Motoori, Ken; Ueda, Takuya; Uchida, Yoshitaka; Chazono, Hideaki; Suzuki, Homare; Ito, Hisao

2005-01-01

The aim of this study was to evaluate the accuracy of technetium-99m (Tc-99m) pertechnetate scintigraphy and magnetic resonance (MR) imaging in the diagnosis of Warthin tumor. Sixteen cases of Warthin tumor and 17 cases of non-Warthin tumor were examined by Tc-99m pertechnetate scintigraphy with lemon juice stimulation and MR imaging, including T1-weighted, T2-weighted, short inversion time inversion recovery, diffusion-weighted, and contrast-enhanced dynamic images. We used the receiver operating characteristic (ROC) curve to evaluate diagnostic accuracy. The mean area under the ROC curves of MR imaging in the diagnosis of Warthin tumor (0.97) was higher than that of Tc-99m pertechnetate scintigraphy (0.88). Magnetic resonance imaging is more useful in the evaluation of Warthin tumor than Tc-99m pertechnetate scintigraphy.

Periodontitis and myocardial hypertrophy.

PubMed

Suzuki, Jun-Ichi; Sato, Hiroki; Kaneko, Makoto; Yoshida, Asuka; Aoyama, Norio; Akimoto, Shouta; Wakayama, Kouji; Kumagai, Hidetoshi; Ikeda, Yuichi; Akazawa, Hiroshi; Izumi, Yuichi; Isobe, Mitsuaki; Komuro, Issei

2017-04-01

There is a deep relationship between cardiovascular disease and periodontitis. It has been reported that myocardial hypertrophy may be affected by periodontitis in clinical settings. Although these clinical observations had some study limitations, they strongly suggest a direct association between severity of periodontitis and left ventricular hypertrophy. However, the detailed mechanisms between myocardial hypertrophy and periodontitis have not yet been elucidated. Recently, we demonstrated that periodontal bacteria infection is closely related to myocardial hypertrophy. In murine transverse aortic constriction models, a periodontal pathogen, Aggregatibacter actinomycetemcomitans markedly enhanced cardiac hypertrophy with matrix metalloproteinase-2 activation, while another pathogen Porphyromonas gingivalis (P.g.) did not accelerate these pathological changes. In the isoproterenol-induced myocardial hypertrophy model, P.g. induced myocardial hypertrophy through Toll-like receptor-2 signaling. From our results and other reports, regulation of chronic inflammation induced by periodontitis may have a key role in the treatment of myocardial hypertrophy. In this article, we review the pathophysiological mechanism between myocardial hypertrophy and periodontitis.

Automated reticle inspection data analysis for wafer fabs

NASA Astrophysics Data System (ADS)

Summers, Derek; Chen, Gong; Reese, Bryan; Hutchinson, Trent; Liesching, Marcus; Ying, Hai; Dover, Russell

2008-10-01

To minimize potential wafer yield loss due to mask defects, most wafer fabs implement some form of reticle inspection system to monitor photomask quality in high-volume wafer manufacturing environments. Traditionally, experienced operators review reticle defects found by an inspection tool and then manually classify each defect as 'pass, warn, or fail' based on its size and location. However, in the event reticle defects are suspected of causing repeating wafer defects on a completed wafer, potential defects on all associated reticles must be manually searched on a layer-by-layer basis in an effort to identify the reticle responsible for the wafer yield loss. This 'problem reticle' search process is a very tedious and time-consuming task and may cause extended manufacturing line-down situations. Often times, Process Engineers and other team members need to manually investigate several reticle inspection reports to determine if yield loss can be tied to a specific layer. Because of the very nature of this detailed work, calculation errors may occur resulting in an incorrect root cause analysis effort. These delays waste valuable resources that could be spent working on other more productive activities. This paper examines an automated software solution for converting KLA-Tencor reticle inspection defect maps into a format compatible with KLA-Tencor's Klarity DefecTM data analysis database. The objective is to use the graphical charting capabilities of Klarity Defect to reveal a clearer understanding of defect trends for individual reticle layers or entire mask sets. Automated analysis features include reticle defect count trend analysis and potentially stacking reticle defect maps for signature analysis against wafer inspection defect data. Other possible benefits include optimizing reticle inspection sample plans in an effort to support "lean manufacturing" initiatives for wafer fabs.

Automated reticle inspection data analysis for wafer fabs

NASA Astrophysics Data System (ADS)

Summers, Derek; Chen, Gong; Reese, Bryan; Hutchinson, Trent; Liesching, Marcus; Ying, Hai; Dover, Russell

2009-04-01

To minimize potential wafer yield loss due to mask defects, most wafer fabs implement some form of reticle inspection system to monitor photomask quality in high-volume wafer manufacturing environments. Traditionally, experienced operators review reticle defects found by an inspection tool and then manually classify each defect as 'pass, warn, or fail' based on its size and location. However, in the event reticle defects are suspected of causing repeating wafer defects on a completed wafer, potential defects on all associated reticles must be manually searched on a layer-by-layer basis in an effort to identify the reticle responsible for the wafer yield loss. This 'problem reticle' search process is a very tedious and time-consuming task and may cause extended manufacturing line-down situations. Often times, Process Engineers and other team members need to manually investigate several reticle inspection reports to determine if yield loss can be tied to a specific layer. Because of the very nature of this detailed work, calculation errors may occur resulting in an incorrect root cause analysis effort. These delays waste valuable resources that could be spent working on other more productive activities. This paper examines an automated software solution for converting KLA-Tencor reticle inspection defect maps into a format compatible with KLA-Tencor's Klarity Defect(R) data analysis database. The objective is to use the graphical charting capabilities of Klarity Defect to reveal a clearer understanding of defect trends for individual reticle layers or entire mask sets. Automated analysis features include reticle defect count trend analysis and potentially stacking reticle defect maps for signature analysis against wafer inspection defect data. Other possible benefits include optimizing reticle inspection sample plans in an effort to support "lean manufacturing" initiatives for wafer fabs.

Automated reticle inspection data analysis for wafer fabs

NASA Astrophysics Data System (ADS)

Summers, Derek; Chen, Gong; Reese, Bryan; Hutchinson, Trent; Liesching, Marcus; Ying, Hai; Dover, Russell

2009-03-01

To minimize potential wafer yield loss due to mask defects, most wafer fabs implement some form of reticle inspection system to monitor photomask quality in high-volume wafer manufacturing environments. Traditionally, experienced operators review reticle defects found by an inspection tool and then manually classify each defect as 'pass, warn, or fail' based on its size and location. However, in the event reticle defects are suspected of causing repeating wafer defects on a completed wafer, potential defects on all associated reticles must be manually searched on a layer-by-layer basis in an effort to identify the reticle responsible for the wafer yield loss. This 'problem reticle' search process is a very tedious and time-consuming task and may cause extended manufacturing line-down situations. Often times, Process Engineers and other team members need to manually investigate several reticle inspection reports to determine if yield loss can be tied to a specific layer. Because of the very nature of this detailed work, calculation errors may occur resulting in an incorrect root cause analysis effort. These delays waste valuable resources that could be spent working on other more productive activities. This paper examines an automated software solution for converting KLA-Tencor reticle inspection defect maps into a format compatible with KLA-Tencor's Klarity DefectTM data analysis database. The objective is to use the graphical charting capabilities of Klarity Defect to reveal a clearer understanding of defect trends for individual reticle layers or entire mask sets. Automated analysis features include reticle defect count trend analysis and potentially stacking reticle defect maps for signature analysis against wafer inspection defect data. Other possible benefits include optimizing reticle inspection sample plans in an effort to support "lean manufacturing" initiatives for wafer fabs.

Structure of Mycobacterium tuberculosis mtFabD, a malonyl-CoA:acyl carrier protein transacylase (MCAT).

PubMed

Ghadbane, Hemza; Brown, Alistair K; Kremer, Laurent; Besra, Gurdyal S; Fütterer, Klaus

2007-10-01

Mycobacteria display a unique and unusual cell-wall architecture, central to which is the membrane-proximal mycolyl-arabinogalactan-peptidoglycan core (mAGP). The biosynthesis of mycolic acids, which form the outermost layer of the mAGP core, involves malonyl-CoA:acyl carrier protein transacylase (MCAT). This essential enzyme catalyses the transfer of malonyl from coenzyme A to acyl carrier protein AcpM, thus feeding these two-carbon units into the chain-elongation cycle of the type II fatty-acid synthase. The crystal structure of M. tuberculosis mtFabD, the mycobacterial MCAT, has been determined to 3.0 A resolution by multi-wavelength anomalous dispersion. Phasing was facilitated by Ni2+ ions bound to the 20-residue N-terminal affinity tag, which packed between the two independent copies of mtFabD.

64Cu-Labeled Trastuzumab Fab-PEG24-EGF Radioimmunoconjugates Bispecific for HER2 and EGFR: Pharmacokinetics, Biodistribution, and Tumor Imaging by PET in Comparison to Monospecific Agents.

PubMed

Kwon, Luke Yongkyu; Scollard, Deborah A; Reilly, Raymond M

2017-02-06

Heterodimerization of EGFR with HER2 coexpressed in breast cancer (BC) promotes tumor growth, and increased EGFR expression is associated with trastuzumab resistance. Our aim was to construct 64 Cu-labeled bispecific radioimmunoconjugates (bsRIC) composed of trastuzumab Fab, which binds HER2 linked through a polyethylene glycol (PEG 24 ) spacer to EGF, and to compare their pharmacokinetic, biodistribution, and tumor imaging characteristics by positron-emission tomography (PET). bsRICs were generated by linking maleimide modified trastuzumab Fab with thiolated EGF through a thioether bond. HER2 and EGFR binding were assessed in vitro in MDA-MB-231 (EGFR mod /HER2 low ), MDA-MB-468 (EGFR high /HER2 neg ), MDA-MB-231-H2N (EGFR mod /HER2 mod ), and SKOV3 (EGFR low /HER2 high ) cells by competition and saturation cell binding assays to estimate the dissociation constant (K d ). The elimination of the 64 Cu-NOTA-trastuzumab Fab-PEG 24 -EGF bsRICs from the blood of Balb/c mice was compared to monospecific 64 Cu-NOTA-trastuzumab Fab and 64 Cu-NOTA-EGF. MicroPET/CT imaging was performed in NOD/SCID mice bearing subcutaneous MDA-MB-468, MDA-MB-231/H2N, or SKOV3 human BC xenografts at 24 and 48 h postinjection (p.i.) of bsRICs. Tumor and normal tissue uptake were quantified by biodistribution studies and compared to monospecific agents. The binding of bsRICs to MDA-MB-231 cells was decreased to 24.5 ± 5.2% by excess EGF, while the binding of bsRICs to SKOV3 cells was decreased to 38.6 ± 5.4% by excess trastuzumab Fab, demonstrating specific binding to both EGFR and HER2. 64 Cu-labeled bsRICs incorporating the PEG 24 spacer were eliminated more slowly from the blood than 64 Cu-bsRICs without the PEG spacer and were cleared much more slowly than 64 Cu-NOTA-Fab or 64 Cu-NOTA-EGF. All three tumor xenografts were visualized by microPET/CT at 24 and 48 h p.i. of bsRICs. Biodistribution studies at 48 h p.i. in NOD/SCID mice with MDA-MB-231/H2N tumors demonstrated significantly

Usefulness of automatic QT dispersion measurement for detecting exercise-induced myocardial ischemia.

PubMed

Takase, Bonpei; Masaki, Nobuyuki; Hattori, Hidemi; Ishihara, Masayuki; Kurita, Akira

2009-06-01

The electrocardiographic index of QT dispersion (QTd) is related to the occurrence of arrhythmia. In patients with suspected or known coronary artery disease, QTd may be affected by exercise. We investigated whether QTd that is automatically calculated by a newly developed computer system could be used as a marker of exercise-induced myocardial ischemia. The design of this study was prospective and observational. Eighty-three consecutive patients were enrolled in this study. Their QTd was measured at rest and after 3 min of exercise during exercise-stress Thallium-201 scintigraphy and compared with conventional ST-segment changes. The patients were classified into 4 groups (normal group, redistribution group, fixed defect group, redistribution with fixed defect group) based on the result of single photon emission computed tomography. As statistical analysis, one-way ANOVA with post-hoc Scheffe's method, receiver-operating characteristics (ROC) and multiple logistic regression analysis were performed. At rest, QTd was significantly greater (p<0.05) in the fixed defect group (52+/-21 ms) and the redistribution with fixed defect group (53+/-20 ms) than in the normal group (32+/-14 ms) and the redistribution group (31+/-16 ms). However, QTd tended to increase after exercise in the redistribution group, while QTd tended to decrease in the normal group, the fixed defect group, and the redistribution with fixed defect group (QTd after exercise, normal group, 28+/-17 ms, redistribution group, 35+/-19 ms, fixed defect group, 43+/-25 ms, redistribution with fixed defect group, 49+/-27 ms). Exercise significantly increased QTcd (RR interval-corrected QT dispersion) in the redistribution group. The best cut-off values of QTd and QTcd obtained from ROC curves for exercise-induced myocardial ischemia were 41.6 ms and 40.4 ms, respectively (Qtd--AUC 0.68, 95%CI 0.53- 0.83 and QTcd--AUC 0.67, 95%CI 0.55-0.80). Using these values as cut-off ones, QTd, QTcd, and conventional ST

Treatment of chronically digoxin-poisoned patients with a newer digoxin immune fab--a retrospective study.

PubMed

Schaeffer, Tammi H; Mlynarchek, Sara L; Stanford, Christopher F; Delgado, João; Holstege, Christopher P; Olsen, Dean; Bogdan, Gregory M

2010-10-01

Digoxin is used in the treatment of patients with cardiac dysfunction, though toxicity sometimes results from the use of this medication. In 1986, the US Food and Drug Administration (FDA) approved a digoxin immune Fab for the treatment of such patients. In 2001, the FDA approved a newer digoxin immune Fab, a digoxin-specific antibody (DSAb) known as DigiFab (Protherics Inc, Brentwood, Tennessee), though minimal literature exists on the clinical effects of this DSAb. To characterize a cohort of patients presenting with chronic digoxin toxicity and to describe the clinical course of these patients with the use of DSAb. A retrospective study included patients with life-threatening cardiotoxicity and serum digoxin level greater than 2 ng/mL who were treated at two US hospitals from 2003 to 2006. Trained investigators abstracted data from patients' medical records and assessed changes in clinical and laboratory parameters at regular intervals (0-4, >4-12, >12-24, and >24-72 hours) after treatment with DSAb. An expert panel reviewed electrocardiogram results to identify life-threatening manifestations of digoxin toxicity before and after DSAb treatment. Efficacy of treatment was assessed as rates of improvement in clinical parameters and cardiotoxic effects. Rates of adverse drug reactions were used to characterize safety. All data were analyzed with descriptive statistics. Fourteen patients (mean [SD] age, 71.3 [10.4] years) were treated for chronic digoxin toxicity. At presentation, 12 patients had a heart rate of less than 45 beats per minute, 1 had third-degree heart block, and 1 had asystole. Mean serum digoxin level was 3.6 ng/mL. Eleven patients had abnormal renal function. After administration of DSAb, clinical parameters improved in all patients. Within 24 hours, cardiotoxicity resolved in 7 of 9 evaluable patients. Two adverse drug reactions possibly related to DigiFab occurred, both of which resolved with conventional measures. Two patients died from

[Stress-ECG is adequate to detect myocardial ischemia: when are additional diagnostic tests needed?].

