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Background: Total hip arthroplasty (THA) in younger patients is projected to increase by a factor of 5 by 2030 and will have important implications for clinical practice, policymaking, and research. This scoping review aimed to synthesize and summarize THA implants' survival, reoperation, and wear rates and identify indications and risk factors for reoperation following THA in patients ≤55 years old. Material and methods: Standardized scoping review methodology was applied. We searched 4 electronic databases (Medline, Embase, CINAHL, and Web of Science) from January 1990 to May 2019. Selection criteria were patients aged ≤55 years, THA survival, reoperation, and/or wear rate reported, a minimum of 20 reoperations included, and minimum level III based on the Oxford Level of Evidence. Two authors independently reviewed the citations, extracted data, and assessed quality. Results: Of the 2255 citations screened, 35 retrospective cohort studies were included. Survival rates for THA at 5 and 20 years were 90%-100% and 60.4%-77.7%, respectively. Reoperation rates at ≤5-year post THA ranged from 1.6% to 5.4% and increased at 10-20 years post THA (8.2%-67%). Common causes for reoperation were aseptic loosening of hip implants, osteolysis, wear, and infection. Higher reoperation and lower survival rates were seen with hip dysplasia and avascular necrosis than with other primary diagnoses. Conclusions: Over time, THA prosthetic survival rates decreased, and reoperation increased in patients ≤55 years. Aseptic loosening of hip implants, osteolysis, wear, and infection were the most frequent reasons for the reoperation.

Recognized in many European countries and Canada as a valid form of therapeutic and educational rehabilitation, conductive education (CE) emphasizes cognitive and motor learning principles for movement reeducation. This article illustrates how CE incorporates motor control and motor learning theories in conjunction with unique facilitation concepts, including rhythmic intention, task series, tailored low-tech equipment, and traditional facilitation concepts such as developmental sequence, manual facilitations, and multimodal interventions.Uniquely, CE brings together task series practice and learning, including a lying program, sitting program, standing program, and walking program, along with activities of daily living within a group treatment model. The conductor uses cadence and rhythmic intention to encourage movement exploration in a scripted plan of care. The participants are active learners and use CE slatted equipment to help support movements. Full participation, to the best of the learners’ ability, is realized with activity modifications made by the conductor.Increased motor control arises through repetition, practice, functional context, and sensory feedback that provide guidance for intention and voluntary movement. Motor control and motor learning theories are foundational principles of CE. Individuals with neurologic injuries, including cerebral palsy, stroke syndrome, Parkinson disease, and traumatic brain injury, can benefit from CE. To date, although research studies cannot objectively compare one person’s movement skills with another’s, new research surrounding motor control and motor learning illustrates and supports the principles and practice of CE. CE is an educational therapy model for teaching and developing new movement skills for individuals with neurologic impairments. This article connects the current science of movement and describes the unique principles involved with CE delivery as an intervention for individuals with neurologic impairments.

Introduction: Clinicians with compassion fatigue (CF) experience behavioral, cognitive, and emotional changes due to repeated exposure to second-hand trauma from the clients with whom they are working. A civic-minded professional possesses the core value of social responsibility. Physical therapy (PT) education programs must balance a focus on developing social responsibility and compassion against the risk of CF. Objective: The objectives of this study were to (1) describe the prevalence of CF in a sample of physical therapists in the early years of practice and (2) to determine whether higher civic-mindedness leads to the development of CF in physical therapists. Method: Three cohorts of recent graduates were administered the Professional Quality of Life (Pro-QOL) survey to measure CF. Thirty-five of 127 surveys sent (27.6% response rate) were completed. Results: A Mann-Whitney U was run to determine differences in the Pro-QOL survey between those scoring high or low in civic-mindedness at graduation. Higher civic-mindedness scores exhibited significantly lower burnout and higher compassion satisfaction. Conclusion: Higher levels of civic-mindedness appear to have a protective effect against developing CF.

