Synthetic antibody gene libraries for in vitro affinity maturation.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Biomedical subjects
C R MacKenzie
Publications and source records attributed to C R MacKenzie.
Structure of a single-chain antibody variable domain (Fv) fragment complexed with a carbohydrate antigen at 1.7-A resolution.
We describe here the 1.7-A resolution structure of a single-chain antibody variable domain (scFv) molecule, based on the carbohydrate-binding antibody Se155-4, complexed with the trisaccharide ligand alpha-D-Gal(1-->2)[alpha-D-Abe(1-->3)]alpha-D-Manp1-->OMe, where Abe is abequose. The scFv expressed in Escherichia coli has the variable region light chain to heavy chain polarity with the domains connected by a 19-residue linker. Although the linker is partially disordered in the crystal, the packing of the molecules suggests a monomeric state of the scFv. The carbohydrate adopts a different conformation about the Man-Gal linkage than was observed previously in the Fab-trisaccharide complex. Instead of a direct hydrogen bond between O2Abe and O2Gal, these two atoms are bridged by a water molecule in the present complex.
Amino Acid Sequence↗
Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display.
A single-chain variable fragment (Fv) version of a murine monoclonal antibody, Se155-4, specific for Salmonella serogroup B O-polysaccharide, was used as a model system for testing monovalent phage display as a route for enhancing the relatively low affinities that typify anti-carbohydrate antibodies. Random single-chain Fv mutant libraries generated by chemical and error-prone polymerase chain reaction methods were panned against the serogroup B lipopolysaccharide. Panning of a randomly mutated heavy chain variable domain library indicated selection for improved serogroup B binders and yielded six mutants, five of which showed wild type activity by enzyme immunoassay. Two of these were apparently selected on the basis of better functional single-chain Fv yield in Escherichia coli. A heavy chain mutation (Ile77-->Thr) in one mutant, 3B1, appeared to have a particularly dramatic effect, resulting in yields of approximately 120 mg/liter of functional periplasmic product. The sixth mutant, 4B2, had complementarity determining region 1 (CDR1) and CDR2 mutations and demonstrated 10-fold improved binding, by enzyme immunoassay, relative to the wild type. Extensive analysis of antigen-antibody interactions indicated that the improved binding properties of 4B2 were attributable to a higher association rate constant and interaction with an epitope that is larger than the trisaccharide recognized by the wild type. None of the mutations involved known trisaccharide contact residues; this was consistent with analysis of wild type and mutant single-chain Fvs by titration microcalorimetry. Examination of the structure indicated that two mutations in the heavy chain CDR2 provided improved surface complementarity between the protein and the extended epitope encompassing 2 additional hexose residues. However, introduction of only the CDR2 mutations into the wild type structure failed to confer the improved binding properties of 4B2, indicating an indirect effect by the more distant mutations. Panning of randomly mutated light chain variable domain and full-length single-chain Fv mutant libraries did not yield mutants with improved assembly or binding properties.
Antibodies, Monoclonal↗
Effect of C lambda-C kappa domain switching on Fab activity and yield in Escherichia coli: synthesis and expression of genes encoding two anti-carbohydrate Fabs.
We have used a strategy of hybrid gene synthesis and constant domain shuffling to construct and functionally express in Escherichia coli genes encoding two anti-carbohydrate Fabs, one specific for a Brucella cell-surface polysaccharide and the second for the human blood group A determinant. Very similar VL amino acid sequences made possible the simultaneous synthesis of the two corresponding genes. A class switching approach was used in Fd and light chain gene assembly. The two independently synthesized VH genes were fused to a previously made sequence encoding the C(gamma 1)1 domain as an alternative to synthesis of the natural C gamma 2b 1 and C mu 1 sequences. The VL genes were initially coupled to a synthetic C kappa gene. When these light chain and the above Fd genes, each preceded by the ompA signal sequence, were expressed from two-cistron DNA, yields of functional periplasmic Fab were low and, in each instance, limited by light chain availability. Replacement of the C kappa domains with a C lambda 1 domain resulted in a significant increase in the amount of soluble periplasmic light chain and functional Fab for both the Brucella and blood group A antibodies. The C kappa and C lambda 1 forms of each of the Brucella and blood group A Fabs, with His5 fusions at the C-termini of the Fd chains, were purified by immobilized metal affinity chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Reduced NK activity correlates with active disease in HIV- patients with multidrug-resistant pulmonary tuberculosis.