PubMed

Baer, F M

2007-09-01

The stress-ECG is the most often adopted and most cost effective initial diagnostic test for the assessment of myocardial ischemia in patients with suspected coronary artery disease (CAD). Prerequisites for the diagnostic usefullness of stress-ECG are a clearly interpretable ST-segment, ability to reach the predicted work load, an intermediate pretest probability for CAD ranging between 10% and 90% and the absence of any contraindications for dynamic exercise. Because of the limited diagnostic sensitivity of about 70%, and a high percentage of patients, who are unable to exercise, a negative stress ECG can definitely not exclude hemodynamically significant CAD. Therefore, stress imaging techniques like myocardial scintigraphy, stress-echocardiography and stress magnetic resonance imaging play a major role in the stepwise diagnostic work-up of patients with suspected CAD. These stress imaging techniques are basically interchangeable since no method is definitely superior to one of the others. However, each method has its specific pros and cons and inherent contraindications. Therefore the choice of the stress imaging method and the form of stress applied should be based on the individual patients characteristics to gain optimal image quality and diagnostic accuracy. Moreover, the decision for one method should take the local availability and institutional expertise of diagnostic centers into account. Although partly substituted by stress imaging techniques the stress-ECG still remains the workhorse for a stepwise diagnostic work-up of patients with suspected CAD.

Resistance Mechanisms and the Future of Bacterial Enoyl-Acyl Carrier Protein Reductase (FabI) Antibiotics

PubMed Central

Yao, Jiangwei; Rock, Charles O.

2016-01-01

Missense mutations leading to clinical antibiotic resistance are a liability of single-target inhibitors. The enoyl-acyl carrier protein reductase (FabI) inhibitors have one intracellular protein target and drug resistance is increased by the acquisition of single-base-pair mutations that alter drug binding. The spectrum of resistance mechanisms to FabI inhibitors suggests criteria that should be considered during the development of single-target antibiotics that would minimize the impact of missense mutations on their clinical usefulness. These criteria include high-affinity, fast on/off kinetics, few drug contacts with residue side chains, and no toxicity. These stringent criteria are achievable by structure-guided design, but this approach will only yield pathogen-specific drugs. Single-step acquisition of resistance may limit the clinical application of broad-spectrum, single-target antibiotics, but appropriately designed pathogen-specific antibiotics have the potential to overcome this liability. PMID:26931811

Impact of FAB classification on predicting outcome in acute myeloid leukemia, not otherwise specified, patients undergoing allogeneic stem cell transplantation in CR1: An analysis of 1690 patients from the acute leukemia working party of EBMT.

PubMed

Canaani, Jonathan; Beohou, Eric; Labopin, Myriam; Socié, Gerard; Huynh, Anne; Volin, Liisa; Cornelissen, Jan; Milpied, Noel; Gedde-Dahl, Tobias; Deconinck, Eric; Fegueux, Nathalie; Blaise, Didier; Mohty, Mohamad; Nagler, Arnon

2017-04-01

The French, American, and British (FAB) classification system for acute myeloid leukemia (AML) is extensively used and is incorporated into the AML, not otherwise specified (NOS) category in the 2016 WHO edition of myeloid neoplasm classification. While recent data proposes that FAB classification does not provide additional prognostic information for patients for whom NPM1 status is available, it is unknown whether FAB still retains a current prognostic role in predicting outcome of AML patients undergoing allogeneic stem cell transplantation. Using the European Society of Blood and Bone Marrow Transplantation registry we analyzed outcome of 1690 patients transplanted in CR1 to determine if FAB classification provides additional prognostic value. Multivariate analysis revealed that M6/M7 patients had decreased leukemia free survival (hazard ratio (HR) of 1.41, 95% confidence interval (CI), 1.01-1.99; P = .046) in addition to increased nonrelapse mortality (NRM) rates (HR, 1.79; 95% CI, 1.06-3.01; P = .028) compared with other FAB types. In the NPM1 wt AML, NOS cohort, FAB M6/M7 was also associated with increased NRM (HR, 2.17; 95% CI, 1.14-4.16; P = .019). Finally, in FLT3-ITD + patients, multivariate analyses revealed that specific FAB types were tightly associated with adverse outcome. In conclusion, FAB classification may predict outcome following transplantation in AML, NOS patients. © 2017 Wiley Periodicals, Inc.

The Fab1/PIKfyve phosphoinositide phosphate kinase is not necessary to maintain the pH of lysosomes and of the yeast vacuole.

PubMed

Ho, Cheuk Y; Choy, Christopher H; Wattson, Christina A; Johnson, Danielle E; Botelho, Roberto J

2015-04-10

Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH < 5 in PIKfyve-inhibited mammalian cells. In addition, quantitative fluorescence microscopy of vacuole-targeted pHluorin, a pH-sensitive GFP variant, indicates that fab1Δ vacuoles are as acidic as wild-type yeast. Importantly, we also employed fluorimetry of vacuoles loaded with cDCFDA, a pH-sensitive dye, to show that both wild-type and fab1Δ vacuoles have a pH < 5.0. In comparison, the vacuolar pH of the V-ATPase mutant vph1Δ or vph1Δ fab1Δ double mutant was 6.1. Although the steady-state vacuolar pH is not affected by PtdIns(3,5)P2 depletion, it may have a role in stabilizing the vacuolar pH during salt shock. Overall, we propose a model in which PtdIns(3,5)P2 does not govern the steady-state pH of vacuoles or lysosomes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The Fab1/PIKfyve Phosphoinositide Phosphate Kinase Is Not Necessary to Maintain the pH of Lysosomes and of the Yeast Vacuole*

PubMed Central

Ho, Cheuk Y.; Choy, Christopher H.; Wattson, Christina A.; Johnson, Danielle E.; Botelho, Roberto J.

2015-01-01

Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH < 5 in PIKfyve-inhibited mammalian cells. In addition, quantitative fluorescence microscopy of vacuole-targeted pHluorin, a pH-sensitive GFP variant, indicates that fab1Δ vacuoles are as acidic as wild-type yeast. Importantly, we also employed fluorimetry of vacuoles loaded with cDCFDA, a pH-sensitive dye, to show that both wild-type and fab1Δ vacuoles have a pH < 5.0. In comparison, the vacuolar pH of the V-ATPase mutant vph1Δ or vph1Δ fab1Δ double mutant was 6.1. Although the steady-state vacuolar pH is not affected by PtdIns(3,5)P2 depletion, it may have a role in stabilizing the vacuolar pH during salt shock. Overall, we propose a model in which PtdIns(3,5)P2 does not govern the steady-state pH of vacuoles or lysosomes. PMID:25713145

The value and throughput of rest Thallium-201/stress Technetium -99m sestamibi dual-isotope myocardial SPECT.

PubMed

Okudan, Berna; Smitherman, Thomas C

2004-06-01

Myocardial perfusion scintigraphy is an established method in cardiology for the diagnosis and evaluation of coronary artery disease (CAD). Thallium-201 and Tc-99m sestamibi myocardial perfusion imaging has been widely accepted as non-invasive diagnostic procedure for detection of CAD, risk stratification and myocardial viability assessment. But, standard Tl-201 redistribution and same day or 2-day rest/stress Tc-99m sestamibi protocols are time-consuming. Hence, the dual isotope rest thallium-201/stress technetium-99m sestamibi gated single-photon emission tomography protocol has gained increasing popularity for these applications. Combining the use of thallium-201 with technetium-99m agents permits optimal image resolution and simultaneous assessment of viability. Dual-isotope imaging may be separate or simultaneous acquisition set-up. The more rapid completion of these studies is appreciated as an advantage by patients, technologists, interpreting and referring physicians, nurses and hospital management. Simultaneous imaging has the potential advantages of precise pixel registration and artifacts, if present, are identical in both thallium and sestamibi, and require only one set of imaging. Also, there are some disadvantages of spillover of activity from the Tc-99m to the Tl-201 window. Fortunately, despite this problem it can be overcome. Separate acquisition dual isotope also has some disadvantages. Difference in defect resolution in attenuation and scatter between T-201 and Tc-99m sestamibi potentially results in interpretation problems. But, studies about cost-effectiveness of dual isotope imaging showed that some selective elimination of the rest studies may decrease the cost of the nuclear procedures and should be considered in the current care health system.

Evaluating Thermodynamic Integration Performance of the New Amber Molecular Dynamics Package and Assess Potential Halogen Bonds of Enoyl-ACP Reductase (FabI) Benzimidazole Inhibitors

PubMed Central

Su, Pin-Chih; Johnson, Michael E.

2015-01-01

Thermodynamic integration (TI) can provide accurate binding free energy insights in a lead optimization program, but its high computational expense has limited its usage. In the effort of developing an efficient and accurate TI protocol for FabI inhibitors lead optimization program, we carefully compared TI with different Amber molecular dynamics (MD) engines (sander and pmemd), MD simulation lengths, the number of intermediate states and transformation steps, and the Lennard-Jones and Coulomb Softcore potentials parameters in the one-step TI, using eleven benzimidazole inhibitors in complex with Francisella tularensis enoyl acyl reductase (FtFabI). To our knowledge, this is the first study to extensively test the new AMBER MD engine, pmemd, on TI and compare the parameters of the Softcore potentials in the one-step TI in a protein-ligand binding system. The best performing model, the one-step pmemd TI, using 6 intermediate states and 1 ns MD simulations, provides better agreement with experimental results (RMSD = 0.52 kcal/mol) than the best performing implicit solvent method, QM/MM-GBSA from our previous study (RMSD = 3.00 kcal/mol), while maintaining similar efficiency. Briefly, we show the optimized TI protocol to be highly accurate and affordable for the FtFabI system. This approach can be implemented in a larger scale benzimidazole scaffold lead optimization against FtFabI. Lastly, the TI results here also provide structure-activity relationship insights, and suggest the para-halogen in benzimidazole compounds might form a weak halogen bond with FabI, which is a well-known halogen bond favoring enzyme. PMID:26666582

Mapping the Binding Interface of VEGF and a Monoclonal Antibody Fab-1 Fragment with Fast Photochemical Oxidation of Proteins (FPOP) and Mass Spectrometry

NASA Astrophysics Data System (ADS)

Zhang, Ying; Wecksler, Aaron T.; Molina, Patricia; Deperalta, Galahad; Gross, Michael L.

2017-05-01

We previously analyzed the Fab-1:VEGF (vascular endothelial growth factor) system described in this work, with both native top-down mass spectrometry and bottom-up mass spectrometry (carboxyl-group or GEE footprinting) techniques. This work continues bottom-up mass spectrometry analysis using a fast photochemical oxidation of proteins (FPOP) platform to map the solution binding interface of VEGF and a fragment antigen binding region of an antibody (Fab-1). In this study, we use FPOP to compare the changes in solvent accessibility by quantitating the extent of oxidative modification in the unbound versus bound states. Determining the changes in solvent accessibility enables the inference of the protein binding sites (epitope and paratopes) and a comparison to the previously published Fab-1:VEGF crystal structure, adding to the top-down and bottom-up data. Using this method, we investigated peptide-level and residue-level changes in solvent accessibility between the unbound proteins and bound complex. Mapping these data onto the Fab-1:VEGF crystal structure enabled successful characterization of both the binding region and regions of remote conformation changes. These data, coupled with our previous higher order structure (HOS) studies, demonstrate the value of a comprehensive toolbox of methods for identifying the putative epitopes and paratopes for biotherapeutic antibodies.

Safety and cost-effectiveness of a clinical protocol implemented to standardize the use of Crotalidae polyvalent immune Fab antivenom at an academic medical center.

PubMed

Weant, Kyle A; Bowers, Rebecca C; Reed, Janelle; Braun, Kristopher A; Dodd, David M; Baker, Stephanie N

2012-05-01

To evaluate the safety and cost-effectiveness of a clinical protocol adopted in June 2006 that included a comprehensive, objective assessment of snake bite envenomations and standardized the use of Crotalidae polyvalent immune Fab antivenom (FabAV). Retrospective medical record review. Academic medical center that serves as the regional level I trauma center. Seventy-five adults treated with FabAV for snake envenomations in the emergency department between June 1, 2003, and June 1, 2009; 30 patients received treatment according to the protocol (treatment group), and 45 patients received treatment that did not adhere to the protocol (control group). Demographic and envenomation characteristics, as well as treatment details, were collected for all patients. In addition, information on quantity of FabAV vials required, length of hospital stay, and length of intensive care unit stay were compared between the treatment and control groups. In the treatment group, significantly fewer vials of FabAV were used (2.5 vs 4.727 vials, p=0.007). This decreased in usage correlated to a cost savings of approximately $2000/patient. Despite no significant difference in the severity of the envenomations between the two groups (p=0.379), the treatment group experienced a significantly shorter hospital length of stay (1.933 vs 2.791 days, p=0.030). No significant difference in the progression to fasciotomy or the development of allergic reactions was noted between the two groups. Use of a clinical protocol related to snake envenomations resulted in approximately two fewer vials of FabAV required for each patient. In addition, the treatment group experienced a shorter hospital length of stay without a corresponding increase in adverse events or envenomation progression. Data show that use of the protocol was cost-effective. The development of institution-specific multidisciplinary protocols regarding snake bite envenomations is recommended. Clinical pharmacists can play a vital role in

Matching the decay half-life with the biological half-life: ImmunoPET imaging with 44 Sc-labeled Cetuximab Fab fragment

DOE PAGES

Chakravarty, Rubel; Goel, Shreya; Valdovinos, Hector F.; ...

2014-11-11

Scandium-44 (t 1/2 = 3.9 h) is a relatively new radioisotope of potential interest for use in clinical positron emission tomography (PET). Herein, we report, for the first time, the room-temperature radiolabeling of proteins with 44Sc for in vivo PET imaging. For this purpose, the Fab fragment of Cetuximab, a monoclonal antibody that binds with high affinity to epidermal growth factor receptor (EGFR), was generated and conjugated with N-[(R)-2-amino-3-( para-isothiocyanato-phenyl)propyl]- trans-(S,S)-cyclohexane-1,2-diamine- N,N,N',N'',N''-pentaacetic acid (CHX-A"-DTPA). The high purity of Cetuximab-Fab was confirmed by SDS-PAGE and mass spectrometry. The potential of the bioconjugate for PET imaging of EGFR expression in human glioblastomamore » (U87MG) tumor-bearing mice was investigated after 44Sc labeling. PET imaging revealed rapid tumor uptake (maximum uptake of ~12% ID/g at 4 h postinjection) of 44Sc–CHX-A"-DTPA–Cetuximab-Fab with excellent tumor-to-background ratio, which might allow for same day PET imaging in future clinical studies. Immunofluorescence staining was conducted to correlate tracer uptake in the tumor and normal tissues with EGFR expression. As a result, this successful strategy for immunoPET imaging of EGFR expression using 44Sc–CHX-''-DTPA–Cetuximab-Fab can make clinically translatable advances to select the right population of patients for EGFR-targeted therapy and also to monitor the therapeutic efficacy of anti-EGFR treatments.« less

The role of the PI(3,5)P2 kinase TbFab1 in endo/lysosomal trafficking in Trypanosoma brucei.