Category: Trauma Introduction/Purpose: Conservative treatment of acute AT ruptures with functional rehabilitation has demonstrated superior results to surgery with equal reported re-rupture rates without the added complications of surgical treatment. The variation in patient selection for functional rehabilitation as well as the duration and type of rehabilitation regime utilised adds to the confusion of this clinically proven method of treatment. Since 2011, all patients presenting to our department with an acute AT rupture have been treated with a standardised functional rehabilitation regime in a Vacoped boot for 8 weeks with immediate weight bearing (the LAMP regime). The aim of this paper is to present ATRS scores and objective measures at 12 months follow up of patients treated with the LAMP regime. Methods: A prospective study of all AT ruptures treated conservatively with the LAMP regime between February 2011 and September 2015 was performed. The LAMP regime consists of the use of a Vacoped boot locked in 30° plantar flexion for the 1st 4 weeks. The boot is then dynamised allowing movement between 15-30° over the following 2 weeks, and finally completely dynamised allowing movement from 0-30° over the final 2 weeks. After a total of 8 weeks treatment, the boot is removed. During treatment, patients are allowed to remove the boot once a day to enable them to shower without putting any weight on the affected leg. Patients who had 12 months follow up data of ATRS scores were included. Subsequent evolution of the service has resulted in objective measures including the calf girth, heel raise height and heel raise repetitions being recorded. Complications of treatment were noted. Results: 442 patients were treated conservatively with the LAMP functional rehabilitation regime. Ultrasound imaging was only performed in 10% of cases where clinical assessment was equivocal. The incidence of a venous thromboembolic event was 5.9% with a re-rupture rate of 2%. 200 patients had 12 months ATRS scores available and 46 patients had ATRS scores as well as objective measures of calf girth, heel raise height and repetitions at 12 months. The average ATRS score was 75.3 at an average follow up of 25 months post injury. Men had significantly better outcomes compared to women. There were statistically significant differences in the muscle girth and heel raise height when compared with the uninjured side but these did not correlate with the ATRS score. Conclusion: This is the largest series of acute AT ruptures treated conservatively with the use of a standardised functional rehabilitation regime demonstrates satisfactory patient reported outcome measures at 12 months follow up. Objective measures demonstrate inferior results when compared to the uninjured side but this does not correlate with the ATRS score

This Tutorial Review acquaints chemists and metallurgists with the properties and industrial applications of methanesulfonic acid (MSA, CH 3 SO 3 H). Over the past quarter-century, MSA has garnered increasing interest as a reagent for green chemistry due to its strong acidity, while circumventing many of the challenges associated with handling concentrated sulfuric acid, hydrochloric acid, or nitric acid. Concentrated MSA is a non-oxidizing reagent, exhibiting high chemical stability against redox reactions and hydrolysis, as well as high thermal stability and limited corrosivity towards construction materials. It is colorless, odorless, and possesses a very low vapor pressure. MSA combines commendable biodegradability with low toxicity. It is extensively utilized as a Brønsted acid catalyst for esterification or alkylation reactions, and is employed in biodiesel production. The high solubility of its metal salts, the high electrical conductivity of its concentrated solutions, coupled with the high electrochemical stability of MSA and its anion, make MSA-based electrolytes beneficial in electrochemical applications. Examples include the electrodeposition of tin-lead solder for electronic applications and the high-speed plating of tin on steel plate for food cans. MSA-based electrolytes are used in redox flow batteries (RFBs). MSA offers a much safer and environmentally friendlier alternative to electrolytes based on fluoroboric or fluorosilicic acid. A novel application area is as a strong acid in extractive metallurgy, where it may contribute to the development of circular hydrometallurgy. MSA is being explored in lithium-ion battery recycling flowsheets, as well as in other applications in the field of metal recovery and refining. However, this review is not solely about the advantages of MSA for green chemistry or clean technologies, as there are also some potential drawbacks. Apart from its higher price compared to regular strong acids, MSA has only minor advantages for applications where sulfuric acid performs well. Since methanesulfonate biodegrades into sulfate, the same emission restrictions as for sulfate should be considered. In conclusion, MSA is the acid of choice for applications where metal sulfates cannot be used due to poor solubility or where concentrated sulfuric acid is too reactive towards organics., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

MaGuCo is a temperate phage isolated from soil collected in Alton, NH, USA, using Arthrobacter globiformis . Its genome is 43,924 base pairs long and contains 63 protein-encoding genes, 44 of which were assigned putative functions. MaCuGo is assigned to cluster AZ2 based on gene content similarity to actinobacteriophages., Competing Interests: The authors declare no conflict of interest.

A hydrometallurgical process is described for conversion of an aqueous solution of lithium chloride into an aqueous solution of lithium hydroxide via a chloride/hydroxide anion exchange reaction by solvent extraction. The organic phase comprises a quaternary ammonium chloride and a hydrophobic phenol in a diluent. The best results were observed for a mixture of the quaternary ammonium chloride Aliquat 336 and 2,6-di- tert -butylphenol (1:1 molar ratio) in the aliphatic diluent Shellsol D70. The solvent extraction process involves two steps. In the first step, the organic phase is contacted with an aqueous sodium hydroxide solution. The phenol is deprotonated, and a chloride ion is simultaneously transferred to the aqueous phase, leading to in situ formation of a quaternary ammonium phenolate in the organic phase. The organic phase, comprising the quaternary ammonium phenolate, is contacted in the second step with an aqueous lithium chloride solution. This contact converts the phenolate into the corresponding phenol by protonation with water extracted to the organic phase, followed by a transfer of hydroxide ions to the aqueous phase and chloride ions to the organic phase. As a result, the aqueous lithium chloride solution is transformed into a lithium hydroxide solution. The process has been demonstrated in continuous counter-current mode in mixer-settlers. Solid battery-grade lithium hydroxide monohydrate was obtained from the aqueous solution by crystallization or by antisolvent precipitation with isopropanol. The process consumes no chemicals other than sodium hydroxide. No waste is generated, with the exception of an aqueous sodium chloride solution., Supplementary Information: The online version contains supplementary material available at 10.1007/s40831-022-00629-2., Competing Interests: Conflict of interestNo conflicts of interest to declare., (© The Author(s) 2022.)