There has been a global increase in the incidence of multidrug-resistant pulmonary tuberculosis (TB). As there are no previous reports of immune function in HIV- patients with multidrug-resistant pulmonary TB, a comprehensive assessment of cellular immunity in this setting was undertaken. This involved a prospective, case-controlled study which included five patients with active multidrug-resistant pulmonary TB and five matched controls with active non-resistant infection, and documented the changes in immune parameters which occurred upon clinical resolution. Patients with multidrug-resistant TB had significantly lower fresh natural killer (NK) cell activity than matched controls with non-resistant pulmonary TB (P < 0.05). This was a specific abnormality, as there were no significant differences in antigen-specific cytotoxicity or lymphocyte proliferation in the case-controlled study. Follow-up assessment of the patients with multidrug-resistant infections indicated that clinical improvement correlated with a moderate increase in NK cell activity. Impaired NK cell function may be involved in the pathogenesis of multidrug-resistant TB.
Adult↗
Preoperative autonomic function abnormalities in patients with diabetes mellitus and patients with hypertension.
BACKGROUND: This study was done to assess whether or not autonomic function abnormalities identified patients with diabetes mellitus as being at risk for postoperative cardiac arrest or death. STUDY DESIGN: We prospectively studied 74 patients with diabetes mellitus and 118 patients with hypertension without diabetes mellitus undergoing elective noncardiac operation. The study was done at an academic medical center between 1982 and 1985. Patients were evaluated preoperatively, monitored intraoperatively by an independent observer and had daily follow-up evaluation for seven days postoperatively, according to a standard surveillance protocol. Outcomes were judged by assessors unaware of the preoperative status and intraoperative course. Patients were interviewed at three and five years postoperatively. RESULTS: Patients with diabetes mellitus with two or more abnormal autonomic function test results did not experience more intraoperative hypotension, nor did they require more intraoperative pressor agents than patients with diabetes mellitus with one or no abnormalities. Seven percent of the patients with diabetes mellitus experienced postoperative cardiorespiratory arrest or death. All of these patients had more than two abnormal autonomic function test results as well as a previous history of either myocardial infarction or cardiomegaly. CONCLUSIONS: While this study obviously requires confirmation in a larger population, the data are sufficiently strong to warrant close postoperative monitoring of all patients with diabetes mellitus who have two or more autonomic abnormalities.
Autonomic Nervous System↗
Simultaneous randomization of antibody CDRs by a synthetic ligase chain reaction strategy.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Probing the combining site of an anti-carbohydrate antibody by saturation-mutagenesis: role of the heavy-chain CDR3 residues.
The carbohydrate-binding site in Fab fragments of an antibody specific for Salmonella serogroup B O-polysaccharide has been probed by site-directed mutagenesis using an Escherichia coli expression system. Of the six hypervariable loops, the CDR3 of the heavy chain was selected for exhaustive study because of its significant contribution to binding-site topography. A total of 90 mutants were produced and screened by an affinity electrophoresis/Western blotting method. Those of particular interest were further characterized by enzyme immunoassay, and on this basis seven of the mutant Fabs were selected for thermodynamic characterization by titration microcalorimetry. With regard to residues that hydrogen bond to ligand through backbone interactions, Gly102H could not be substituted, while several side chains could be introduced at Gly100H and Tyr103H with relatively little effect on antigen binding. There was, however, a preference for nonpolar side chains at position 103H. Substitution of His101H with carboxylate and amide side chains gave mutants with binding affinities approaching that of the wild type; complete side-chain removal by mutation to Gly was tolerated with a 10-fold reduction in binding constant. Analysis of binding by titration microcalorimetry revealed some dramatic thermodynamic changes hidden by the similarity of the binding constants. Similar effects were observed with residue changes in an Arg-Asp salt-bridge at the base of the loop. These results indicate that alterations to higher affinity anti-carbohydrate antibodies are characterized by an enthalpy-entropy compensation factor which allows for fundamental changes in the nature of the binding interactions but impedes engineering for increases in affinity.