PubMed

Gilden, Julia K; Umaer, Khan; Kruzel, Emilia K; Hecht, Oliver; Correa, Renan O; Mansfield, John M; Bangs, James D

2017-06-01

Protein trafficking through endo/lysosomal compartments is critically important to the biology of the protozoan parasite Trypanosoma brucei, but the routes material may take to the lysosome, as well as the molecular factors regulating those routes, remain incompletely understood. Phosphoinositides are signaling phospholipids that regulate many trafficking events by recruiting specific effector proteins to discrete membrane subdomains. In this study, we investigate the role of one phosphoinositide, PI(3,5)P 2 in T. brucei. We find a low steady state level of PI(3,5)P 2 in bloodstream form parasites comparable to that of other organisms. RNAi knockdown of the putative PI(3)P-5 kinase TbFab1 decreases the PI(3,5)P 2 pool leading to rapid cell death. TbFab1 and PI(3,5)P 2 both localize strongly to late endo/lysosomes. While most trafficking functions were intact in TbFab1 deficient cells, including both endocytic and biosynthetic trafficking to the lysosome, lysosomal turnover of an endogenous ubiquitinylated membrane protein, ISG65, was completely blocked suggesting that TbFab1 plays a role in the ESCRT-mediated late endosomal/multivesicular body degradative pathways. Knockdown of a second component of PI(3,5)P 2 metabolism, the PI(3,5)P 2 phosphatase TbFig4, also resulted in delayed turnover of ISG65. Together, these results demonstrate an essential role for PI(3,5)P 2 in the turnover of ubiquitinylated membrane proteins and in trypanosome endomembrane biology. Copyright © 2017 Elsevier B.V. All rights reserved.

Pilot study testing the effect of physical training over the myocardial perfusion and quality of life in patients with primary microvascular angina.

PubMed

de Carvalho, Eduardo Elias Vieira; Santi, Giovani Luiz; Crescêncio, Júlio César; de Oliveira, Luciano Fonseca Lemos; dos Reis, Daniela Caetano Costa; Figueiredo, Alexandre Baldini; Pintya, Antonio Osvaldo; Lima-Filho, Moyses Oliveira; Gallo-Júnior, Lourenço; Marin-Neto, José Antonio; Simões, Marcus Vinícius

2015-02-01

Primary microvascular angina (PMA) is a common clinical condition associated to negative impact on quality of life (QOL) and reduced physical capacity. This study aimed at evaluating the effects of aerobic physical training (APT) on myocardial perfusion, physical capacity, and QOL in patients with PMA. We investigated 12 patients (53.8 ± 9.7 years old; 7 women) with PMA, characterized by angina, angiographycally normal coronary arteries, and reversible perfusion defects (RPDs) detected on (99m)Tc-sestamibi-SPECT myocardial perfusion scintigraphy (MPS). At baseline and after 4 month of APT, the patients underwent MPS, cardiopulmonary test, and QOL questionnaire. Stress-rest MPS images were visually analyzed by attributing semi-quantitative scores (0 = normal; 4 = absent uptake), using a 17-segment left ventricular model. Summed stress, rest, and difference scores (SDS) were calculated. In comparison to the baseline, in the post-training we observed a significant increase in peak-VO2 (19.4 ± 4.8 and 22.1 ± 6.2 mL·kg(-1)·minute(-1), respectively, P = .01), reduction of SDS (10.1 ± 8.8 and 2.8 ± 4.9, P = .008), and improvement in QOL scores. Physical training in patients with PMA is associated with reduction of myocardial perfusion abnormalities, increasing of physical capacity, and improvement in QOL. The findings of this hypothesis-generating study suggest that APT can be a valid therapeutic option for patients with PMA.

Lack of correlation between extended pH monitoring and scintigraphy in the evaluation of infants with gastroesophageal reflux

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tolia, V.; Calhoun, J.A.; Kuhns, L.R.

1990-05-01

Sixty-nine infants younger than 1 year of age, with symptoms of persistent vomiting, recurrent choking, apnea, persistent cough, or stridor, were evaluated for gastroesophageal reflux. All infants underwent extended intraesophageal pH monitoring for 16 to 24 hours as well as gastroesophageal scintigraphy with technetium 99m sulfur colloid to study the correlation between the two tests. Forty-eight infants exhibited reflux with extended pH monitoring whereas 46 infants showed reflux with scintigraphy. However, the diagnosis of reflux in individual patients by extended pH monitoring corresponded poorly with the diagnosis of reflux in the same patients by scintigraphy. Similarly, no correlation was observedmore » between extended pH monitoring and scintigraphy results, whether expressed as percent gastric emptying or as gastroesophageal reflux ratio. We conclude that extended pH monitoring and scintigraphy measure different pathophysiologic phenomena and detect reflux under different conditions. The ability of these tests to detect reflux may be complementary and they may be of greatest value when used together to enhance the sensitivity and specificity of the diagnostic evaluation. Extended pH monitoring and scintigraphy should not be used interchangeably to monitor gastroesophageal reflux.« less

Perioperative Assessment of Myocardial Deformation

PubMed Central

Duncan, Andra E.; Alfirevic, Andrej; Sessler, Daniel I.; Popovic, Zoran B.; Thomas, James D.

2014-01-01

Evaluation of left ventricular performance improves risk assessment and guides anesthetic decisions. However, the most common echocardiographic measure of myocardial function, the left ventricular ejection fraction (LVEF), has important limitations. LVEF is limited by subjective interpretation which reduces accuracy and reproducibility, and LVEF assesses global function without characterizing regional myocardial abnormalities. An alternative objective echocardiographic measure of myocardial function is thus needed. Myocardial deformation analysis, which performs quantitative assessment of global and regional myocardial function, may be useful for perioperative care of surgical patients. Myocardial deformation analysis evaluates left ventricular mechanics by quantifying strain and strain rate. Strain describes percent change in myocardial length in the longitudinal (from base to apex) and circumferential (encircling the short-axis of the ventricle) direction and change in thickness in the radial direction. Segmental strain describes regional myocardial function. Strain is a negative number when the ventricle shortens longitudinally or circumferentially and is positive with radial thickening. Reference values for normal longitudinal strain from a recent meta-analysis using transthoracic echocardiography are (mean ± SD) −19.7 ± 0.4%, while radial and circumferential strain are 47.3 ± 1.9 and −23.3 ± 0.7%, respectively. The speed of myocardial deformation is also important and is characterized by strain rate. Longitudinal systolic strain rate in healthy subjects averages −1.10 ± 0.16 sec−1. Assessment of myocardial deformation requires consideration of both strain (change in deformation), which correlates with LVEF, and strain rate (speed of deformation), which correlates with rate of rise of left ventricular pressure (dP/dt). Myocardial deformation analysis also evaluates ventricular relaxation, twist, and untwist, providing new and noninvasive methods to

Direct Evidence that Myocardial Insulin Resistance following Myocardial Ischemia Contributes to Post-Ischemic Heart Failure

PubMed Central

Fu, Feng; Zhao, Kun; Li, Jia; Xu, Jie; Zhang, Yuan; Liu, Chengfeng; Yang, Weidong; Gao, Chao; Li, Jun; Zhang, Haifeng; Li, Yan; Cui, Qin; Wang, Haichang; Tao, Ling; Wang, Jing; Quon, Michael J; Gao, Feng

2015-01-01

A close link between heart failure (HF) and systemic insulin resistance has been well documented, whereas myocardial insulin resistance and its association with HF are inadequately investigated. This study aims to determine the role of myocardial insulin resistance in ischemic HF and its underlying mechanisms. Male Sprague-Dawley rats subjected to myocardial infarction (MI) developed progressive left ventricular dilation with dysfunction and HF at 4 wk post-MI. Of note, myocardial insulin sensitivity was decreased as early as 1 wk after MI, which was accompanied by increased production of myocardial TNF-α. Overexpression of TNF-α in heart mimicked impaired insulin signaling and cardiac dysfunction leading to HF observed after MI. Treatment of rats with a specific TNF-α inhibitor improved myocardial insulin signaling post-MI. Insulin treatment given immediately following MI suppressed myocardial TNF-α production and improved cardiac insulin sensitivity and opposed cardiac dysfunction/remodeling. Moreover, tamoxifen-induced cardiomyocyte-specific insulin receptor knockout mice exhibited aggravated post-ischemic ventricular remodeling and dysfunction compared with controls. In conclusion, MI induces myocardial insulin resistance (without systemic insulin resistance) mediated partly by ischemia-induced myocardial TNF-α overproduction and promotes the development of HF. Our findings underscore the direct and essential role of myocardial insulin signaling in protection against post-ischemic HF. PMID:26659007

Dissection of epitope-specific mechanisms of neutralization of influenza virus by intact IgG and Fab fragments.

PubMed

Williams, James A; Gui, Long; Hom, Nancy; Mileant, Alexander; Lee, Kelly K

2017-12-20

The neutralizing antibody (nAb) response against the influenza virus's hemagglutinin (HA) fusion glycoprotein is important for preventing viral infection, but we lack a comprehensive understanding of the mechanisms by which these antibodies act. Here we investigated the effect of nAb binding and the role of IgG bivalency on inhibition of HA function for nAbs targeting distinct HA epitopes. HC19 targets the receptor-binding pocket at HA's distal end, while FI6v3 binds primarily to the HA2 fusion subunit towards the base of the stalk. Surprisingly, HC19 inhibited HA's ability to induce lipid mixing by preventing structural rearrangement of HA under fusion activating conditions. These results suggest that nAbs such as HC19 not only act by blocking receptor binding, but also inhibit key late-stage HA conformational changes required for fusion. Intact HC19 IgG was also shown to crosslink separate virus particles, burying large proportions of HA within aggregates where they are blocked from interacting with target membranes; Fabs yielded no such aggregation and displayed weaker neutralization than IgG, emphasizing the impact of bivalency on the ability to neutralize virus. In contrast, the stem-targeting nAb FI6v3 did not aggregate particles. The Fab was significantly less effective than IgG in preventing both membrane disruption and fusion. We infer that inter-spike crosslinking within a given particle by FI6v3 IgG may be critical to its potent neutralization, as no significant neutralization occurred with Fabs. These results demonstrate that IgG bivalency enhances HA inhibition through functionally important modes not evident in pared down Fab-soluble HA structures. IMPORTANCE The influenza virus's hemagglutinin (HA) fusion glycoprotein mediates entry into target cells and is the primary antigenic target of neutralizing antibodies (nAbs). Our current structural understanding of mechanisms of Ab-mediated neutralization largely relies on high resolution characterization

Clinical utility of bone scintigraphy in patients with limb pain of suspected musculoskeletal origin

PubMed Central

Ferrari, Robert

2015-01-01

Objective To determine the clinical utility of bone scintigraphy in patients with limb pain of suspected musculoskeletal origin. Material and Methods All patients aged ≥18 years who were referred for diagnosis and management of limb pain were diagnosed on the basis of history, physical examination, and investigations excluding bone scintigraphy. After the presumptive diagnosis was made (the pre-test diagnosis), all subjects underwent bone scintigraphy, or if they had a previous bone scintigram for their pain condition, the results of that scintigram were reviewed. Then, the pre-test diagnosis was reviewed in light of the bone scintigraphy findings and repeat clinical assessment as needed. The post-test diagnosis was considered either as unchanged diagnosis or changed diagnosis for the region or regions of interest. Results There were 118 females (54.8%) and 97 males (45.2%). The mean age of the entire group was 36±8.1 years (range: 18–87 years). The mean duration of the symptoms was 17.4±11.2 months (range: 1–264 months). Of the 215 subjects, 212 had a bone scintigram. Of these 212 subjects, none had a changed diagnosis. Conclusion In the evaluation of limb pain of suspected musculoskeletal origin, scintigraphy is unlikely to alter the pre-test diagnosis or affect treatment decisions after history, physical examination, and non-scintigraphic investigations. The clinical utility of scinitigraphy in this setting is low. PMID:27708914

Relating gastric scintigraphy and symptoms to motility capsule transit and pressure findings in suspected gastroparesis

PubMed Central

Hasler, W. L.; May, K. P.; Wilson, L. A.; Van Natta, M.; Parkman, H. P.; Pasricha, P. J.; Koch, K. L.; Abell, T. L.; McCallum, R. W.; Nguyen, L. A.; Snape, W. J.; Sarosiek, I.; Clarke, J. O.; Farrugia, G.; Calles-Escandon, J.; Grover, M.; Tonascia, J.; Lee, L. A.; Miriel, L.; Hamilton, F. A.

2018-01-01

Background Wireless motility capsule (WMC) findings are incompletely defined in suspected gastroparesis. We aimed to characterize regional WMC transit and contractility in relation to scintigraphy, etiology, and symptoms in patients undergoing gastric emptying testing. Methods A total of 209 patients with gastroparesis symptoms at NIDDK Gastroparesis Consortium centers underwent gastric scintigraphy and WMCs on separate days to measure regional transit and contractility. Validated questionnaires quantified symptoms. Key Results Solid scintigraphy and liquid scintigraphy were delayed in 68.8% and 34.8% of patients; WMC gastric emptying times (GET) were delayed in 40.3% and showed 52.8% agreement with scintigraphy; 15.5% and 33.5% had delayed small bowel (SBTT) and colon transit (CTT) times. Transit was delayed in ≥2 regions in 23.3%. Rapid transit was rarely observed. Diabetics had slower GET but more rapid SBTT versus idiopathics (P ≤ .02). GET delays related to greater scintigraphic retention, slower SBTT, and fewer gastric contractions (P ≤ .04). Overall gastroparesis symptoms and nausea/vomiting, early satiety/fullness, bloating/distention, and upper abdominal pain subscores showed no relation to WMC transit. Upper and lower abdominal pain scores (P ≤ .03) were greater with increased colon contractions. Constipation correlated with slower CTT and higher colon contractions (P = .03). Diarrhea scores were higher with delayed SBTT and CTT (P ≤ .04). Conclusions & Inferences Wireless motility capsules define gastric emptying delays similar but not identical to scintigraphy that are more severe in diabetics and relate to reduced gastric contractility. Extragastric transit delays occur in >40% with suspected gastroparesis. Gastroparesis symptoms show little association with WMC profiles, although lower symptoms relate to small bowel or colon abnormalities. PMID:28872760

Molecular dynamics and docking simulations as a proof of high flexibility in E. coli FabH and its relevance for accurate inhibitor modeling

NASA Astrophysics Data System (ADS)

Pérez-Castillo, Yunierkis; Froeyen, Matheus; Cabrera-Pérez, Miguel Ángel; Nowé, Ann

2011-04-01

Bacterial β-ketoacyl-acyl carrier protein synthase III (FabH) has become an attractive target for the development of new antibacterial agents which can overcome the increased resistance of these pathogens to antibiotics in clinical use. Despite several efforts have been dedicated to find inhibitors for this enzyme, it is not a straightforward task, mainly due its high flexibility which makes difficult the structure-based design of FabH inhibitors. Here, we present for the first time a Molecular Dynamics (MD) study of the E. colil FabH enzyme to explore its conformational space. We compare the flexibility of this enzyme for the unliganded protein and an enzyme-inhibitor complex and find a correspondence between our modeling results and the experimental evidence previously reported for this enzyme. Furthermore, through a 100 ns MD simulation of the unliganded enzyme we extract useful information related to the concerted motions that take place along the principal components of displacement. We also establish a relation between the presence of water molecules in the oxyanion hole with the conformational stability of structural important loops. Representative conformations of the binding pocket along the whole trajectory of the unliganded protein are selected through cluster analysis and we find that they contain a conformational diversity which is not provided by the X-ray structures of ecFabH. As a proof of this last hypothesis, we use a set of 10 FabH inhibitors and show that they cannot be correctly modeled in any available X-ray structure, while by using our set of conformations extracted from the MD simulations, this task can be accomplish. Finally, we show the ability of short MD simulations for the refinement of the docking binding poses and for MM-PBSA calculations to predict stable protein-inhibitor complexes in this enzyme.