Background: The demands on healthcare professionals caring for families grappling with a life-limiting condition in an unborn or newly born child can be overwhelming. Clinicians working in emergency/trauma, hospice, and pediatric settings are already at high risk for burnout and compassion fatigue, which can leave healthcare institutions increasingly vulnerable to poor retention, absenteeism, and waning quality of care. The provision of exemplary palliative care requires a cohesive interdisciplinary team of seasoned professionals resilient to daily challenges. In September 2019, the American College of Gynecology, in a committee opinion, published standard of care guidelines for perinatal palliative care. This has created an impetus for exceptional caregiving and a greater demand for both physician and interdisciplinary healthcare provider education, training, and ongoing support that promotes truly beneficent care for pregnant patients confronted with life-limiting fetal conditions. Methods: A scoping review of the research literature was conducted in order to distinguish the barriers and facilitators of professional resiliency in perinatal palliative care. PubMed, Medline, CINAHL, and EBSCO Psychology & Behavioral Sciences Collections were systematically reviewed. Because of the paucity of studies specific to perinatal palliative care, several interviews of nurses and physicians in that field were conducted and analyzed for content distinctly pertaining to personal practices or workplace factors that support or hinder professional resiliency. Results: The research indicated that medical professionals often cite a lack of knowledge, inexperience using effective communication skills related to perinatal palliative care and bereavement, challenges with interdisciplinary collaboration, misconceptions about the role and function of palliative care in the perinatal or neonatal settings, moral distress, and workload challenges as encumbrances to professional satisfaction. Strategic implementation of facility-wide bereavement care training, effective communication modalities, and evidenced-based practical applications are critical components for a thriving perinatal palliative care team. Authentic formal and informal debriefing, peer mentoring, adequate caseloads, robust provider self-care practices, exceptional relational efficacy, and cultural and spiritual humility can foster personal growth and even vicarious resilience for perinatal palliative care professionals. Conclusions: Support should be strategic and multifaceted. The onus to implement salient measures to cultivate resilience in the perinatal palliative caregiver should not be only upon the individuals themselves but also upon prevailing regulatory governing bodies and healthcare institutions., (Copyright © 2020 Grauerholz, Fredenburg, Jones and Jenkins.)

Our previous studies showed that Fgf8 mutations can cause Kallmann syndrome (KS), a form of congenital hypogonadotropic hypogonadism, in which patients do not undergo puberty and are infertile. Interestingly, some KS patients also have agenesis of the corpus callosum (ACC) suggesting that KS pathology is not limited to reproductive function. Here, we asked whether FGF8 dysfunction is the underlying cause of ACC in some KS patients. Indeed, early studies in transgenic mice with Fgf8 mutations reported the presence of failed or incomplete corpus callosum formation. Additional studies in transgenic mice showed that FGF8 function most likely prevents the prenatal elimination of glial fibrillary acidic protein (GFAP)-immunoreactive (IR) glial cells in the indusium griseum (IG) and midline zipper (MZ), two anterior-dorsal midline regions required for corpus callosum formation (i.e., between embryonic days (E) 15.5-18.5). Here, we tested the hypothesis that FGF8 function is critical for the survival of the GFAP-IR midline glial cells. First, we measured the incidence of apoptosis in the anterior-dorsal midline region in Fgf8 hypomorphic mice during embryonic corpus callosum formation. Second, we quantified the GFAP expression in the anterior-dorsal midbrain region during pre- and postnatal development, in order to study: 1) how Fgf8 hypomorphy disrupts prenatal GFAP-IR midline glial cell development, and 2) whether Fgf8 hypomorphy continues to disrupt postnatal GFAP-IR midline glial cell development. Our results indicate that perinatal FGF8 signaling is important for the timing of the onset of anterior-dorsal Gfap expression in midline glial cells suggesting that FGF8 function regulates midline GFAP-IR glial cell development, which when disrupted by Fgf8 deficiency prevents the formation of the corpus callosum. These studies provide an experimentally-based mechanistic explanation as to why corpus callosum formation may fail in KS patients with deficits in FGF signaling., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Quality-based care is a hallmark of physical therapy. Treatment effectiveness must be evident to patients, managers, employers, and funders. Quality indicators (QIs) are tools that specify the minimum acceptable standard of practice. They are used to measure health care processes, organizational structures, and outcomes that relate to aspects of high-quality care of patients. Physical therapists can use QIs to guide clinical decision making, implement guideline recommendations, and evaluate and report treatment effectiveness to key stakeholders, including third-party payers and patients. Rehabilitation managers and senior decision makers can use QIs to assess care gaps and achievement of benchmarks as well as to guide quality improvement initiatives and strategic planning. This article introduces the value and use of QIs to guide clinical practice and health service delivery specific to physical therapy. A framework to develop, select, report, and implement QIs is outlined, with total joint arthroplasty rehabilitation as an example. Current initiatives of Canadian and American physical therapy associations to develop tools to help clinicians report and access point-of-care data on patient progress, treatment effectiveness, and practice strengths for the purpose of demonstrating the value of physical therapy to patients, decision makers, and payers are discussed. Suggestions on how physical therapists can participate in QI initiatives and integrate a quality-of-care approach in clinical practice are made., (© 2016 American Physical Therapy Association.)

