Search

Background: The healthcare sector contributes directly to global warming and environmental decline. This is partly due to disproportionately large waste generation compared to other sectors and the environmental consequences of medical waste incineration. Waste separation and recycling decrease the total waste generation of the theatre complex, decreasing the cost of waste disposal, and if properly implemented, can generate revenue. Waste separation is not performed uniformly, and no recycling programmes exist in the theatre complexes of the academic hospitals in Johannesburg. Potentially recyclable anaesthetic waste is not identified in our setting. Methods: Recyclable anaesthetic waste items were identified. Anaesthetic waste was collected after every anaesthetic case. General and medical waste were weighed respectively and inspected for correct separation. Recyclable items were separated from general waste and weighed. Results: A total of 107.6 kg of anaesthetic waste was evaluated. Per anaesthetic case, 74.6% (65.0-84.2%) was medical waste, and 25.4% (15.8-35.0%) was general waste. Of the general waste, 68.8% (57.7-78.8%) was recyclable. Only 6.8% of medical and 61.4% of general waste bags inspected were correctly separated. Within each medical waste bag, 6.9% (2.3-15.5%) of waste was incorrectly placed general waste. Similarly, each general waste bag contained 6% (0-21.6%) incorrectly placed medical waste. Waste generated per surgical discipline was significantly different. Conclusion: Correct waste separation, a key step in decreasing the burden of healthcare waste, was poor. The study demonstrated that most general anaesthetic waste is recyclable. [ABSTRACT FROM AUTHOR]

Background: The anaesthetic chart is an important component of a patient’s health record in the perioperative period. Studies have shown that anaesthetic charts are often incomplete. The adequacy of chart completion in the anaesthetic department at Chris Hani Baragwanath Academic Hospital (CHBAH) has never been quantified. An audit was done, and the charts were assessed for adequacy of completion. Methods: This audit assessed adequacy of completion of anaesthetic charts for 2019. Using a peer-reviewed checklist adapted from guidelines by the Australian and New Zealand College of Anaesthetists (ANZCA) and the South African Society of Anaesthesiologists (SASA). A sample of 333 charts was audited to assess adequacy of completion. To eliminate sampling bias, a stratified sampling method was used. Results: Completeness was defined as a chart scoring 100%. None of the charts, however, scored 100% and the overall median score was 77%. Charts were subdivided into three groups. Those scoring 75–99% (n = 212), those scoring 50–74% (n = 121) and those that were less than 50% complete (n = 0). Patient category (adult vs paediatrics), time of shift (day vs night) and type of anaesthetic were audited and compared as factors that could affect chart completeness. The only factor that had a statistically significant difference in chart completeness was the patient category, where adult chart completion scored higher compared to paediatric charts, with a p-value < 0.0074. Conclusion: The audited charts scored higher than charts in previous audits done both locally and internationally. Some important aspects of the charts were poorly documented. Ongoing audits and training on chart completion can potentially improve the adequacy of completion and should be part of the academic programme. Better documentation could potentially lead to improved perioperative patient care and mitigate medicolegal risks. [ABSTRACT FROM AUTHOR]

Prehabilitation consists of planned interventions implemented prior to surgery with the aim of increasing functional reserve and creating surgical resilience to positively impact a patient's surgical outcome. [ABSTRACT FROM AUTHOR]

Patients with heart failure have increased risk for adverse cardiac events including death, following non-cardiac surgery. The necessity of surgery needs to be determined along with appropriate preoperative evaluation and risk stratification. The anaesthetic should be tailored to specific haemodynamic goals with careful fluid management and appropriate use of vasoactive therapies. [ABSTRACT FROM AUTHOR]

β-blockers bind selectively to beta-adrenergic receptors and interfere with catecholamines provoking β-responses on the heart and smooth muscles of the airways and blood vessels. To block or not to block. [ABSTRACT FROM AUTHOR]