Evaluating thermodynamic integration performance of the new amber molecular dynamics package and assess potential halogen bonds of enoyl-ACP reductase (FabI) benzimidazole inhibitors.

PubMed

Su, Pin-Chih; Johnson, Michael E

2016-04-05

Thermodynamic integration (TI) can provide accurate binding free energy insights in a lead optimization program, but its high computational expense has limited its usage. In the effort of developing an efficient and accurate TI protocol for FabI inhibitors lead optimization program, we carefully compared TI with different Amber molecular dynamics (MD) engines (sander and pmemd), MD simulation lengths, the number of intermediate states and transformation steps, and the Lennard-Jones and Coulomb Softcore potentials parameters in the one-step TI, using eleven benzimidazole inhibitors in complex with Francisella tularensis enoyl acyl reductase (FtFabI). To our knowledge, this is the first study to extensively test the new AMBER MD engine, pmemd, on TI and compare the parameters of the Softcore potentials in the one-step TI in a protein-ligand binding system. The best performing model, the one-step pmemd TI, using 6 intermediate states and 1 ns MD simulations, provides better agreement with experimental results (RMSD = 0.52 kcal/mol) than the best performing implicit solvent method, QM/MM-GBSA from our previous study (RMSD = 3.00 kcal/mol), while maintaining similar efficiency. Briefly, we show the optimized TI protocol to be highly accurate and affordable for the FtFabI system. This approach can be implemented in a larger scale benzimidazole scaffold lead optimization against FtFabI. Lastly, the TI results here also provide structure-activity relationship insights, and suggest the parahalogen in benzimidazole compounds might form a weak halogen bond with FabI, which is a well-known halogen bond favoring enzyme. © 2015 Wiley Periodicals, Inc.

Bone scintigraphy in children with cat scratch disease.

PubMed

Donoso, Gilda; Paulsen, Cesar; Riquelme, Paulina; Lobo, Gabriel; Gutierrez, Daniela; Perez, Andrés; Jiménez, César

2013-12-01

The objective of this study was to evaluate the degree and incidence of bone involvement in patients with cat scratch disease. Patients admitted between 2004 and 2011 at the pediatric department for cat scratch disease and a positive serology for Bartonella henselae were identified. Only those having undergone a bone scintigraphy (BS) were included in this retrospective study. Sixteen girls and 8 boys with a mean age of 7 years were studied. Bone scintigraphy was positive in 6 (25%), but only 2 had bone pain. Axial involvement was present in all 6 patients, and appendicular lesions in 3 of them. Three patients had a BS control, with improvement or normalization after treatment with antibiotics. Bone involvement occurs infrequently in patients with cat scratch disease and is not always associated with specific signs. Cat scratch disease must be suspected in patients with fever of unknown origin presenting multifocal lesions on BS.

Prognostic value of cardiac sympathetic nerve activity evaluated by [123I]m-iodobenzylguanidine imaging in patients with ST-segment elevation myocardial infarction.

PubMed

Kasama, Shu; Toyama, Takuji; Sumino, Hiroyuki; Kumakura, Hisao; Takayama, Yoshiaki; Minami, Kazutomo; Ichikawa, Shuichi; Matsumoto, Naoya; Sato, Yuichi; Kurabayashi, Masahiko

2011-01-01

Many studies have shown that cardiac sympathetic nerve activity evaluated by [(123)I]m-iodobenzylguanidine ([(123)I]MIBG) scintigraphic study during a stable period is useful for determining the prognosis of patients with chronic heart failure. To examine whether results of this imaging method performed 3 weeks after the onset of ST-segment elevation myocardial infarction (STEMI) are a reliable prognostic marker for patients with STEMI. The study analysed findings for 213 consecutive patients with STEMI undergoing [(123)I]MIBG scintigraphy. The left ventricular (LV) end-diastolic and end-systolic volume and LV ejection fraction (EF) were determined by left ventriculography or echocardiography 3 weeks after the onset of STEMI. The delayed total defect score, heart-to-mediastinum ratio and washout rate (WR) were also determined from [(123)I]MIBG scintigraphy at the same time. Of the 213 patients, 46 experienced major adverse cardiac events (MACE) during the study. The median follow-up period was 982 days. Patients were divided into an event-free group (n = 167; 78.4%) and a MACE group (n = 46; 21.6%). The LV and [(123)I]MIBG scintigraphic parameters in the event-free group were better than those in the MACE group. Multivariate Cox regression analyses revealed that WR was a significant predictor of MACE along with oral nicorandil (ATP-sensitive potassium channel opener) treatment and undergoing percutaneous coronary intervention. On Kaplan-Meier analysis, the event-free rate of patients with a WR<40% was significantly higher than that in patients with a WR ≥ 40% (p<0.001). Even when confined to patients with LVEF>45%, WR was a predictor of MACE, pump failure death, cardiac death and progression of heart failure in patients with STEMI. WR evaluated by [(123)I]MIBG scintigraphy 3 weeks after the onset of STEMI is a significant predictor of MACE in patients with STEMI, independent of LVEF.

Charge-mediated Fab-Fc interactions in an IgG1 antibody induce reversible self-association, cluster formation, and elevated viscosity.

PubMed

Arora, Jayant; Hu, Yue; Esfandiary, Reza; Sathish, Hasige A; Bishop, Steven M; Joshi, Sangeeta B; Middaugh, C Russell; Volkin, David B; Weis, David D

Concentration-dependent reversible self-association (RSA) of monoclonal antibodies (mAbs) poses a challenge to their pharmaceutical development as viable candidates for subcutaneous delivery. While the role of the antigen-binding fragment (Fab) in initiating RSA is well-established, little evidence supports the involvement of the crystallizable fragment (Fc). In this report, a variety of biophysical tools, including hydrogen exchange mass spectrometry, are used to elucidate the protein interface of such non-covalent protein-protein interactions. Using dynamic and static light scattering combined with viscosity measurements, we find that an IgG1 mAb (mAb-J) undergoes RSA primarily through electrostatic interactions and forms a monomer-dimer-tetramer equilibrium. We provide the first direct experimental mapping of the interface formed between the Fab and Fc domains of an antibody at high protein concentrations. Charge distribution heterogeneity between the positively charged interface spanning complementarity-determining regions CDR3H and CDR2L in the Fab and a negatively charged region in C H 3/Fc domain mediates the RSA of mAb-J. When arginine and NaCl are added, they disrupt RSA of mAb-J and decrease the solution viscosity. Fab-Fc domain interactions between mAb monomers may promote the formation of large transient antibody complexes that ultimately cause increases in solution viscosity. Our findings illustrate how limited specific arrangements of amino-acid residues can cause mAbs to undergo RSA at high protein concentrations and how conserved regions in the Fc portion of the antibody can also play an important role in initiating weak and transient protein-protein interactions.

Value of positive myocardial technetium-99m-pyrophosphate scintigraphy in the noninvasive diagnosis of cardiac amyloidosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wizenberg, T.A.; Muz, J.; Sohn, Y.H.

1982-04-01

Ten consecutive patients with tissue-proven amyloidosis, seven of whom presented with congestive heart failure, were found to exhibit intense diffuse uptake of technetium-99m-pyrophosphate (Tc-99m-PYP) on cardiac radionuclide imaging. The patients exhibited echocardiographic and systolic time interval abnormalities suggesting combined restrictive and congestive cardiomyopathic changes. On M-mode echocardiograms, there was symmetrically increased thickness of the interventricular septum and left ventricular (LV) posterior wall in diastole (10 of 10), decreased fractional shortening of the LV minor axis diameter in systole (eight of nine), and decreased percent thickening of the LV minor axis diameter in systole (eight of nine) and LV posterior wallmore » (10 of 10) in systole. Three patients demonstrated enlarged LV end-diastolic diameter. All 10 patients had abnormal PEP/LVET and eight had shortened LVETI. When combined with noninvasive tests of LV performance, positive myocardial pyrophosphate (PYP) scanning provides a new and useful adjunct in the diagnosis of amyloid heart disease.« less

Slow-Onset Inhibition of the FabI Enoyl Reductase from Francisella Tularensis: Residence Time and In Vivo Activity

PubMed Central

Lu, Hao; England, Kathleen; Ende, Christopher am; Truglio, James J.; Luckner, Sylvia; Reddy, B. Gopal; Marlenee, Nikki; Knudson, Susan E.; Knudson, Dennis L.; Bowen, Richard A.; Kisker, Caroline; Slayden, Richard A.; Tonge, Peter J.

2009-01-01

Francisella tularensis is a highly virulent and contagious gram-negative intracellular bacterium that causes the disease tularemia in mammals. The high infectivity and the ability of the bacterium to survive for weeks in a cool, moist environment have raised the possibility that this organism could be exploited deliberately as a potential biological weapon. Fatty acid biosynthesis (FAS-II) is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterials. The FAS-II enoyl reductase ftuFabI has been cloned and expressed, and a series of diphenyl ethers have been identified that are subnanomolar inhibitors of the enzyme with MIC90 values as low as 0.00018 μg/ml. The existence of a linear correlation between the Ki and MIC values strongly suggests that the antibacterial activity of the diphenyl ethers results from direct inhibition of ftuFabI within the cell. The compounds are slow onset inhibitors of ftuFabI, and the residence time of the inhibitors on the enzyme correlates with their in vivo activity in a mouse model of tularemia infection. Significantly, the rate of breakdown of the enzyme-inhibitor complex is a better predictor of in vivo activity than the overall thermodynamic stability of the complex, a concept that has important implications for the discovery of novel chemotherapeutics that normally rely on equilibrium measurements of potency. PMID:19206187

The Response Regulator YycF Inhibits Expression of the Fatty Acid Biosynthesis Repressor FabT in Streptococcus pneumoniae

PubMed Central

Mohedano, Maria L.; Amblar, Mónica; de la Fuente, Alicia; Wells, Jerry M.; López, Paloma

2016-01-01

The YycFG (also known as WalRK, VicRK, MicAB, or TCS02) two-component system (TCS) is highly conserved among Gram-positive bacteria with a low G+C content. In Streptococcus pneumoniae the YycF response regulator has been reported to be essential due to its control of pcsB gene expression. Previously we showed that overexpression of yycF in S. pneumoniae TIGR4 altered the transcription of genes involved in cell wall metabolism and fatty acid biosynthesis, giving rise to anomalous cell division and increased chain length of membrane fatty acids. Here, we have overexpressed the yycFG system in TIGR4 wild-type strain and yycF in a TIGR4 mutant depleted of YycG, and analyzed their effects on expression of proteins involved in fatty acid biosynthesis during activation of the TCS. We demonstrate that transcription of the fab genes and levels of their products were only altered in the YycF overexpressing strain, indicating that the unphosphorylated form of YycF is involved in the regulation of fatty acid biosynthesis. In addition, DNA-binding assays and in vitro transcription experiments with purified YycF and the promoter region of the FabTH-acp operon support a direct inhibition of transcription of the FabT repressor by YycF, thus confirming the role of the unphosphorylated form in transcriptional regulation. PMID:27610104

Dual Constant Domain-Fab: A novel strategy to improve half-life and potency of a Met therapeutic antibody.

PubMed

Cignetto, Simona; Modica, Chiara; Chiriaco, Cristina; Fontani, Lara; Milla, Paola; Michieli, Paolo; Comoglio, Paolo M; Vigna, Elisa

2016-06-01

The kinase receptor encoded by the Met oncogene is a sensible target for cancer therapy. The chimeric monovalent Fab fragment of the DN30 monoclonal antibody (MvDN30) has an odd mechanism of action, based on cell surface removal of Met via activation of specific plasma membrane proteases. However, the short half-life of the Fab, due to its low molecular weight, is a severe limitation for the deployment in therapy. This issue was addressed by increasing the Fab molecular weight above the glomerular filtration threshold through the duplication of the constant domains, in tandem (DCD-1) or reciprocally swapped (DCD-2). The two newly engineered molecules showed biochemical properties comparable to the original MvDN30 in vitro, acting as full Met antagonists, impairing Met phosphorylation and activation of downstream signaling pathways. As a consequence, Met-mediated biological responses were inhibited, including anchorage-dependent and -independent cell growth. In vivo DCD-1 and DCD-2 showed a pharmacokinetic profile significantly improved over the original MvDN30, doubling the circulating half-life and reducing the clearance. In pre-clinical models of cancer, generated by injection of tumor cells or implant of patient-derived samples, systemic administration of the engineered molecules inhibited the growth of Met-addicted tumors. Copyright © 2016 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Recovery of BMIPP uptake and regional wall motion in insulin resistant patients following angioplasty for acute myocardial infarction.

PubMed

Fujino, Takayuki; Ishii, Yoshinao; Takeuchi, Toshiharu; Hirasawa, Kunihiko; Tateda, Kunihiko; Kikuchi, Kenjiro; Hasebe, Naoyuki

2003-09-01

The effect of insulin resistance (IR) on the fatty acid metabolism of myocardium, and therefore on the recovery of left ventricular (LV) wall motion, has not been established in patients with acute myocardial infarction (AMI). A total of consecutive 58 non-diabetic AMI patients who had successfully undergone emergency coronary angioplasty were analyzed retrospectively. They were categorized into 2 groups, normal glucose tolerance (NGT) and impaired glucose tolerance (IGT), based on a 75-g oral glucose tolerance test (OGTT). The parameters of OGTT, myocardial scintigraphy (n=58) (thallium-201 (Tl) and iodine-123-beta-methyl-iodophenylpentadecanoic acid (BMIPP)) and left ventriculography (n=24) were compared in the 2 groups after reperfusion (acute phase) and 3-4 weeks after the AMI (chronic phase). The insulin resistance (IR), estimated by the serum concentration of insulin at 120 min (IRI 120') of the OGTT and by the HOMA (the homeostasis model assessment) index, was higher in the IGT group than in NGT group. An inverse correlation was found between the recovery of regional LV wall motion in the ischemic lesion and the IRI 120' and HOMA index. Although the recovery of BMIPP uptake from the acute to the chronic phase was higher in the IGT group, it was only correlated with the degree of IRI 120', not with the HOMA. IR accompanied by IGT can negatively influence the recovery of regional LV wall motion.