Objective: To assess the effectiveness of total contact insoles (TCI) in patients with plantar fasciitis (PF)., Methods: A double-blind randomized controlled trial was carried out with intention-to-treat analysis. Seventy-four patients were randomly allocated to use a TCI made of ethylene vinyl acetate (study group, n = 37) or a flat insole (control group, n = 37). The following assessment tools were used: visual analog scale for pain while walking and at rest, Medical Outcomes Study Short Form-36 (SF-36) for quality of life, Foot Function Index and Foot Health Status Questionnaire for foot function, 6-min walk test (6MWT), and baropodometer FootWalk Pro for plantar pressure analysis. The groups were evaluated by a blinded assessor at baseline and after 45, 90, and 180 days., Results: The groups were homogeneous for the majority of variables at baseline. The over-time comparisons show a statistical difference between the groups for pain while walking (p = 0.008) and the 6MWT (p = 0.010). Both groups showed significant improvements in pain at rest, foot function, and some quality of life variables (physical functioning, bodily pain, vitality, and social functioning), with no significant statistical differences between them. The baropodometer recorded no changes from the use of the insoles., Conclusion: A TCI can be used to reduce pain while walking and to increase walking distance in individuals with PF.

Background: Clinicians are faced with using the current best evidence to make treatment decisions, yet synthesis of knowledge translation (KT) strategies that influence professional practice behaviors in rehabilitation disciplines remains largely unknown., Purpose: The purposes of this study were: (1) to examine the state of science for KT strategies used in the rehabilitation professions (physical therapy, occupational therapy, speech-language pathology), (2) to identify the methodological approaches utilized in studies exploring KT strategies, and (3) to report the extent that KT interventions are described., Data Sources: Eight electronic databases (MEDLINE, CINAHL, ERIC, PASCAL, EMBASE, IPA, Scopus, and CENTRAL) were searched from January 1985 to May 2013 using language (English) restriction., Study Selection: Eligibility criteria specified articles evaluating interventions or strategies with a primary purpose of translating research or enhancing research uptake into clinical practice., Data Extraction: Two reviewers independently screened the titles and abstracts, reviewed full-text articles, performed data extraction, and performed quality assessment. The published descriptions of the KT interventions were compared with the Workgroup for Intervention Development and Evaluation Research's (WIDER) Recommendations to Improve the Reporting of the Content of Behaviour Change Interventions., Data Synthesis: Of a total of 2,793 articles located and titles and abstracts screened, 26 studies were included in the systematic review. Eighteen articles reported interventions that used a multicomponent KT strategy. Education-related components were the predominant KT intervention regardless of whether it was a single or multicomponent intervention. Few studies used reminders or audit and feedback intervention (n=3). Only one study's primary outcome measure was an economic evaluation. No clear delineation of the effect on KT strategies was seen., Limitations: Diverse studies were included; however, the heterogeneity of the studies was not conducive to pooling the data., Conclusions: The modest-to-low methodological quality assessed in the studies underscores the gaps in KT strategies used in rehabilitation and highlights the need for rigorously designed studies that are well reported., (© 2015 American Physical Therapy Association.)