Background: Endotracheal tube (ETT) cuff pressure commonly exceeds the recommended range of 20-30 cm H2O during anaesthesia. A set volume of air will not deliver the same cuff pressure in each patient and the pressure exerted by the ETT cuff can lead to complications, with either over- or under-inflated cuffs. These can include a sore throat and cough, aspiration, volume loss during positive pressure ventilation, nerve palsies, tracheomalacia and tracheal stenosis. No objective means of ETT cuff pressure monitoring is available in the operating theatres of Charlotte Maxeke Johannesburg Academic Hospital (CMJAH) and Chris Hani Baragwanath Academic Hospital (CHBAH). The ETT cuff pressure of patients undergoing general anaesthesia is therefore unknown. Method: ETT cuff pressure of 96 adult patients undergoing general anaesthesia without nitrous oxide at CMJAH and CHBAH was measured by one researcher. A RUSCH Endotest™ manometer was used to measure ETT cuff pressure in size 7.0 - 8.5 mm ETTs. The cuff inflation technique that was used by the anaesthetist was also documented. Results: The mean ETT cuff pressure recorded was 47.5 cm H2O (range 10-120 cm H2O). ETT cuff pressures exceeded 30 cm H2O in 64.58% of patients. Only 18.75% of patients had ETT cuff pressures within the recommended range of 20-30 cm H2O. There was no statistically significant difference between the ETT cuff pressures measured at the two hospitals. Minimal occlusive volume was the most frequent technique used to inflate the ETT cuff (37.5%); this was followed by inflating the ETT cuff with a predetermined volume of air in 31.25% of cases and palpation of the pilot balloon (27.08%). There was no statistically significant difference between the ETT cuff pressure measured and the inflation technique used by the anaesthetist. Conclusion: ETT cuff pressures of the majority of patients undergoing general anaesthesia at two academic hospitals were higher than the recommended range. ETT cuff pressure should routinely be measured using a manometer. [ABSTRACT FROM AUTHOR]

Cross-linking of the T-cell receptor (CD3) induces activation of tyrosine kinases and the subsequent phosphorylation of intracellular protein substrates. We examined whether early events in signal transduction through CD3 or CD3 x CD4 receptor ligation were altered in aged murine T-lymphocytes. Both calcium mobilization and tyrosine phosphorylation of phospholipase C gamma 1 (PLC gamma 1) were decreased in T-lymphocytes from old mice. In addition, there was less tyrosine phosphorylation of a 35/36 kDa protein both in whole cell lysates and in PLC gamma 1 immunoprecipitates from old mice. This 35/36 kDa phosphoprotein binds specifically to the SH2 domains of PLC gamma 1. Using a fusion protein containing the SH2 domains of PLC gamma 1 and human IgG1 heavy chain, we identified three additional proteins that bind to the SH2 domains which were tyrosine phosphorylated following CD3 x CD4 ligation to a lesser degree with age. The tyrosine phosphorylation of two phosphoproteins binding to a fusion protein consisting of the SH2 domains of GAP (ras GTPase-activating protein) and human IgG1 heavy chain was also reduced with aging. The observed binding to SH2 domains was thiol redox sensitive. Thus, decreases in antioxidants with age may be responsible for inhibitory effects on PLC gamma 1-phosphatidylinositol signaling through redox regulation of tyrosine phosphoproteins. [ABSTRACT FROM AUTHOR]

Background : Proximal humeral bone deficiency in revision shoulder arthroplasty is an emerging and challenging problem as the use of reverse shoulder arthroplasty (RSA) increases. This paper presents a technical note discussing our detailed preoperative planning steps, surgical techniques, and their rationale in carrying out the use of an allograft prosthetic composite (APC) to address proximal humeral bone deficiency in revision RSA. The outcomes of this technique are also presented. This paper also presents a systematic review to further discuss the existing literature on RSA with APCs. Methods : The preoperative surgical planning and the surgical technique employed to execute proximal humeral reconstruction using APC during revision arthroplasty are discussed in the technical note. The preliminary clinical and radiological results of five patients who underwent revision shoulder arthroplasty with proximal humeral reconstruction using APCs are presented. The PRISMA guidelines were followed to perform the systematic review. A systematic search using PubMed, Embase, and Cochrane databases was conducted. All studies involving RSA and APCs were pooled, and the data were extracted and analyzed. Results : A total of 14 studies were eligible for inclusion in the systematic review, with a total of 255 patients and a mean follow-up of 57 months. All studies in the systematic review and the patients included in the author's case series showed improvements in the level of pain, range of motion, function, and satisfaction. Graft incorporation in the systematic review was 84%. Conclusions : Based on the available literature and the results of our case series, the use of an APC construct is a viable option for proximal humeral bone deficiency in revision shoulder arthroplasty.