Experimental myocardial infarction

PubMed Central

Kumar, Raj; Joison, Julio; Gilmour, David P.; Molokhia, Farouk A.; Pegg, C. A. S.; Hood, William B.

1971-01-01

The hemodynamic effects of tachycardia induced by atrial pacing were investigated in left ventricular failure of acute and healing experimental myocardial infarction in 20 intact, conscious dogs. Myocardial infarction was produced by gradual inflation of a balloon cuff device implanted around the left anterior descending coronary artery 10-15 days prior to the study. 1 hr after acute myocardial infarction, atrial pacing at a rate of 180 beats/min decreased left ventricular end-diastolic pressure from 19 to 8 mm Hg and left atrial pressure from 17 to 12 mm Hg, without change in cardiac output. In the healing phase of myocardial infarction 1 wk later, atrial pacing decreased left ventricular end-diastolic pressure from 17 to 9 mm Hg and increased the cardiac output by 37%. This was accompanied by evidence of peripheral vasodilation. In two dogs with healing anterior wall myocardial infarction, left ventricular failure was enhanced by partial occlusion of the circumflex coronary artery. Both the dogs developed pulmonary edema. Pacing improved left ventricular performance and relieved pulmonary edema in both animals. In six animals propranolol was given after acute infarction, and left ventricular function deteriorated further. However the pacing-induced augmentation of cardiac function was unaltered and, hence, is not mediated by sympathetics. The results show that the spontaneous heart rate in left ventricular failure of experimental canine myocardial infarction may be less than optimal and that maximal cardiac function may be achieved at higher heart rates. Images PMID:4395910

A case of reversible dilated cardiomyopathy after alpha-interferon therapy in a patient with renal cell carcinoma.

PubMed

Kuwata, Akiko; Ohashi, Masuo; Sugiyama, Masaya; Ueda, Ryuzo; Dohi, Yasuaki

2002-12-01

A 47-year-old man with renal cell carcinoma underwent nephrectomy, and postoperative chemotherapy was performed with recombinant alpha-interferon. Five years later, he experienced dyspnea during physical exertion. An echocardiogram revealed dilatation and systolic dysfunction of the left ventricle, and thallium-201 myocardial scintigraphy showed diffuse heterogeneous perfusion. We diagnosed congestive heart failure because of cardiomyopathy induced by alpha-interferon therapy. Withdrawal of interferon therapy and the combination of an angiotensin-converting enzyme inhibitor, diuretics, and digitalis improved left ventricular systolic function. Furthermore, myocardial scintigraphy using [123I] beta-methyl-p-iodophenylpentadecanoic acid (123I-BMIPP) or [123 I]metaiodobenzylguanidine (123I-MIBG) revealed normal perfusion after the improvement of congestive heart failure. This is a rare case of interferon-induced cardiomyopathy that resulted in normal myocardial images in 123I-BMIPP and 123I-MIBG scintigrams after withdrawal of interferon therapy.

ROC analysis of diagnostic performance in liver scintigraphy.

PubMed

Fritz, S L; Preston, D F; Gallagher, J H

1981-02-01

Studies on the accuracy of liver scintigraphy for the detection of metastases were assembled from 38 sources in the medical literature. An ROC curve was fitted to the observed values of sensitivity and specificity using an algorithm developed by Ogilvie and Creelman. This ROC curve fitted the data better than average sensitivity and specificity values in each of four subsets of the data. For the subset dealing with Tc-99m sulfur colloid scintigraphy, performed for detection of suspected metastases and containing data on 2800 scans from 17 independent series, it was not possible to reject the hypothesis that interobserver variation was entirely due to the use of different decision thresholds by the reporting clinicians. Thus the ROC curve obtained is a reasonable baseline estimate of the performance potentially achievable in today's clinical setting. Comparison of new reports with these data is possible, but is limited by the small sample sizes in most reported series.

Bacterial production and structure-functional validation of a recombinant antigen-binding fragment (Fab) of an anti-cancer therapeutic antibody targeting epidermal growth factor receptor.

PubMed

Kim, Ji-Hun; Sim, Dae-Won; Park, Dongsun; Jung, Tai-Geun; Lee, Seonghwan; Oh, Taeheun; Ha, Jong-Ryul; Seok, Seung-Hyeon; Seo, Min-Duk; Kang, Ho Chul; Kim, Young Pil; Won, Hyung-Sik

2016-12-01

Fragment engineering of monoclonal antibodies (mAbs) has emerged as an excellent paradigm to develop highly efficient therapeutic and/or diagnostic agents. Engineered mAb fragments can be economically produced in bacterial systems using recombinant DNA technologies. In this work, we established recombinant production in Escherichia coli for monovalent antigen-binding fragment (Fab) adopted from a clinically used anticancer mAB drug cetuximab targeting epidermal growth factor receptor (EGFR). Recombinant DNA constructs were designed to express both polypeptide chains comprising Fab in a single vector and to secrete them to bacterial periplasmic space for efficient folding. Particularly, a C-terminal engineering to confer an interchain disulfide bond appeared to be able to enhance its heterodimeric integrity and EGFR-binding activity. Conformational relevance of the purified final product was validated by mass spectrometry and crystal structure at 1.9 Å resolution. Finally, our recombinant cetuximab-Fab was found to have strong binding affinity to EGFR overexpressed in human squamous carcinoma model (A431) cells. Its binding ability was comparable to that of cetuximab. Its EGFR-binding affinity was estimated at approximately 0.7 nM of Kd in vitro, which was quite stronger than the binding affinity of natural ligand EGF. Hence, the results validate that our construction could serve as an efficient platform to produce a recombinant cetuximab-Fab with a retained antigen-binding functionality.

Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type

NASA Technical Reports Server (NTRS)

He, X. M.; Ruker, F.; Casale, E.; Carter, D. C.

1992-01-01

The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 degrees. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

An Alternate, Egg-Free Radiolabeled Meal Formulation for Gastric-Emptying Scintigraphy.

PubMed

Garrigue, Philippe; Bodin-Hullin, Aurore; Gonzalez, Sandra; Sala, Quentin; Guillet, Benjamin

2017-07-01

Tc-radiolabeled scrambled eggs (SEs) are most often used as the ingested solid phase for gastric-emptying scintigraphy, leading egg-reluctant patients to avoid the examination. We formulated and validated 2 egg-free alternate meals, in the absence of any commercialized formulation: chocolate mug cake (MC) and scrambled tofu (ST). Six healthy volunteers underwent gastric-emptying scintigraphy after ingesting Tc-radiolabeled MC, ST, or SE. Gastric retention indexes did not change significantly between formulations (% of overtime variation to SE: MC 7.75% ± 7.1%, ST 7.17% ± 5.8%; P = 0.6618, not statistically significant), suggesting MC and ST as interesting egg-free alternatives.

[Lung scintigraphy].

PubMed

Schümichen, Carl; Schmidt, Matthias; Krause, Thomas

2018-06-01

The S1 guideline for lung scintigraphy has been updated and extended in order to emphasize the advantages oft the method in detecting acute pulmonary embolism (PE) in the periphery oft the lung (subsegmental PE), in underlying subacute and chronic pulmonary disorders, as well as in detecting chronic LE (CTEPH). Method of choice is ventilation / perfusion (V/P) SPECT or V/P SPECT/CT with even higher specificity. Because of its high sensitivity, a threshold (V/P mismatch in at least one segment or two subsegments) is introduced to avoid overtreatment. In case of a change in the therapeutic approach (observation only instead of anticoaculation) the threshold can be omitted. New data concerning the clinical and therapeutical impact of subsegmental PE are included, the chapters open questions have been extented. Other indications for V/P SPECT (secondary diagnoses, abnormalities in pulmonary perfusion, prediction of postoperative lung function) are presented with new data. Schattauer GmbH.

The bionomics of the cottonwood leaf beetle, Chrysomela scripta Fab., on tissue culture hybrid poplars

Treesearch

T.R. Burkot; D.M. Benjamin

1977-01-01

Tissue culture methods are applied to poplars of the Aigeiros group in attempts to overcome premature decline thought to be associated with viral infections. Hybrid selections from such cultures outplanted in 1975 at the F. G. Wilson Nursery in Boscobel, Wisconsin subsequently were severely infested by the Cottonwood Leaf Beetle, Chrysomela scripta Fab. Beetle...

GingisKHAN™ protease cleavage allows a high-throughput antibody to Fab conversion enabling direct functional assessment during lead identification of human monoclonal and bispecific IgG1 antibodies

PubMed Central

Moelleken, Jörg; Gassner, Christian; Lingke, Sabine; Tomaschek, Simone; Tyshchuk, Oksana; Lorenz, Stefan; Mølhøj, Michael

2017-01-01

ABSTRACT The determination of the binding strength of immunoglobulins (IgGs) to targets can be influenced by avidity when the targets are soluble di- or multimeric proteins, or associated to cell surfaces, including surfaces introduced from heterogeneous assays. However, for the understanding of the contribution of a second drug-to-target binding site in molecular design, or for ranking of monovalent binders during lead identification, affinity-based assessment of the binding strength is required. Typically, monovalent binders like antigen-binding fragments (Fabs) are generated by proteolytic cleavage with papain, which often results in a combination of under- and over-digestion, and requires specific optimization and chromatographic purification of the desired Fabs. Alternatively, the Fabs are produced by recombinant approaches. Here, we report a lean approach for the functional assessment of human IgG1s during lead identification based on an in-solution digestion with the GingisKHAN™ protease, generating a homogenous pool of intact Fabs and Fcs and enabling direct assaying of the Fab in the digestion mixture. The digest with GingisKHAN™ is highly specific and quantitative, does not require much optimization, and the protease does not interfere with methods typically applied for lead identification, such as surface plasmon resonance or cell-based assays. GingisKHAN™ is highly suited to differentiate between affinity and avidity driven binding of human IgG1 monoclonal and bispecific antibodies during lead identification. PMID:28805498

Relevance of spontaneous fabT mutations to a streptococcal toxic shock syndrome to non-streptococcal toxic shock syndrome transition in the novel-type Streptococcus pyogenes isolates that lost a salRK.

PubMed

Tatsuno, Ichiro; Okada, Ryo; Matsumoto, Masakado; Hata, Nanako; Matsui, Hideyuki; Zhang, Yan; Isaka, Masanori; Hasegawa, Tadao

2016-05-01

Streptococcus pyogenes is a causative agent of streptococcal toxic shock syndrome (STSS). Mutations in covR/S or rgg, negative regulators, can reportedly modulate the severity of infection in this pathogen. Recently, we showed that the regions encoding the SalR-SalK, a two-component regulatory system, were deleted in some emm 1-type isolates (named as 'novel-type'). In this study, the two novel 'STSS' isolates 10-85stss and 11-171stss were more virulent than the two novel 'non-STSS' isolates 11O-2non and 11T-3non when examined using a mouse model of invasive infection. Genome-sequencing experiments using the three strains 10-85stss , 11-171stss , and 11O-2non detected only one single nucleotide polymorphism that causes a non-synonymous mutation in fabT encoding a transcriptional regulator in strain 11O-2non . Loss of fabT reduced the high level of virulence observed in the STSS isolates to that in the non-STSS isolates, and introduction of an intact fabT compensated the lower virulence of 11O-2non , suggesting that the mutation in fabT, but not in covR/S or rgg, is involved in the differential virulence among the novel-type clinical isolates. This type of non-synonymous fabT mutation was also identified in 12 non-STSS isolates (including 11O-2non and 11T-3non ), and most of those 12 isolates showed impaired FabT function. © 2016 APMIS. Published by John Wiley & Sons Ltd.

Microvascular autonomic dysfunction may justify false-positive stress myocardial perfusion imaging in patients with liver cirrhosis undergoing liver transplantation.

PubMed

Senzolo, M; Bassanello, M; Graziotto, A; Zucchetta, P; Cillo, U; Maraglino, G; Loreno, M; Bellotto, F; Davià, G; Burra, P

2008-01-01

Up to 15% of liver transplant candidates have asymptomatic coronary artery diseases, which increase the risk of cardiac complications during and after transplantation. The aim of this study was to prospectively investigate the usefulness of an integrated cardiological approach in cirrhotic patients undergoing liver transplantation. Twenty-four consecutive patients undergoing evaluation for liver transplantation were studied by assessing risk factors for coronary artery diseases, electrocardiogram with QTc interval determination, chest X-ray, echocardiography, 24-hour Holter monitor, myocardial perfusion scintigraphy (99mTc)MIBI-GSPECT at rest and after dipyridamole infusion. Cardiac (123)I-metaiodobenzylguanidine (MIBG) scan and coronarography were performed in patients with myocardial perfusion defects. Twenty three of 24 patients underwent successful liver transplantation; one patient died on the waiting list. Before liver transplantation, 29% of patients were diabetic and 41% were smokers. Eleven of 24 patients had a prolonged QTc interval, and 3/24 had positive myocardioscintigraphy after dipyridamole infusion: in two coronarography was negative, while the (123)I-MIBG washout was altered. No cardiac events were recorded during the short-and long-term follow-up after surgery. Predictive value of positive cardiac (99mTc)MIBI-GSPECT in patients with liver cirrhosis is low, and this may be due to alterations of cardiac microvascular tone as showed by cardiac (123)I-MIBG scan.

Isolation of a human anti-epidermal growth factor receptor Fab antibody, EG-19-11, with subnanomolar affinity from naïve immunoglobulin repertoires using a hierarchical antibody library system.

PubMed

Hur, Byung-ung; Yoon, Jae-bong; Liu, Li-Kun; Cha, Sang-hoon

2010-11-30

Specific antibodies that possess a subnanomolar affinity are very difficult to obtain from human naïve immunoglobulin repertoires without the use of lengthy affinity optimization procedures. Here, we designed a hierarchical phage-displayed antibody library system to generate an enormous diversity of combinatorial Fab fragments (6×10(17)) and attempted to isolate high-affinity Fabs against the human epidermal growth factor receptor (EGFR). A primary antibody library, designated HuDVFab-8L, comprising 4.5×10(9) human naïve heavy chains and eight unspecified human naïve light chains was selected against the EGFR-Fc protein by biopanning, and four anti-EGFR Fab clones were isolated. Because one of the Fab clones, denoted EG-L2-11, recognized a native EGFR expressed on A431 cells, the heavy chain of the Fab was shuffled with a human naïve light chain repertoire with a diversity of 1.4×10(8) and selected a second time against the EGFR-Fc protein again. One EG-L2-11 variant, denoted EG-19-11, recognized an EGFR epitope that was almost the same as that bound by cetuximab and had a K(D) of approximately 540 pM for soluble EGFR, which is about 7-fold higher than that of the FabC225 derived from cetuximab. This variant was also internalized by A431 cells, likely via receptor-mediated endocytosis, and it efficiently inhibited EGF-mediated tyrosine phosphorylation of the EGFR. These results demonstrate that the use of our hierarchical antibody library system is advantageous in generating fully human antibodies especially with a therapeutic purpose. Copyright © 2010 Elsevier B.V. All rights reserved.