Background: The US Public Health Service (USPHS) recommends tobacco cessation counseling (TCC) training for all health care professionals. Within physical therapist practice, smoking can have adverse effects on treatment outcomes in all body systems. In addition, people with physical disabilities have a higher smoking prevalence than the general population, creating a strong need for tobacco cessation among physical therapy clientele. Therefore, TCC training is an important component of entry-level physical therapist education., Objective: The aims of this study were: (1) to determine need for TCC training within entry-level physical therapist education and (2) to identify potential barriers to implementation of USPHS guidelines in the academic environment., Design: A descriptive cross-sectional survey was conducted., Methods: Directors or academic coordinators of clinical education from entry-level physical therapist programs (N=204) were surveyed using an online instrument designed specifically for this study. Data regarding program and faculty characteristics, tobacco-related training content, and faculty opinions toward TCC in both physical therapist practice and education were analyzed descriptively., Results: The response rate was 71%. A majority (60%) of programs indicated inclusion of tobacco-related training, most commonly 1 to 2 hours in duration, and of these programs, 40% trained students in the implementation of USPHS clinical guidelines for TCC., Limitations: Data analyses were constrained by limited or missing data in some areas. A single faculty member completed the survey for each program., Conclusions: There is a need for TCC training in entry-level physical therapist education. Inclusion may be facilitated by addressing perceived barriers toward TCC as a component of physical therapist practice and promoting the relevance of TCC as it relates to intended outcomes of physical therapy interventions., (© 2014 American Physical Therapy Association.)

Sequence diversity in the human antibody repertoire is generated in two steps: by the combinatorial assembly of V gene segments and by somatic hypermutation. Here, we have characterised these processes for the lambda (lambda) light chain using a library of 7600 lambda cDNA clones from peripheral blood lymphocytes. By hybridisation and sequencing we found that most lambda chains are derived from the cluster of V(lambda) segments closest to the J(lambda)-C(lambda) pairs and that there is considerable variation in the use of individual V(lambda) segments (ranging from 0.02% to 27%): three of the 30 functional V(lambda) segments encode half the expressed V(lambda) repertoire. As a result of these biases, sequence diversity in the primary repertoire is focused at the centre of the antigen binding site. By contrast, somatic hypermutation spreads diversity to the periphery. Comparison with the human kappa (kappa) light chain indicates that both kappa and lambda use the same strategy for searching sequence space and have almost identical patterns of diversity in the mature antibody repertoire.

Background: A human single chain Fv (scFv) specific for human carcinoembryonic antigen (CEA) has been isolated from a 2.0 x 10(9) phage display library from unimmunised human donors. The dissociation constant of the scFv has been measured by surface plasmon resonance (SPR) and found to be 7.7 x 10(-9) M, with an off-rate component of 6.2 x 10(-3) s-1. In order to investigate directly whether increased affinity leads to improved targeting of CEA-positive tumours, this scFv has been affinity matured by both targeted mutagenesis of the CDRs of heavy and light chains, and by light chain shuffling., Study Design: A partial randomisation scheme, biased towards amino acids commonly found as somatic mutations of germline antibody sequences, was used for directed diversification of VH and VL CDR3s. Diversification of the entire VL region was also introduced by light chain shuffling of the parental anti-CEA scFv. Selection of the mutagenised repertoires was carried out to enrich for antibodies with a reduced koff., Results: Sequencing the selected clones identified a number of amino acid changes in the VH CDR3, one of which gave a four-fold reduction in koff. Stringent selection of the light chain shuffled library resulted in several clones with a two- to three-fold reduction in koff. It has been possible to combine the selected changes from both mutagenesis approaches by using the mutagenised heavy chain and a light chain derived by shuffling to give a human scFv with a dissociation constant for human CEA of 6.0 x 10(-10) M., Conclusion: A panel of human anti-CEA scFvs has been generated with differing dissociation constants for antigen, which will allow the correlation between tumour targeting efficiency in relation to binding affinity to be assessed directly. The scFv panel will be valuable in the optimisation of human antibodies for immunotherapy.

It is difficult to raise antibodies against haptens and antigens that are unstable under the physiological conditions of the serum. Here we have used a phage antibody library to isolate antibody fragments against oxygen sensitive reduced flavin, by selection of the phage under anaerobic and reducing conditions at pH 5 and a pre-elution step with the oxidized flavin. The binding of the reduced hapten to one of the antibody fragments was characterised by time-resolved polarised fluorescence, and shown to be highly specific for the reduced flavin.

In the human immune system, antibodies with high affinities for antigen are created in two stages. A diverse primary repertoire of antibody structures is produced by the combinatorial rearrangement of germline V gene segments and antibodies are selected from this repertoire by binding to the antigen. Their affinities are then improved by somatic hypermutation and further rounds of selection. We have dissected the sequence diversity created at each stage in response to a wide range of antigens. In the primary repertoire, diversity is focused at the centre of the binding site. With somatic hypermutation, diversity spreads to regions at the periphery of the binding site that are highly conserved in the primary repertoire. We propose that evolution has favoured this complementarity as an efficient strategy for searching sequence space and that the germline V gene families evolved to exploit the diversity created by somatic hypermutation.