Deep learning (DL) in orthopaedics has gained significant attention in recent years. Previous studies have shown that DL can be applied to a wide variety of orthopaedic tasks, including fracture detection, bone tumour diagnosis, implant recognition, and evaluation of osteoarthritis severity. The utilisation of DL is expected to increase, owing to its ability to present accurate diagnoses more efficiently than traditional methods in many scenarios. This reduces the time and cost of diagnosis for patients and orthopaedic surgeons. To our knowledge, no exclusive study has comprehensively reviewed all aspects of DL currently used in orthopaedic practice. This review addresses this knowledge gap using articles from Science Direct, Scopus, IEEE Xplore, and Web of Science between 2017 and 2023. The authors begin with the motivation for using DL in orthopaedics, including its ability to enhance diagnosis and treatment planning. The review then covers various applications of DL in orthopaedics, including fracture detection, detection of supraspinatus tears using MRI, osteoarthritis, prediction of types of arthroplasty implants, bone age assessment, and detection of joint-specific soft tissue disease. We also examine the challenges for implementing DL in orthopaedics, including the scarcity of data to train DL and the lack of interpretability, as well as possible solutions to these common pitfalls. Our work highlights the requirements to achieve trustworthiness in the outcomes generated by DL, including the need for accuracy, explainability, and fairness in the DL models. We pay particular attention to fusion techniques as one of the ways to increase trustworthiness, which have also been used to address the common multimodality in orthopaedics. Finally, we have reviewed the approval requirements set forth by the US Food and Drug Administration to enable the use of DL applications. As such, we aim to have this review function as a guide for researchers to develop a reliable DL application for orthopaedic tasks from scratch for use in the market., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: The use of reverse shoulder arthroplasty as a primary and revision implant is increasing. Advances in implant design and preoperative surgical planning allow the management of complex glenoid defects. As the demand for treating severe bone loss increases, custom allograft composites are needed to match the premorbid anatomy. Baseplate composite structural allografts are used in patients with eccentric and centric defects to restore the glenoid joint line. Preserving bone stock is important in younger patients where a revision surgery is expected. The aim of this article is to present the assessment, planning, and indications of femoral head allografting for bony defects of the glenoid. Methods: The preoperative surgical planning and the surgical technique to execute the plan with a baseplate composite graft are detailed. The preliminary clinical and radiological results of 29 shoulders which have undergone this graft planning and surgical technique are discussed. Clinical outcomes included visual analogue score of pain (VAS), American Shoulder and Elbow Surgeons score (ASES), Constant-Murley score (CS), satisfaction before and after operation, and active range of motion. Radiological outcomes included graft healing and presence of osteolysis or loosening. Results: The use of composite grafts in this series has shown excellent clinical outcomes, with an overall graft complication rate in complex bone loss cases of 8%. Conclusion: Femoral head structural allografting is a valid and viable surgical option for glenoid bone defects in reverse shoulder arthroplasty.