[Utility of methoxy isobutyl isonitrile (MIBI) scintigraphy, ultrasound and computerized axial tomography in preoperative topographic diagnosis of hiperparathyroidism].

PubMed

Gómez Palacios, Angel; Gómez Zábala, Jesús; Gutiérrez, María Teresa; Expósito, Amaya; Barrios, Borja; Zorraquino, Angel; Taibo, Miguel Angel; Iturburu, Ignacio

2006-12-01

1. To assess the sensitivity of scintigraphy using methoxy isobutyl isonitrile (MIBI). 2. To compare its resolution with that of ultrasound (US) and computerized axial tomography (CAT). 3. To use its diagnostic reliability to determine whether selective approaches can be used to treat hyperparathyroidism (HPT). A study of 76 patients who underwent surgery for HPT between 1996 and 2005 was performed. MIBI scintigraphy and cervical US were used for whole-body scanning in all patients; CAT was used in 47 patients. Intraoperative and postoperative biopsies were used for final evaluation of the tests, after visualization and surgical extirpation. The results of scintigraphy were positive in 65 patients (85.52%). The diagnosis was correct in all of the single images. Multiple images were due to hyperplasia and parathyroid adenomas with thyroid disease (5.2%). Three images, incorrectly classified as negative (3.94%), were positive. The sensitivity of US was 63% and allowed detection of three MIBI-negative adenomas (4%). CAT was less sensitive (55%), but detected a further three MIBI-negative adenomas (4%). 1. The sensitivity of MIBI reached 89.46%. In the absence of thyroid nodules, MIBI diagnosed 100% of single lesions. Pathological thyroid processes produced false-positive results (5.2%) and there were diagnostic errors (4%). 2. MIBI scintigraphy was more sensitive than US and CAT. 3. Positive, single image scintigraphy allows a selective cervical approach. US and CAT may help to save a further 8% of patients (with negative scintigraphy).

Characterization of the complex formed between a potent neutralizing ovine-derived polyclonal anti-TNFα Fab fragment and human TNFα

PubMed Central

Abbott, W. Mark; Snow, Melanie; Eckersley, Sonia; Renshaw, Jonathan; Davies, Gareth; Norman, Richard A.; Ceuppens, Peter; Slootstra, Jerry; Benschop, Joris J.; Hamuro, Yoshitomo; Lee, Jessica E.; Newham, Peter

2013-01-01

TNFα (tumour necrosis factor α) is an early mediator in the systemic inflammatory response to infection and is therefore a therapeutic target in sepsis. AZD9773 is an ovine-derived, polyclonal anti-TNFα Fab fragment derived from a pool of serum and currently being developed as a treatment for severe sepsis and septic shock. In the present study, we show that although AZD9773 has a modest affinity for TNFα in a binding assay, the Ki in a cell-based assay is approximately four orders of magnitude lower. We show using SEC (size exclusion chromatography) that the maximum size of the complex between AZD9773 and TNFα is consistent with approximately 12 Fabs binding to one TNFα trimer. A number of approaches were taken to map the epitopes recognized by AZD9773. These revealed that a number of different regions on TNFα are involved in binding to the polyclonal Fab. The data suggest that there are probably three epitopes per monomer that are responsible for most of the inhibition by AZD9773 and that all three can be occupied at the same time in the complex. We conclude that AZD9773 is clearly demonstrated to bind to multiple epitopes on TNFα and suggest that the polyclonal nature may account, at least in part, for the very high potency observed in cell-based assays. PMID:23863106

Miscellaneous indications in bone scintigraphy: metabolic bone diseases and malignant bone tumors.

PubMed

Cook, Gary J R; Gnanasegaran, Gopinath; Chua, Sue

2010-01-01

The diphosphonate bone scan is ideally suited to assess many global, focal or multifocal metabolic bone disorders and there remains a role for conventional bone scintigraphy in metabolic bone disorders at diagnosis, investigation of complications, and treatment response assessment. In contrast, the role of bone scintigraphy in the evaluation of primary malignant bone tumors has reduced with the improvement of morphologic imaging, such as computed tomography and magnetic resonance imaging. However, an increasing role for (18)F-fluorodeoxyglucose positron emission tomography and positron emission tomography/computed tomography is emerging as a functional assessment at diagnosis, staging, and neoadjuvant treatment response assessment.

Slow-Onset Inhibition of the FabI Enoyl Reductase from Francisella tularensis: Residence Time and in Vivo Activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lu, H.; England, K; Ende, C

2009-01-01

Francisella tularensis is a highly virulent and contagious Gram-negative intracellular bacterium that causes the disease tularemia in mammals. The high infectivity and the ability of the bacterium to survive for weeks in a cool, moist environment have raised the possibility that this organism could be exploited deliberately as a potential biological weapon. Fatty acid biosynthesis (FAS-II) is essential for bacterial viability and has been validated as a target for the discovery of novel antibacterials. The FAS-II enoyl reductase ftuFabI has been cloned and expressed, and a series of diphenyl ethers have been identified that are subnanomolar inhibitors of the enzymemore » with MIC90 values as low as 0.00018 ?g mL-1. The existence of a linear correlation between the Ki and MIC values strongly suggests that the antibacterial activity of the diphenyl ethers results from direct inhibition of ftuFabI within the cell. The compounds are slow-onset inhibitors of ftuFabI, and the residence time of the inhibitors on the enzyme correlates with their in vivo activity in a mouse model of tularemia infection. Significantly, the rate of breakdown of the enzyme-inhibitor complex is a better predictor of in vivo activity than the overall thermodynamic stability of the complex, a concept that has important implications for the discovery of novel chemotherapeutics that normally rely on equilibrium measurements of potency.« less

Molecular modeling and simulation of FabG, an enzyme involved in the fatty acid pathway of Streptococcus pyogenes.

PubMed

Shafreen, Rajamohmed Beema; Pandian, Shunmugiah Karutha

2013-09-01

Streptococcus pyogenes (SP) is the major cause of pharyngitis accompanied by strep throat infections in humans. 3-keto acyl reductase (FabG), an important enzyme involved in the elongation cycle of the fatty acid pathway of S. pyogenes, is essential for synthesis of the cell-membrane, virulence factors and quorum sensing-related mechanisms. Targeting SPFabG may provide an important aid for the development of drugs against S. pyogenes. However, the absence of a crystal structure for FabG of S. pyogenes limits the development of structure-based drug designs. Hence, in the present study, a homology model of FabG was generated using the X-ray crystallographic structure of Aquifex aeolicus (PDB ID: 2PNF). The modeled structure was refined using energy minimization. Furthermore, active sites were predicted, and a large dataset of compounds was screened against SPFabG. The ligands were docked using the LigandFit module that is available from Discovery Studio version 2.5. From this list, 13 best hit ligands were chosen based on the docking score and binding energy. All of the 13 ligands were screened for Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) properties. From this, the two best descriptors, along with one descriptor that lay outside the ADMET plot, were selected for molecular dynamic (MD) simulation. In vitro testing of the ligands using biological assays further substantiated the efficacy of the ligands that were screened based on the in silico methods. Copyright © 2013 Elsevier Inc. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Massuger, L.F.; Kenemans, P.; Claessens, R.A.

The safety and diagnostic accuracy of immunoscintigraphy with the indium-111-labeled monoclonal antibody OV-TL 3 F(ab')2({sup 111}In-OV-TL 3 F(ab')2) for diagnosis and follow-up of ovarian cancer was prospectively studied in 31 patients. Planar and SPECT scintigraphy were performed up to 4 days after i.v. injection of 140 MBq {sup 111}In-OV-TL 3 F(ab')2. Surgical evaluation was possible in 22 out of 31 patients. Imaging results were compared with X-ray computed tomography, ultrasound, and CA 125 serum level using the histologically confirmed surgical findings as a gold standard. Apart from a transient rash observed in two patients, no other immediate or delayed adversemore » reactions were observed. Within the surgically evaluated group, ovarian cancer lesions were detected in 16 out of 17 patients (94%). Of 45 distinct tumor deposits found at operation, 67% were detected and localized with immunoscintigraphy while X-ray computed tomography and ultrasound visualized 53% and 23%, respectively.« less

The effect of captopril and losartan on the electrophysiology of myocardial cells of myocardial ischemia rats.

PubMed

Shi, Xiangmin; Shan, Zhaoling; Yuan, Hongtao; Guo, Hongyang; Wang, Yutang

2014-01-01

This study aims to investigate the effect of captopril and losartan on the electrophysiology of myocardial cells parameters in ventricular vulnerable period and effective refractory period of myocardial ischemia rats. 96 wistar rats were enrolled in the study and divided into six groups: Captopril myocardial ischemia group, losartan myocardial ischemia group, myocardial ischemia control group, captopril normal group, losartan normal group and normal control group (n=16). We observed morphological changes of myocardial tissue in each group. The cardiac electrophysiological parameters in effective refractory period of each group were measured. Creatine kinase (CK), alanine aminotransferase (GOT), lactate dehydrogenase (LDH), the expression of Cardiotrophin 1 (CT-1) and malonaldehyde (MDA) were detected. Compared the losartan and captopril group with the control group, (P<0.05). Losartan and captopril can shorten the ventricular vulnerable period of the normal group and ischemic group. There was no interaction effect between losartan and captopril group and the acute myocardial ischemia group. The effect of losartan and captopril on time window in ventricular vulnerable period showed that compared with the control group (P<0.05). Losartan and captopril had a significant effect on prolonged effective refractory period of normal and ischemic rats. There was no interaction effect between losartan and captopril group and the acute myocardial ischemia group. Compared with the myocardial ischemia control group, CK, GOT, LDH and MDA decreased in captopril and losartan myocardial ischemia groups (P<0.05). Losartan and captopril had a significant effect on prolonged effective refractory period and shorten ventricular vulnerable period, they can also effectively prevent arrhythmias.

Myocardialization of the cardiac outflow tract

NASA Technical Reports Server (NTRS)

van den Hoff, M. J.; Moorman, A. F.; Ruijter, J. M.; Lamers, W. H.; Bennington, R. W.; Markwald, R. R.; Wessels, A.

1999-01-01

During development, the single-circuited cardiac tube transforms into a double-circuited four-chambered heart by a complex process of remodeling, differential growth, and septation. In this process the endocardial cushion tissues of the atrioventricular junction and outflow tract (OFT) play a crucial role as they contribute to the mesenchymal components of the developing septa and valves in the developing heart. After fusion, the endocardial ridges in the proximal portion of the OFT initially form a mesenchymal outlet septum. In the adult heart, however, this outlet septum is basically a muscular structure. Hence, the mesenchyme of the proximal outlet septum has to be replaced by cardiomyocytes. We have dubbed this process "myocardialization." Our immunohistochemical analysis of staged chicken hearts demonstrates that myocardialization takes place by ingrowth of existing myocardium into the mesenchymal outlet septum. Compared to other events in cardiac septation, it is a relatively late process, being initialized around stage H/H28 and being basically completed around stage H/H38. To unravel the molecular mechanisms that are responsible for the induction and regulation of myocardialization, an in vitro culture system in which myocardialization could be mimicked and manipulated was developed. Using this in vitro myocardialization assay it was observed that under the standard culture conditions (i) whole OFT explants from stage H/H20 and younger did not spontaneously myocardialize the collagen matrix, (ii) explants from stage H/H21 and older spontaneously formed extensive myocardial networks, (iii) the myocardium of the OFT could be induced to myocardialize and was therefore "myocardialization-competent" at all stages tested (H/H16-30), (iv) myocardialization was induced by factors produced by, most likely, the nonmyocardial component of the outflow tract, (v) at none of the embryonic stages analyzed was ventricular myocardium myocardialization-competent, and finally

Local sympathetic denervation attenuates myocardial inflammation and improves cardiac function after myocardial infarction in mice

PubMed Central

Ziegler, Karin A; Ahles, Andrea; Wille, Timo; Kerler, Julia; Ramanujam, Deepak; Engelhardt, Stefan

2018-01-01

Abstract Aims Cardiac inflammation has been suggested to be regulated by the sympathetic nervous system (SNS). However, due to the lack of methodology to surgically eliminate the myocardial SNS in mice, neuronal control of cardiac inflammation remains ill-defined. Here, we report a procedure for local cardiac sympathetic denervation in mice and tested its effect in a mouse model of heart failure post-myocardial infarction. Methods and results Upon preparation of the carotid bifurcation, the right and the left superior cervical ganglia were localized and their pre- and postganglionic branches dissected before removal of the ganglion. Ganglionectomy led to an almost entire loss of myocardial sympathetic innervation in the left ventricular anterior wall. When applied at the time of myocardial infarction (MI), cardiac sympathetic denervation did not affect acute myocardial damage and infarct size. In contrast, cardiac sympathetic denervation significantly attenuated chronic consequences of MI, including myocardial inflammation, myocyte hypertrophy, and overall cardiac dysfunction. Conclusion These data suggest a critical role for local sympathetic control of cardiac inflammation. Our model of myocardial sympathetic denervation in mice should prove useful to further dissect the molecular mechanisms underlying cardiac neural control. PMID:29186414

[Somatostatin receptor scintigraphy in medullary thyroid carcinomas, GEP and carcinoid tumors].

PubMed

Eising, E G; Farahati, J; Bier, D; Knust, E J; Reiners, C

1995-02-01

For this study, 24 patients with medullary thyroid cancer (MTC) and 10 with carcinoid-/GEP-tumours underwent scintigraphy with 123I-Tyr3-octreotide or 111In-DTPA-D-Phe1-octreotide (Octreoscan) or 99mTc-V-DMSA. Calcitonin and CEA were elevated in MTC patients, the other had tumour lesions on CT. Octreoscan-scintigraphy was positive in 68% of all suspicious cases. On the other hand, 123I-Tyr3-octreotide showed only rarely positive results. 99mTc-V-DMSA-scans in MTC patients were positive in 23%. Liver metastases could be seen only with Octreoscan in the non-MTC-group. These results showed better sensitivity of 111In-labelled octreotide.

Association of hyperfunctioning thyroid adenoma with thyroid cancer presenting as "trapping only" nodule at 99mTcO4- scintigraphy.

PubMed

dell'Erba, L; Gerundini, P; Caputo, M; Bagnasco, M

2003-11-01

Rarely may a non-hyperfunctioning thyroid nodule present as "hot" at Technetium-99m pertechnetate (99mTcO4-) and "cold" at radioiodine scintigraphy at late acquisitions. We report the case of a hyperthyroid female patient whose 99mTcO4- scintigraphy showed two "hot" nodules, whereas Iodide-131 (131I-) revealed a lack of indicator uptake by the larger, and intense uptake by the smaller nodule. The patient underwent surgery: histology demonstrated that the larger nodule, mismatched at pertechnetate vs iodine scintigraphy, was a papillary carcinoma. Our suggestion is to perform thyroid scintigraphy with radioiodine in hyperthyroid patients with more than one nodule concentrating pertechnetate, especially when an ultrasonographic pattern possibly suspect for malignancy is present.