Antibody fragments of moderate affinity (approximately microM) can be isolated from repertoires of approximately 10(8) immunoglobulin genes by phage display and rounds of selection with antigen, and the affinities improved by further rounds of mutation and selection. Here, as an alternative strategy, we attempted to isolate high affinity human antibodies directly from large repertoires. We first created highly diverse repertoires of heavy and light chains entirely in vitro from a bank of human V gene segments and then, by recombination of the repertoires in bacteria, generated a large (close to 6.5 x 10(10)) synthetic repertoire of Fab fragments displayed on filamentous phage. From this repertoire we isolated Fab fragments which bound to a range of different antigens and haptens, and with affinities comparable with those of antibodies from a secondary immune response in mice (up to 4 nM). Although the VH-26 (DP-47) segment was the most commonly used segment in both artificial and natural repertoires, there were also major differences in the pattern of segment usage. Such comparisons may help dissect the contributions of biological mechanisms and structural features governing V gene usage in vivo.

We describe a process for the identification of mRNAs within single cells, as demonstrated with the immunoglobulin (Ig) variable region (V) genes of two mouse hybridoma cell lines and the bcr-abl fusion gene of the human K562 myeloid leukaemia line. The cells were fixed and permeabilised, the mRNA reverse transcribed to cDNA and the cDNA amplified by the polymerase chain reaction (PCR). After using fluorescent PCR primers, the amplified DNA could be detected within the cells as demonstrated by confocal fluorescence microscopy and flow cytometry. Furthermore the amplified Ig VH and VL DNA could be assembled within the same cell using suitable PCR primers. We detected no cross-contamination of amplified DNA between cells: the DNA isolated from mixtures of two hybridoma cell lines (B1-8 and NQ10/12.5) treated to in-cell PCR and assembly, was shown by cloning to correspond to the combinations of VH and VL genes of the parent hybridomas. We forsee diverse applications of in-cell assembly by PCR, especially for the analysis of the combinations of chains of rearranged Ig or T cell receptor (TCR) V-genes in a population of cells, and the construction of human antibodies from the V-genes of immune B-lymphocytes.

The serum half-lives of a wild-type recombinant mouse monoclonal antibody of the IgG2b isotype and a mutant antibody differing from the wild-type antibody by a single amino acid substitution introduced into the CH2 domain, the replacement of Asn 297 by Ala to delete the conserved site of heavy chain glycosylation, were determined in the rat. The biological half-life of the aglycosyl Asn 297-Ala mutant recombinant antibody (4.8 days) was significantly shorter than that of the normally glycosylated wild-type antibody (7.4 days) by enzyme immunoassay. A similar difference between the biological half-lives of 125I-labelled aglycosyl and wild-type antibodies (2.9 and 4.0 days, respectively) was determined by gamma counting. Analysis of serum samples demonstrated that both recombinant antibodies were present in the circulation predominantly as intact monomeric IgG and revealed no differences that could account for the more rapid elimination of the aglycosyl antibody. The results of this investigation indicate that the carbohydrate residues contribute only in part to the survival of IgG in vivo and suggest that the diminished half-life of the aglycosyl antibody is due to increased catabolism in the extravascular tissues.

The serum half-lives of three recombinant mouse monoclonal antibodies, differing radically in their ability to bind to Clq or FcRI but only minimally in structure, were determined in the BALB/c mouse following intravenous administration. The wild-type antibody, a chimaeric antibody comprising variable domains binding 3-iodo-4-hydroxy-5-nitrophenylacetate and constant domains of the mouse IgG2b isotype, was eliminated from the bloodstream with biphasic kinetics: alpha-phase, 0.5 days; beta-phase, 7.0 days. The alpha- and beta-phase half-lives of mutant recombinant antibodies with single amino acid substitutions, either Glu 235-Leu allowing binding to the mouse FcRI, or Lys 322-Ala reducing Clq binding 30-fold, were indistinguishable from those of the wild-type antibody demonstrating that the biological half-life of intact mouse IgG is independent of the ability to bind Clq or FcRI. The major implication of the present study is that IgG molecules which have been genetically engineered to eliminate interaction with other components of the immune system should retain the long half-life typical of natural antibodies.

Important mammalian defensive functions such as phagocytosis are triggered in leukocytes by the interaction of the Fc region of IgG with cell surface receptors (Fc gamma R). The CH2 domain of IgG has been implicated previously as the site of interaction with human and mouse Fc gamma R. This domain was mapped for interaction with mouse Fc gamma R11 expressed by the macrophage-like cell line P388D1, using two panels of a total of 32 site-directed mutants of mouse IgG2b and chimeric human IgG3 monoclonal antibodies. Two potential binding sites have been identified: one in or within the vicinity of the lower hinge site on IgG for human Fc gamma R1, and one within the binding site on IgG for Clq. The three mutant IgGs (Gly 237----Ala, Asn 297----Ala, and Glu 318----Ala) which do not interact in complexed form also fail to bind as monomers. A 1H NMR study of the three non-binding monomeric mutants suggests that the mutations are largely site-specific, indicating that IgG interacts with mouse Fc gamma R11 at two regions within the CH2 domain. This interaction dictates phagocytosis mediated by Fc gamma R11 of the P388D1 cell line.