Amino Acid Sequence↗
An organizational model for developing multidisciplinary clinical research in the academic medical center.
OBJECTIVE: To assess the impact of a new organizational model designed to stimulate multidisciplinary clinical research. METHODS: We conducted a prospective, 3 1/2-year followup of a research training program for residents, fellows, faculty, nurses, and allied health professionals in rheumatology and orthopedic surgery. Program components included a multidisciplinary clinical research conference, a clinical research methods curriculum, consultations, a patient registry, and regular meetings of a Research Methodology Core group. Measures included participation in each program component and the number of new investigators who developed funded clinical research projects. RESULTS: The multidisciplinary clinical research conference was attended by 369 new health professionals; 218 professionals participated in at least one of the courses; and 280 consultations were provided to 108 professionals. Thirteen new investigators developed 17 new grant proposals, of which 14 were externally funded. Investigators who successfully procured funding for new projects demonstrated significantly more participation in program components compared with those who did not (P < 0.001 overall). CONCLUSION: Participation in the program was significantly correlated with the development of new prospective patient-based studies. We conclude that our model has the potential to foster such research in other settings.
Academic Medical Centers↗
A walking education program for patients with osteoarthritis of the knee: theory and intervention strategies.
Walking is an ideal physical activity for patients with osteoarthritis because it can help to improve functional status without exacerbating pain or necessitating an increase in the use of medication. Although patient education programs designed to encourage walking have become increasingly important in the management of this condition, there is no single synthesis or other published source of theory and intervention strategies to guide the practitioner in the development of such programs. This paper describes a hospital-based patient education program designed to enhance the functional capacity of who have osteoarthritis of the knee by encouraging the adoption and maintenance of walking. The paper describes the goals, objectives, process, and impact of the program; the principal theoretical model utilized in the design of the program; and how concepts and intervention strategies from the literatures on patient compliance and patient education, exercise compliance, behavioral psychology, and relapse prevention have been operationalized and integrated in a walking program that we have evaluated for safety and efficacy.
Adult↗
Supervised fitness walking in patients with osteoarthritis of the knee. A randomized, controlled trial.
OBJECTIVE: To assess the effect of a program of supervised fitness walking and patient education on functional status, pain, and use of medication in patients with osteoarthritis of the knee. DESIGN: An 8-week randomized, controlled trial. SETTING: Inpatient and outpatient services of an orthopedic hospital in an academic medical center. PATIENTS: A total of 102 patients with a documented diagnosis of primary osteoarthritis of one or both knees participated in the study. Data were obtained on 47 of 51 intervention patients and 45 of 51 control patients. INTERVENTIONS: An 8-week program of supervised fitness walking and patient education or standard routine medical care. MEASUREMENTS: Patients were evaluated and outcomes assessed before and after the intervention using a 6-minute test of walking distance and scores on the physical activity, arthritis impact, pain, and medication subscales of the Arthritis Impact Measurement Scale (AIMS). RESULTS: Patients randomly assigned to the walking program had a 70-meter increase in walking distance relative to their baseline assessment, which represents an improvement of 18.4% (95% Cl, 9.8% to 27.0%). In contrast, controls showed a 17-meter decrease in walking distance relative to their baseline assessment (P less than 0.001). Improvements in functional status as measured by the AIMS physical activity subscale were also observed in the walking group but not in the control group (P less than 0.001); patients assigned to the walking program improved 39% (Cl, 15.6% to 60.4%). Although changes in scores on the arthritis impact subscale were similar in the two groups (P = 0.093), the walking group experienced a decrease in arthritis pain of 27% (Cl, 9.6% to 41.4%) (P = 0.003). Medication use was less frequent in the walking group than in the control group at the post-test (P = 0.08). CONCLUSIONS: A program of supervised fitness walking and patient education can improve functional status without worsening pain or exacerbating arthritis-related symptoms in patients with osteoarthritis of the knee.