[Screening with angiographic images prior to (99m)Tc-HMPAO labelled leukocyte scintigraphy in the diagnosis of periprosthetic infection].

PubMed

Granados, U; Fuster, D; Soriano, A; García, S; Bori, G; Martínez, J C; Mayoral, M; Perlaza, P; Tomás, X; Pons, F

2015-01-01

To evaluate the impact of the angioscintigrapy of the three phase bone scan as screening method to rule out infection of the hip and knee prosthesis prior to performing the (99m)Tc-HMPAO leukocyte scintigraphy. A total of 120 (70 women, 50 men; mean age 71±11years) with clinical suspicion of hip (n=63) or knee (n=57) infection of the prosthesis and clinical suspicion of infection were evaluated prospectively. All patients underwent three-phase bone scan (angioscintigraphy, vascular and bone phase) and (99m)Tc-HMPAO-labelled white blood cell scintigraphy. Final diagnosis of infection was made by microbiological documentation or clinical follow-up for at least 12months. Eighteen out of 120 patients were diagnosed of infection of hip prosthesis (n=10) or knee prosthesis (n=8). The angioscintigraphy was positive in 15/18 infected cases and in 21/102 of the non-infected cases with a sensitivity of 83%, specificity of 79% and negative predictive value of 97%. Sensitivity and specificity of (99m)Tc-HMPAO leukocyte scintigraphy were 72% and 95%, respectively. If the leukocyte labeled scintigraphies had been used exclusively for patients with positive angioscintigraphy, this would have saved up to 70% of the (99m)Tc-HMPAO leukocyte scintigraphies performed. There were no cases of infection with positive labeled leukocyte scintigraphy and negative angioscintigraphy. Angioscintigraphy (blood flow phase of bone scan) is a useful technique for screening for hip and knee joint prosthesis infection, significantly reducing the need for (99m)Tc-HMPAO leukocyte scintigraphy without affecting the sensitivity of the technique. Copyright © 2014 Elsevier España, S.L.U. and SEMNIM. All rights reserved.

Importance of the lung perfusion scintigraphy in single lung transplantation.

PubMed

Rodríguez Mesa, N V; Guerrero Cancio, M C; Cordero Jiménez, M D; Alvarez Velázquez, I K

2012-01-01

Lung perfusion scintigraphy (LPS) with (99m)Tc-MAA gives valuable information about patients who will undergo a single lung transplantation. This technique makes it possible to evaluate and quantify the relative function of both lungs to select the organ to be transplanted. Once the surgery has been performed, the LPS represents a diagnostic method to study the status of the transplanted organ. Two patients who underwent single lung transplantation were studied in our hospital. In both cases, a pre-operative LPS was performed before surgery for selection of the organ to be transplanted and the scintigraphy study was performed a few months after transplantation to establish the perfusion function of the transplanted lung. Copyright © 2011 Elsevier España, S.L. y SEMNIM. All rights reserved.

Panic attack triggering myocardial ischemia documented by myocardial perfusion imaging study. A case report

PubMed Central

2012-01-01

Background Chest pain, a key element in the investigation of coronary artery disease is often regarded as a benign prognosis when present in panic attacks. However, panic disorder has been suggested as an independent risk factor for long-term prognosis of cardiovascular diseases and a trigger of acute myocardial infarction. Objective Faced with the extreme importance in differentiate from ischemic to non-ischemic chest pain, we report a case of panic attack induced by inhalation of 35% carbon dioxide triggering myocardial ischemia, documented by myocardial perfusion imaging study. Discussion Panic attack is undoubtedly a strong component of mental stress. Patients with coronary artery disease may present myocardial ischemia in mental stress response by two ways: an increase in coronary vasomotor tone or a sympathetic hyperactivity leading to a rise in myocardial oxygen consumption. Coronary artery spasm was presumed to be present in cases of cardiac ischemia linked to panic disorder. Possibly the carbon dioxide challenge test could trigger myocardial ischemia by the same mechanisms. Conclusion The use of mental stress has been suggested as an alternative method for myocardial ischemia investigation. Based on translational medicine objectives the use of CO2 challenge followed by Sestamibi SPECT could be a useful method to allow improved application of research-based knowledge to the medical field, specifically at the interface of PD and cardiovascular disease. PMID:22999016

Panic attack triggering myocardial ischemia documented by myocardial perfusion imaging study. A case report.

PubMed

Soares-Filho, Gastão Luiz Fonseca; Mesquita, Claudio Tinoco; Mesquita, Evandro Tinoco; Arias-Carrión, Oscar; Machado, Sergio; González, Manuel Menéndez; Valença, Alexandre Martins; Nardi, Antonio Egidio

2012-09-21

Chest pain, a key element in the investigation of coronary artery disease is often regarded as a benign prognosis when present in panic attacks. However, panic disorder has been suggested as an independent risk factor for long-term prognosis of cardiovascular diseases and a trigger of acute myocardial infarction. Faced with the extreme importance in differentiate from ischemic to non-ischemic chest pain, we report a case of panic attack induced by inhalation of 35% carbon dioxide triggering myocardial ischemia, documented by myocardial perfusion imaging study. Panic attack is undoubtedly a strong component of mental stress. Patients with coronary artery disease may present myocardial ischemia in mental stress response by two ways: an increase in coronary vasomotor tone or a sympathetic hyperactivity leading to a rise in myocardial oxygen consumption. Coronary artery spasm was presumed to be present in cases of cardiac ischemia linked to panic disorder. Possibly the carbon dioxide challenge test could trigger myocardial ischemia by the same mechanisms. The use of mental stress has been suggested as an alternative method for myocardial ischemia investigation. Based on translational medicine objectives the use of CO2 challenge followed by Sestamibi SPECT could be a useful method to allow improved application of research-based knowledge to the medical field, specifically at the interface of PD and cardiovascular disease.

Tuning the specificity of a Two-in-One Fab against three angiogenic antigens by fully utilizing the information of deep mutational scanning.

PubMed

Koenig, Patrick; Sanowar, Sarah; Lee, Chingwei V; Fuh, Germaine

Monoclonal antibodies developed for therapeutic or diagnostic purposes need to demonstrate highly defined binding specificity profiles. Engineering of an antibody to enhance or reduce binding to related antigens is often needed to achieve the desired biologic activity without safety concern. Here, we describe a deep sequencing-aided engineering strategy to fine-tune the specificity of an angiopoietin-2 (Ang2)/vascular endothelial growth factor (VEGF) dual action Fab, 5A12.1 for the treatment of age-related macular degeneration. This antibody utilizes overlapping complementarity-determining region (CDR) sites for dual Ang2/VEGF interaction with K D in the sub-nanomolar range. However, it also exhibits significant (K D of 4 nM) binding to angiopoietin-1, which has high sequence identity with Ang2. We generated a large phage-displayed library of 5A12.1 Fab variants with all possible single mutations in the 6 CDRs. By tracking the change of prevalence of each mutation during various selection conditions, we identified 35 mutations predicted to decrease the affinity for Ang1 while maintaining the affinity for Ang2 and VEGF. We confirmed the specificity profiles for 25 of these single mutations as Fab protein. Structural analysis showed that some of the Fab mutations cluster near a potential Ang1/2 epitope residue that differs in the 2 proteins, while others are up to 15 Å away from the antigen-binding site and likely influence the binding interaction remotely. The approach presented here provides a robust and efficient method for specificity engineering that does not require prior knowledge of the antigen antibody interaction and can be broadly applied to antibody specificity engineering projects.

Fab antibodies capable of blocking T cells by competitive binding have the identical specificity but a higher affinity to the MHC-peptide-complex than the T cell receptor.

PubMed

Neumann, Frank; Sturm, Christine; Hülsmeyer, Martin; Dauth, Nina; Guillaume, Philippe; Luescher, Immanuel F; Pfreundschuh, Michael; Held, Gerhard

2009-08-15

In transplant rejection, graft versus host or autoimmune diseases T cells are mediating the pathophysiological processes. Compared to unspecific pharmacological immune suppression specific inhibition of those T cells, that are involved in the disease, would be an alternative and attractive approach. T cells are activated after their T cell receptor (TCR) recognizes an antigenic peptide displayed by the Major Histocompatibility Complex (MHC). Molecules that interact with MHC-peptide-complexes in a specific fashion should block T cells with identical specificity. Using the model of the SSX2 (103-111)/HLA-A*0201 complex we investigated a panel of MHC-peptide-specific Fab antibodies for their capacity blocking specific T cell clones. Like TCRs all Fab antibodies reacted with the MHC complex only when the SSX2 (103-111) peptide was displayed. By introducing single amino acid mutations in the HLA-A*0201 heavy chain we identified the K66 residue as the most critical binding similar to that of TCRs. However, some Fab antibodies did not inhibit the reactivity of a specific T cell clone against peptide pulsed, artificial targets, nor cells displaying the peptide after endogenous processing. Measurements of binding kinetics revealed that only those Fab antibodies were capable of blocking T cells that interacted with an affinity in the nanomolar range. Fab antibodies binding like TCRs with affinities on the lower micromolar range did not inhibit T cell reactivity. These results indicate that molecules that block T cells by competitive binding with the TCR must have the same specificity but higher affinity for the MHC-peptide-complex than the TCR.

⁸⁹Zr-Labeled Versus ¹²⁴I-Labeled αHER2 Fab with Optimized Plasma Half-Life for High-Contrast Tumor Imaging In Vivo.

PubMed

Mendler, Claudia T; Gehring, Torben; Wester, Hans-Jürgen; Schwaiger, Markus; Skerra, Arne

2015-07-01

Immuno-PET imaging of the tumor antigen HER2/neu allows for the noninvasive detection and monitoring of oncogene expression; such detection and monitoring are of prognostic value in patients with breast cancer. Compared with the full-size antibody trastuzumab, smaller protein tracers with more rapid blood clearance permit higher imaging contrast at earlier time points. Antigen-binding fragments (Fabs) of antibodies with moderately prolonged circulation achieved through the genetic fusion with a long, conformationally disordered chain of the natural amino acids Pro, Ala, and Ser (PASylation)-a biologic alternative to chemical conjugation with polyethylene glycol, PEG-offer a promising tracer format with improved pharmacokinetics for in vivo imaging. Recently, the transition metal radionuclide (89)Zr has attracted increasing interest for immuno-PET studies, complementing the conventional halogen radionuclide (124)I. To allow direct comparison of these 2 radioactive labels for the same protein tracer, the recombinant αHER2 Fab fused with 200 Pro, Ala, and Ser (PAS200) residues was either conjugated with (124)I via an iodination reagent or coupled with deferoxamine (Df) and complexed with (89)Zr. After confirmation of the stability of both radioconjugates and quality control in vitro, immuno-PET and biodistribution studies were performed with CD1-Foxn1(nu) mice bearing HER2-positive human tumor xenografts. (89)Zr⋅Df-Fab-PAS200 and (124)I-Fab-PAS200 showed specific tumor uptake of 11 and 2.3 percentage injected dose per gram 24 h after injection, respectively; both led to high tumor-to-blood (3.6 and 4.4, respectively) and tumor-to-muscle (20 and 43, respectively) ratios. With regard to off-target accumulation, overt (124)I activity was seen in the thyroid, as expected, whereas high kidney uptake was evident for (89)Zr; the latter was probably due to glomerular filtration and reabsorption of the protein tracer in proximal tubular cells. Both (89)Zr- and (124)I

Effective prophylaxis of influenza A virus pneumonia in mice by topical passive immunotherapy with polyvalent human immunoglobulins or F(ab')2 fragments.

PubMed

Ramisse, F; Deramoudt, F X; Szatanik, M; Bianchi, A; Binder, P; Hannoun, C; Alonso, J M

1998-03-01

The effectiveness of polyvalent plasma-derived human immunoglobulins (IVIG) in passive immunotherapy of influenza virus pneumonia was assessed, using the Strain Scotland (A/Scotland/74 (H3N2)) adapted to BALB/c mice by repeated lung passages. Haemagglutinin antibodies in two batches of IVIG at 10 mg/ml had a titre of 1/16. Intravenous injection of 1000-5000 microg of IVIG, 3 h after infection, gave 60-70% protection, whereas intranasal injection of 25-50 microg protected 90% of mice infected with a lethal dose of influenza virus. F(ab')2 fragments were at least as protective as intact IVIG, suggesting that complement or Fcgamma receptor-bearing cells were not required. Topical passive immunotherapy with IVIG or F(ab')2 gave protection up to 8 h after infection, but not at 24 h, suggesting that anti-influenza A antibodies in IVIG, delivered locally, are only effective at early stages of the infectious process. The potential value of topical administration of IVIG or F(ab')2 fragments for influenza A pneumonia prophylaxis was further demonstrated by the protective effects of their intranasal administration 24 h before challenge.

Thyrotoxicosis-induced acute myocardial infarction due to painless thyroiditis.

PubMed

Kim, Hee Jin; Jung, Tae Sik; Hahm, Jong Ryeal; Hwang, Seok-Jae; Lee, Sang Min; Jung, Jung Hwa; Kim, Soo Kyoung; Chung, Soon Il

2011-10-01

Thyrotoxicosis influences cardiovascular hemodynamics and can induce coronary vasospasm. Patients with thyrotoxicosis-induced acute myocardial infarction (AMI) are unusual and almost all reported cases have been associated with Graves' disease. Patients with painless thyroiditis show a thyrotoxic phase during the early stages. Here we describe a very rare case of thyrotoxicosis with painless thyroiditis-induced AMI. A 35-year-old Korean man visited the emergency room for a 2-hour duration of typical AMI chest pain. The patient did not have any coronary artery disease (CAD) risk factors. The electrocardiogram showed 3 mm of ST-segment elevation in leads II, III, and aVF, which is consistent with inferior AMI. We immediately treated the patient with aspirin, clopidogrel, and nitroglycerine and performed emergent coronary angiography. Coronary angiography showed normal coronary arteries without any stenotic lesions. Consistent with AMI, cardiac enzyme levels of serum creatine kinase (CK), CK-MB, and troponin-I were also elevated. Laboratory findings showed thyrotoxicosis without any thyroid autoantibodies. A 99m-technetium scintigraphy showed markedly decreased thyroid uptake compatible with thyroiditis. We treated the patient with calcium channel blockers and nitrates. The patient spontaneously recovered normal thyroid function after 6 weeks of observation and did not complain of chest pain. Thyrotoxicosis due to painless thyroiditis provoked AMI in a young man who had no atherosclerotic coronary lesions and no CAD risk factors.

FMLP provokes coronary vasoconstriction and myocardial ischemia in rabbits

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gillespie, M.N.; Booth, D.C.; Friedman, B.J.