Cellular receptors for IgG (Fc gamma R) mediate important protective functions. By using site-specific mutants of a chimeric antibody (mouse V H domain and L chain; human IgG3 C H domains), we have demonstrated that human Fc gamma RI interacts with a site in the lower hinge of human IgG (residues 234 to 237) and that this interaction dictates Fc gamma RI-mediated superoxide generation. Mutations at position 235 resulted in the most profound reductions in Fc gamma RI recognition. We have also mapped an interaction site for Fc gamma RII to the same region; however, mutations at position 234 and 237 resulted in the greatest reductions in Fc gamma RII recognition. The two receptors appear to recognize overlapping but nonidentical sites on the lower hinge of IgG. Deviations from the optimal motif 234-Leu-Leu-Gly-Gly-237 may then explain the human IgG subclass specificity profile for human Fc gamma RI and Fc gamma RII.

The variable domains of an antibody consist of a beta-sheet framework with hypervariable regions (or complementarity-determining regions--CDRs) which fashion the antigen-binding site. Here we attempted to determine whether the antigen-binding site could be transplanted from one framework to another by grafting the CDRs. We substituted the CDRs from the heavy-chain variable region of mouse antibody B1-8, which binds the hapten NP-cap (4-hydroxy-3-nitrophenacetyl caproic acid; KNP-cap = 1.2 microM), for the corresponding CDRs of a human myeloma protein. We report that in combination with the B1-8 mouse light chain, the new antibody has acquired the hapten affinity of the B1-8 antibody (KNP-cap = 1.9 microM). Such 'CDR replacement' may offer a means of constructing human monoclonal antibodies from the corresponding mouse monoclonal antibodies.

For shotgun cloning into M13 vectors, a double-stranded cassette of synthetic oligonucleotides containing a SmaI site within the two halves of an EcoK site, has been introduced into the vector M13mp8. Cloning of blunt end DNA into the SmaI site destroys the EcoK site, and recombinants are therefore preferentially selected on transfection into a K strain of E.coli. For deletion mutagenesis using synthetic oligonucleotides, an M13 vector with four copies of the EcoK cassette has been made to facilitate the joining of lacZ or a Factor Xa cleavage site to any protein reading frame.

Using a chemical quench device, the rate of synthesis of carbamyl aspartate from the substrates aspartate and carbamyl phosphate was followed as a function of the time between mixing the enzyme with substrates and quenching with trichloroacetic acid. This function, which is linear at long times, shows (at 4 degrees C) a transient lag phase of product of roughly 10 ms. However, when the catalytic subunit (in which the enzymatic activity is desensitized) is used instead of the enzyme, the lag disappears. Therefore the lag seems to be associated with the control functions of the enzyme, i.e. to represent the allosteric transition involved in substrate-substrate (homotropic) co-operativity. Thus the relaxation time for the activation process is roughly 10 ms. The implications of these results are examined.

The result of binding the effectors ATP and CTP to aspartate transcarbamylase was studied by X-ray solution scattering. Binding of substrate analogues produces a substantial change in the solution scattering curve, allowing us to monitor the proportion of the different quaternary structure states present in solution. In the initial solution this ratio was made roughly unity by adding either carbamyl phosphate and succinate, or N-(phosphonacetyl)-L-aspartate (PALA). ATP or CTP were then added, and their effect on the proportion of the different quaternary structure states was followed. When using carbamyl phosphate and succinate (weakly bound), ATP or CTP had a clear effect, as observed previously by monitoring the sedimentation rate (Changeux et al., 1968). However, when PALA (strongly bound) was used, the effect of CTP was very much smaller, and that of ATP was undetectable. This result supports the explanation by Tauc et al. (1982), that nucleotides act mostly through changing the affinity of the active sites for substrate, and only to a small extent by directly modifying the quaternary structure equilibrium in the case of CTP.

Human blood neutrophils manifested markedly decreased motility following exposure to the horseradish peroxidase (HRP)/H2O2/halide system in vitro. These cells were protected from this inhibitory effect (of the HRP/H2O2/halide system) by inclusion of concentrations in the reaction system of ascorbate, cysteinee, levamisole and thiamine which stimulate neutrophil migration and inhibit activity of the HRP/H2O2/halide system. The reversible nature of the oxidative inhibition of migration was demonstrated by exposing neutrophils to the HRP/H2O2/halide system for 15 min followed by washing to remove the components of the peroxidative system, and subsequent addition of ascorbate, cystein, levamisole, thiamine and the reducing agent, dithiothreitol. Neutrophils so treated completely recovered normal or increased motility induced by the leucoattractants endotoxin-activated serum or synthetic chemotactic tripeptide f-met-leu-phe. This reversible loss of migratory responsiveness following exposure of neutrophils to the HRP/H2O2/halide system was not associated with decreased cell viability or adherence. However, membrane oxidation was accompanied by increased uptake of radiolabelled f-met-leu-phe and degranulation. The increased leucoattractant uptake was decreased by ascorbate, levamisole and thiamine. These agents also prevented oxidation of the neutrophil membrane by the HRP/H2O2/halide system as measured indirectly by inhibition of iodination.