Adult↗
Laryngeal involvement in systemic lupus erythematosus.
Laryngeal involvement in systemic lupus erythematosus (SLE) can range from mild ulcerations, vocal cord paralysis, and edema to necrotizing vasculitis with airway obstruction. In this report, four cases showing the range of severity of this disease manifestation are presented, accompanied by a comprehensive review of the literature. The clinical course of 97 patients with laryngeal involvement with SLE are reviewed, of whom 28% had laryngeal edema and 11% had vocal cord paralysis. In the majority of cases, symptoms such as hoarseness, dyspnea, and vocal cord paralysis resolved with corticosteroid therapy. Other, less common causes of this entity included subglottic stenosis, rheumatoid nodules, inflammatory mass lesions, necrotizing vasculitis, and epiglottitis. The clinical presentation of laryngeal involvement in patients with SLE follows a highly variable course, ranging from an asymptomatic state to severe, life-threatening upper airway compromise. With its unpredictable course and multiple causations, this complication remains a diagnostic and therapeutic challenge to physicians involved in the care of patients with SLE.
Adult↗
Predicting postoperative pulmonary complications. Is it a real problem?
To identify predictors of postoperative pulmonary complications, a population of 278 patients, mainly hypertensive and diabetic patients undergoing elective general surgery was studied; 60% of the patients underwent abdominal surgery. Of the 278 patients, 6% had postoperative pulmonary complications: 3% had radiographic evidence of infiltrates or segmental atelectasis and 3% had clinical evidence of atelectasis. Among the two thirds of patients undergoing abdominal surgery, only patients with underlying asthma or chronic bronchitis were at increased risk. Generally, patients with better exercise tolerance by self-report, walking distance, or cardiovascular classification had lower rates. Pulmonary function tests did not help to delineate patients at higher risk of postoperative pulmonary complications. Simple clinical information provided as much data about the patients' risk as pulmonary function tests. Many of these complications occurred in patients who sustained other types of postoperative morbidity, suggesting that predicting and preventing postoperative cardiac morbidity may be the best approach to reducing postoperative pulmonary morbidity.
Aged↗
Bacterial expression and secretion of various single-chain Fv genes encoding proteins specific for a Salmonella serotype B O-antigen.
Active single-chain Fv molecules encoded by synthetic genes have been expressed and secreted to the periplasm of Escherichia coli using the ompA secretory signal. Four different constructs were developed to investigate the effects of peptide linker design and VL-VH orientation on expression, secretion, and binding to a Salmonella O-polysaccharide antigen. Peptide linker sequences derived from the elbow regions of the Fab molecule were used alone or in combination with the flexible (GGGGS)2 sequence. VL and VH domain order in the single chain molecules had a profound effect on the level of secretion but hardly influenced total expression levels, which were approximately 50 mg/liter, chiefly in the form of inclusion bodies. With VL in the NH2-terminal position, the amount of secreted product obtained was 2.4 mg/liter, but when VH occupied this position the yield was less than 5% of this value. Enzyme immunoassays of the four products showed domain order and linker sequence affected antigen binding by less than an order of magnitude. Attempts to express active Fv from dicistronic DNA were unsuccessful, but active Fv was obtained from single-chain Fv by enzymic cleavage at a site in the elbow linker peptide. The thermodynamic binding parameters of intact and cleaved single-chain Fvs determined by titration microcalorimetry were similar to those of bacterially produced Fab and mouse IgG.