Recent pathological studies of coronary arteries from humans with suspected coronary spasm have revealed an augmented intramural burden of inflammatory cells. To test the hypothesis than inappropriate activation of inflammatory cells participates in the evolution of coronary vasospasm, the present experiment employed a newly developed coronary arteriographic technique for use in pentobarbital-anesthetized rabbits to evaluate the coronary vasomotor actions of the nonselective inflammatory cell stimulant, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP). In 10 of 10 animals, selective left intracoronary injection of 200 ng fMLP evoked profound left coronary narrowing accompanied in all cases by ST segment deviation and dysrhythmias. Thallium-201 scintigraphy demonstrated hypoperfusion ofmore » the left ventricular free wall and septum supplied by the spastic coronary artery. The fMLP-induced epicardial vasoconstriction, ischemic electrocardiogram (ECG) changes, and thallium perfusion defects were reversed by intravenous nitroglycerin. Neither the right coronary artery nor its distribution were influenced by left coronary injection of fMLP. Additional experiments in isolated, salt solution-perfused rabbit hearts demonstrated that fMLP failed to exert direct coronary vasoconstrictor effects. These observations indicate that the nonselective inflammatory cell stimulant, fMLP, provokes arteriographically demonstrable coronary spasm with attendant myocardial hypoperfusion and ischemic ECG changes in anesthetized rabbits. Such a model may be useful in exploring the dynamic role of inflammatory cells in development of coronary spasm.« less

Structure of a human monoclonal antibody Fab fragment against gp41 of human immunodeficiency virus type 1

NASA Technical Reports Server (NTRS)

He, Xiao M.; Rueker, Florian; Casale, Elena; Carter, Daniel C.

1992-01-01

The three-dimensional structure of a human monoclonal antibody (Fab), which binds specifically to a major epitope of the transmembrane protein gp41 of the human immunodeficiency virus type 1, has been determined by crystallographic methods to a resolution of 2.7 A. It has been previously determined that this antibody recognizes the epitope SGKLICTTAVPWNAS, belongs to the subclass IgG1 (kappa), and exhibits antibody-dependent cellular cytotoxicity. The quaternary structure of the Fab is in an extended conformation with an elbow bend angle between the constant and variable domains of 175 deg. Structurally, four of the hypervariable loops can be classified according to previously recognized canonical structures. The third hypervariable loops of the heavy (H3) and light chain (L3) are structurally distinct. Hypervariable loop H3, residues 102H-109H, is unusually extended from the surface. The complementarity-determining region forms a hydrophobic binding pocket that is created primarily from hypervariable loops L3, H3, and H2.

Future cardiac events in patients with ischemic ECG changes during adenosine infusion as a myocardial stress agent and normal cardiac scan.

PubMed

Amer, Hamid; Niaz, Khalid; Hatazawa, Jun; Gasmelseed, Ahmed; Samiri, Hussain Al; Al Othman, Maram; Hammad, Mai Al

2017-11-01

We sought to determine the prognostic importance of adenosine-induced ischemic ECG changes in patients with normal single-photon emission computed tomography myocardial perfusion images (MPI). We carried out a retrospective analysis of 765 patients undergoing adenosine MPI between January 2013 and January 2015. Patients with baseline ECG abnormalities and/or abnormal scan were excluded. Overall, 67 (8.7%) patients had ischemic ECG changes during adenosine infusion in the form of ST depression of 1 mm or more. Of these, 29 [43% (3.8% of all patients)] had normal MPI (positive ECG group). An age-matched and sex-matched group of 108 patients with normal MPI without ECG changes served as control participants (negative ECG group). During a mean follow-up duration of 33.3±6.1 months, patients in the positive ECG group did not have significantly more adverse cardiac events than those in the negative ECG group. One (0.9%) patient in the negative ECG group had a nonfatal myocardial infarction (0.7% annual event rate after a negative MPI). Also in this group, two (1.8%) patients admitted with a diagnosis of CAD where they have been ruled out by angiography. A fourth case in this, in the negative ECG group, was admitted because of heart failure that proved to be secondary to a pulmonary cause and not CAD. A case only in the positive ECG group was admitted as a CAD that was ruled out by coronary angiography. Patients with normal myocardial perfusion scintigraphy in whom ST-segment depression develops during adenosine stress test appear to have no increased risk for future cardiac events compared with similar patients without ECG evidence of ischemia.

Predictive value of bone scintigraphy for the detection of joint involvement in Behçet's disease: Dermatologists' perspectives.

PubMed

Seo, Jimyung; Lee, Minseok; Choi, Min Ju; Zheng, Zhenlong; Cho, Arthur; Bang, Dongsik; Kim, Do Young

2015-01-01

Behçet's disease (BD) is a multisystemic inflammatory disease with articular involvement. Non-specific arthralgia without objective signs of arthritis, such as swelling or effusion, is common in such patients. Thus, an accurate diagnosis of joint involvement may be challenging for dermatologists. To evaluate the validity of (99m)Tc-methylene diphosphonate (Tc-99m-MDP) bone scintigraphy for joint involvement assessment in patients with BD. In 211 patients with BD who had scintigraphic evaluations due to joint symptoms, agreement between bone scintigraphy findings and clinically evaluated joint complaints was retrospectively assessed using Cohen's kappa (κ) statistic. A patient subset (n = 104) showing agreement between joint complaints and scintigraphy results was re-evaluated by a rheumatologist to determine the level of diagnostic specificity attained by combining bone scintigraphy with clinical examinations of dermatologists. The total kappa value (211 patients) was 0.604, indicating fair agreement between joint complaints and scintigraphy results. Individual analysis of eleven joint categories revealed statistically significant correlations for wrist (κ = 0.677), shoulder (κ = 0.661), and foot joints (κ = 0.618). Of the 104 referrals to a rheumatologist, 95 (91.34%) were confirmed as having BD-associated articular involvement. Joint acral areas (e.g., foot, hand, wrist and shoulder) that had the highest kappa value correlations also ranked highest in diagnostic specificity. Bone scintigraphy presents a simple and useful option for dermatologists to assess joint involvement in BD patients, especially for specific anatomic sites.

Detection of right ventricular pressure overloading by thallium-201 myocardial scintigraphy. Results in 57 patients with chronic respiratory diseases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Weitzenblum, E.; Moyses, B.; Dickele, M.C.

1984-02-01

The diagnostic value of thallium 201 (/sup 201/Tl) myocardial imaging was studied in 57 patients with chronic respiratory diseases, most with COPD (n . 46), by comparing the results to hemodynamic findings. In healthy subjects, the right ventricle (RV) is not visualized; therefore, any recorded activity of the RV was considered as indicating RV hypertrophy due to RV pressure overloading (RVPO). RV activity was graded from 0 (no activity) to 3 (activity greater than or equal to that of the left ventricle). Patients were divided into three groups according to the level of the pulmonary artery mean pressure (PPA): PPAmore » less than or equal to 20 mm Hg (no pulmonary arterial hypertension (PAH) ) . group 1, n . 20; PPA ranging from 21 to 30 mm Hg (mild to moderate PAH) . group 2, n . 20; PPA greater than 30 mm Hg (marked PAH) . group 3, n . 17. RV was visualized in 14 patients in group 3 (82 percent) and in 13 patients in group 2 (65 percent). For all patients with PAH (2 + 3) the sensitivity of /sup 201/Tl imaging for the diagnosis of RVPO was of 73 percent, higher than that of ECG and echocardiography (both 51 percent). The sensitivity of /sup 201/Tl, even if moderate (65 percent) was better than that of ECG (30 percent) or echo (40 percent) in patients with mild-to-moderate PAH (group 2). A high RV activity (grade 3) was observed in only three patients. The specificity of this method (obtained from results in group 1) was of 80 percent vs 89 percent for echo and 100 percent for ECG. These results suggest that 201Tl myocardial imaging is a rather sensitive method and could be of interest for the noninvasive diagnosis of RVPO in COPD patients.« less

Fab MAbs specific to HA of influenza virus with H5N1 neutralizing activity selected from immunized chicken phage library.

PubMed

Pitaksajjakul, Pannamthip; Lekcharoensuk, Porntippa; Upragarin, Narin; Barbas, Carlos F; Ibrahim, Madiha Salah; Ikuta, Kazuyoshi; Ramasoota, Pongrama

2010-05-14

Hemagglutinin protein (HA) was considered to be the primary target for monoclonal antibody production. This protein not only plays an important role in viral infections, but can also be used to differentiate H5N1 virus from other influenza A viruses. Hence, for diagnostic and therapeutic applications, it is important to develop anti-HA monoclonal antibody (MAb) with high sensitivity, specificity, stability, and productivity. Nine unique Fab MAbs were generated from chimeric chicken/human Fab phage display library constructed from cDNA derived from chickens immunized with recombinant hemagglutinin protein constructed from H5N1 avian influenza virus (A/Vietnam/1203/04). The obtained Fab MAbs showed several characteristics for further optimization and development-three clones were highly specific to only H5N1 virus. This finding can be applied to the development of H5N1 diagnostic testing. Another clone showed neutralization activity that inhibited H5N1 influenza virus infection in Madin-Darby canine kidney (MDCK) cells. In addition, one clone showed strong reactivity with several of the influenza A virus subtypes tested. The conversion of this clone to whole IgG is a promising study for a cross-neutralization activity test. Copyright (c) 2010 Elsevier Inc. All rights reserved.

Effect of Polysorbate 20 and Polysorbate 80 on the Higher-Order Structure of a Monoclonal Antibody and Its Fab and Fc Fragments Probed Using 2D Nuclear Magnetic Resonance Spectroscopy.

PubMed

Singh, Surinder M; Bandi, Swati; Jones, David N M; Mallela, Krishna M G

2017-12-01

We examined how polysorbate 20 (PS20; Tween 20) and polysorbate 80 (PS80; Tween 80) affect the higher-order structure of a monoclonal antibody (mAb) and its antigen-binding (Fab) and crystallizable (Fc) fragments, using near-UV circular dichroism and 2D nuclear magnetic resonance (NMR). Both polysorbates bind to the mAb with submillimolar affinity. Binding causes significant changes in the tertiary structure of mAb with no changes in its secondary structure. 2D 13 C- 1 H methyl NMR indicates that with increasing concentration of polysorbates, the Fab region showed a decrease in crosspeak volumes. In addition to volume changes, PS20 caused significant changes in the chemical shifts compared to no changes in the case of PS80. No such changes in crosspeak volumes or chemical shifts were observed in the case of Fc region, indicating that polysorbates predominantly affect the Fab region compared to the Fc region. This differential effect of polysorbates on the Fab and Fc regions was because of the lesser thermodynamic stability of the Fab compared to the Fc. These results further indicate that PS80 is the preferred polysorbate for this mAb formulation, because it offers higher protection against aggregation, causes lesser structural perturbation, and has weaker binding affinity with fewer binding sites compared to PS20. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

Comparison of Tc-99m GSA scintigraphy and CT volumetry for evaluation in portal vein embolization.

PubMed

Kono, Yumiko; Kariya, Shuji; Komemushi, Atsushi; Nakatani, Miyuki; Yoshida, Rie Yagi; Suzuki, Satoshi; Ha-Kawa, Sung Kil; Utsunomiya, Keita; Ueno, Yasuhiro; Satoi, Sohei; Kaibori, Masaki; Kon, Masanori; Tanigawa, Noboru

2014-08-01

To determine the correlation of the rate of change of each future remnant liver (FRL) before and after portal vein embolization (PVE), by CT volumetry and Tc-99m galactosyl human serum albumin scintigraphy (GSA scintigraphy). From December 2007 to July 2012, ten patients underwent PVE before hepatic resection. CT volumetry and GSA scintigraphy were performed before and after PVE. The FRL was divided at Cantlie's line for CT volumetry, and volume change rates before and after PVE were calculated. The maximum removal rate (Rmax) was calculated using a radiopharmacokinetic model in GSA scintigraphy. The FRL Rmax change rates before and after PVE were calculated. The correlation between the volume change rates and the Rmax change rates was analyzed. The FRL volume change rate was 1.28 ± 0.26 (mean ± SD); the FRL hypertrophied in all patients significantly (p = 0.005). The FRL Rmax change rate was 1.66 ± 0.75; excluding one patient, there was significant FRL Rmax increase (p = 0.022). Although both increased significantly, no correlation between the volume change rate and the Rmax change rate was observed. No correlation was observed between the FRL volume rate and the Rmax rate.

Comparison of personnel radiation dosimetry from myocardial perfusion scintigraphy: Technetium-99m-sestamibi versus thallium-201

DOE Office of Scientific and Technical Information (OSTI.GOV)

Culver, C.M.; Dworkin, H.J.

1993-07-01

The whole-body and hand radiation doses to our technical staff were retrospectively compared for three distinct 4-mo periods when either 201TI or 99mTc-sestamibi were exclusively used for stress myocardial perfusion imaging. During the initial 4-mo period when 99mTc-sestamibi replaced 201TI, the mean whole-body film badge readings increased from 100 to 450 microSv/mo (p < 0.001) for nuclear medicine technologists (n = 10) and from 240 to 560 microSv/mo (p < 0.05) for radiopharmacy technologists (n = 2). Mean TLD readings to the hands also increased, although the differences were not statistically significant for the nuclear medicine technologists. Noninvasive cardiology staffmore » were monitored with film badges and the mean whole-body film badge reading, when 99mTc-sestamibi was the imaging agent, was 360 microSv per month. Radiation reduction methods that decreased radiation exposure to staff were utilized. The most effective included the use of a lead face shield and lead lined storage container in the noninvasive imaging area, handling spills by shielding instead of decontamination and methods to reduce time spent in close proximity to the patient.« less

Myocardial contusion following nonfatal blunt chest trauma

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kumar, S.A.; Puri, V.K.; Mittal, V.K.

1983-04-01

Currently available diagnostic techniques for myocardial contusion following blunt chest trauma were evaluated. We investigated 30 patients prospectively over a period of 1 year for the presence of myocardial contusion. Among the 30 patients, eight were found to have myocardial contusion on the basis of abnormal electrocardiograms, elevated creatine phosphokinase MB fraction (CPK-MB), and positive myocardial scan. Myocardial scan was positive in seven of eight patients (87.5%). CPK-MB fraction was elevated in four of eight patients (50%). Definitive electrocardiographic changes were seen in only two of eight patients (25%). It appears that myocardial scan using technetium pyrophosphate and CPK-MB fractionmore » determinations are the most reliable aids in diagnosis of myocardial contusion following blunt chest trauma.« less

Functional imaging of SDHx-related head and neck paragangliomas: comparison of 18F-fluorodihydroxyphenylalanine, 18F-fluorodopamine, 18F-fluoro-2-deoxy-D-glucose PET, 123I-metaiodobenzylguanidine scintigraphy, and 111In-pentetreotide scintigraphy.

PubMed

King, Kathryn S; Chen, Clara C; Alexopoulos, Dimitrios K; Whatley, Millie A; Reynolds, James C; Patronas, Nicholas; Ling, Alexander; Adams, Karen T; Xekouki, Paraskevi; Lando, Howard; Stratakis, Constantine A; Pacak, Karel

2011-09-01