For the first time, the structural change associated with an allosteric transition has been monitored by X-ray solution scattering. The kinetics of the quaternary structure change of aspartate transcarbamylase were first slowed by using acetyl phosphate instead of carbamyl phosphate, and by the presence of 10% or 30% ethylene glycol. At 6.5 degrees C, the quaternary structure change was found to have a time constant of about 11 seconds. This appears to be larger than that obtained for the switching of homotropic co-operativity, measured by chemical quench under the same conditions.

The membrane-traversing subunit c parallel from the F0 part of the ATP synthase molecule has been studied in chloroform/methanol by high-resolution 1H n.m.r. Various one-dimensional and two-dimensional techniques have been used for assignment purposes, some NOE connectivities were established and some 3JHN alpha coupling constants were measured from spin--echo experiments. The effects of varying pH, solvent composition, lanthanide concentration and temperature have been investigated. Evidence is presented that the molecule has extensive alpha-helical segments, and the hairpin structure suggested by other groups is supported by our n.m.r. data. Only one ionizable group, assigned to the C-terminal carboxyl, is observed to titrate in the pH range 2 to 10; so the conserved residue, Asp61, which binds dicyclohexylcarbodiimide, presumably has (at least in this solvent system) an abnormally high pK value.

We have designed a set of oligonucleotide primers to amplify the cDNA of mouse immunoglobulin heavy and light chain variable domains by the polymerase chain reaction. The primers incorporate restriction sites that allow the cDNA of the variable domains to be force-cloned for sequencing and expression. Here we have applied the technique to clone and sequence the variable domains of five hybridoma antibodies and to express a mouse-human chimeric antibody that binds to the human mammary carcinoma line MCF-7. The technique should also lead to the cloning of antigen-binding specificities directly from immunoglobulin genes.

In antibodies, a heavy and a light chain variable domain, VH and VL, respectively, pack together and the hypervariable loops on each domain contribute to binding antigen. We find, however, that isolated VH domains with good antigen-binding affinities can also be prepared. Using the polymerase chain reaction, diverse libraries of VH genes were cloned from the spleen genomic DNA of mice immunized with either lysozyme or keyhole-limpet haemocyanin. From these libraries, VH domains were expressed and secreted from Escherichia coli. Binding activities were detected against both antigens, and two VH domains were characterized with affinities for lysozyme in the 20 nM range. Isolated variable domains may offer an alternative to monoclonal antibodies and serve as the key to building high-affinity human antibodies. We suggest the name 'single domain antibodies (dAbs)' for these antigen binding demands.

The antimicrobial oxidative system (myeloperoxidase (MPO), H2O2 and a halide) produced by stimulated PMNLs is simulated in vitro using horseradish peroxidase (HRP), H2O2 and NaI. Ascorbate, thiamine, levamisole and cysteine prevent and reverse the PMNL motility inhibiting effects of the HRP/H2O2/NaI system. The ability of these agents to protect the PMNL specifically from the known iodinating and oxidising abilities of this system was investigated. All four agents protect the PMNL from iodination by HRP/H2O2/NaI. However, only ascorbate and thiamine are able to reverse this process after it has occurred. Thiamine is seen on thin layer chromatography followed by autoradiography to be iodinated by this system. Ascorbate, thiamine and cysteine are able to protect the neutrophil sulfhydryl groups from oxidation by the system. One can therefore conclude that ascorbate and cysteine protect neutrophil motility from inhibition by the HRP/H2O2/NaI system by acting as reducing agents which maintain the neutrophil sulfhydryl groups. Thiamine also acts as a reducing agent, though not as effectively as ascorbate or cysteine. In addition, thiamine protects the PMNL from iodination by a competitive mechanism. The mechanism of levamisole protection is less clear but may involve scavenging of free radicals generated by the HRP/H2O2/NaI system. Protease enzymes, glycolysis and adherence are found not to be target sites for the PMNL motility inhibiting effects of the HRP/H2O2/NaI system. Further, increasing concentrations of the synthetic leukoattractant FMLP were shown to increase the auto-iodination of PMNLs without addition of extraneous peroxidase or peroxide. This data was compared with optimal FMLP concentrations for chemotaxis.