Amino Acid Sequence↗
Hip fracture in older persons. Does self-efficacy-based intervention have a role in rehabilitation?
Over 250,000 patients are hospitalized each year in the United States for treatment of a fractured hip, resulting in considerable medical, social, and economic costs to society. Most hip fractures occur among older persons who have fallen. The consequences of hip fractures due to falls by older persons include loss of confidence and the accompanying deterioration in global functional capacity for independent living. This article reviews the problem of hip fracture in the elderly and suggests that intervention approaches designed to enhance personal self-efficacy through patient education may have an important role to play in rehabilitation after hip fracture.
Aged↗
Synthesis and expression in Escherichia coli of cistronic DNA encoding an antibody fragment specific for a Salmonella serotype B O-antigen.
A 1460-bp DNA encoding the two chains of the antigen-binding fragment (Fab) portion of a monoclonal antibody have been chemically synthesized and expressed in Escherichia coli. The antibody, Se155-4, is specific for a Salmonella serogroup B O-antigen and its crystal structure is under investigation. The genes were synthesized according to a strategy that allows for easy manipulation in genetic engineering studies of the Fab-binding site. Each gene is preceded by the ompA secretory signal and a ribosome-binding site, and has been expressed from the two-cistron DNA under the control of the lac promoter. Active Fab of 50 kDa with an inter-chain disulfide bond has been isolated from the periplasm of E. coli in a one-step affinity purification in high yield (2 micrograms/ml of cells). The bacterially produced Fab is as active as purified mouse Fab in antigen-binding and competitive immunoassays. This is the first example of a completely synthetic Fab gene and provides an ideal system to probe the nature of antigen binding by anti-carbohydrate antibodies.
Amino Acid Sequence↗
Relating patient characteristics at the time of admission to outcomes of hospitalization.
In clinical practice, physicians informally address patient characteristics such as illness severity, comorbidity, functional status and stability when considering prognosis and diagnostic or therapeutic interventions. These same attributes, more formally measured, have been used as measures of casemix in clinical research to classify patients into similar risk strata. To determine whether physician estimates of illness severity, function status and stability were predictive of morbidity, mortality, length of stay and average daily ancillary charges, a cohort of 604 patients was studied. The predictive ability of the patient characteristics were found to be outcome specific. Illness severity was a significant predictor of in-hospital morbidity and mortality, length of stay and charges (p less than 0.001). Functional status was predictive of in-hospital morbidity and mortality, 1 year mortality, length of stay and charges (p less than 0.01). Physician estimates of stability were predictive only of morbidity (p less than 0.01) and comorbidity was only a preditor of 1 year mortality (p less than 0.001). Stratifying patients on the basis of specific clinical characteristics determined at the time of admission will be useful in studies of patient outcomes and resource utilization.
Aged↗
Scanning of key residues in antibody binding sites by two saturation-mutagenesis approaches.
The complementarity-determining region 3 of the heavy chain (CDRH3) generally contributes the most to antibody-antigen binding. His101H in CDRH3 of the antibody Se155-4, which is specific for a trisaccharide epitope of Salmonella serotype B O-antigen, was mutated systematically into all nineteen other amino acids by a double mutation approach. Enzyme immunoassay (EIA) and affinity chromatography showed that the Asn, Gln, Gly and Ser mutants exhibited moderate to strong activity. Some mutants, such as Thr and Pro, had weak binding activity, while the acidic and hydrophobic amino acid substitutions resulted in complete loss of activity. A second mutation approach which randomly changed a selected residue into all other nineteen amino acids, while precluding wild-type transformants, is also described.
Amino Acid Sequence↗