Background: Restoration of the glenoid joint line in shoulder arthroplasty is important for implant positioning and function. Medialization of the glenohumeral joint line due to glenoid bone loss is commonly encountered in primary and revision of shoulder arthroplasty albeit the direction and location of bone loss varies with different pathology. Three-Dimensional (3D) planning software has assisted in preoperative planning of complex glenoid deformities. However, limited literature is available defining a reliable 3D method to evaluate the glenoid joint line preoperatively., Aims: The purpose of this study is to identify a set of reliable scapular landmarks to be used as reference points to measure the premorbid glenoid joint line using 3D segmented models of healthy scapulae., Methods: Bilateral computed tomography scans from 79 patients eligible for primary stabilization procedures were retrospectively selected from our institutional surgical database (mean age 35 ± 10 years, 58 males and 21 females). 3D models of the contralateral healthy scapulae were created via computed tomography scan segmentation using Mimics 24.0 software (Materialise, Leuven, Belgium). Anatomical landmarks were identified using 3-Matic 16.0 software (Materialise, Leuven, Belgium). The distance between identified landmarks and a sagittal plane created on the deepest point of the glenoid was recorded for each scapula and reliability of each landmark was assessed. Inter- and intra-observer reliabilities were also evaluated using intraclass correlation coefficients (ICCs)., Results: Four landmarks showed statistically significant results: the scapular notch (SN), the centroid of the coracoid (CC), a point on the most medial border of the scapula in line with the scapular spine (TS), and the most lateral point of the acromion (AL). The mean (± standard deviation) joint line measured from the SN, CC, TS and AL were 28.36 ± 2.97 mm, 11.66 ± 2.07 mm, 107.52 ± 8.1 mm, and 29.72 ± 4.46 mm, respectively. Inter-observer reliability analysis for SN, TS, and AL showed excellent agreement with ICC values of 0.966, 0.997, and 0.944, respectively, and moderate agreement for CC with ICC of 0.728., Conclusion: The results from this study assist in estimating joint line medialization preoperatively and in planning its subsequent restoration. A set of reliable landmarks can be used as references to estimate the premorbid glenoid joint line preoperatively., (© 2023 The Author(s).)

Revision shoulder arthroplasty is increasing with the number of primary shoulder replacements rising globally. Complex primary and revisions of shoulder arthroplasties pose specific challenges for the surgeon, which must be addressed preoperatively and intraoperatively. This article aimed to present strategies for the management of revision of shoulder arthroplasties through a single-stage approach. Preoperatively, patient factors, such as age, comorbidities, and bone quality, should be considered. The use of planning software can aid in accurately evaluating implants in situ and predict bony anatomy that will remain after explantation during the revision surgery. The planning from such software can then be executed with the help of mixed reality technology to allow accurate implant placement. Single-stage revision is performed in two steps (debridement as first step, implantation and reconstruction as the second step), guided by the following principles: adequate debridement while preserving key soft tissue attachments (i.e., rotator cuff, pectoralis major, latissimus dorsi, deltoid), restoration of glenoid joint line using bone grafting, restoration of humeral length, reconstruction and/or reattachment of soft tissues, and strict compliance with the postoperative antibiotic regimen. Preliminary results of single-stage revision shoulder arthroplasty show improvement in patient outcomes (mean 1 year), successful treatment of infection for those diagnosed with periprosthetic joint infection, and improved cost-benefit parameters for the healthcare system.

Joint motion calculated using multi-body models and inverse kinematics presents many advantages over direct marker-based calculations. However, the sensitivity of the computed kinematics is known to be partly caused by the model and could also be influenced by the participants' anthropometry and sex. This study aimed to compare kinematics computed from an anatomical shoulder model based on medical images against a scaled-generic model and quantify the effects of anatomical errors and participants' anthropometry on the calculated joint angles. Twelve participants have had planar shoulder movements experimentally captured in a motion lab, and their shoulder anatomy imaged using an MRI scanner. A shoulder multi-body dynamics model was developed for each participant, using both an image-based approach and a scaled-generic approach. Inverse kinematics have been performed using the two different modelling procedures and the three different experimental motions. Results have been compared using Bland-Altman analysis of agreement and further analysed using multi-linear regressions. Kinematics computed via an anatomical and a scaled-generic shoulder models differed in average from 3.2 to 5.4 degrees depending on the task. The MRI-based model presented smaller limits of agreement to direct kinematics than the scaled-generic model. Finally, the regression model predictors, including anatomical errors, sex, and BMI of the participant, explained from 41 to 80% of the kinematic variability between model types with respect to the task. This study highlighted the consequences of modelling precision, quantified the effects of anatomical errors on the shoulder kinematics, and showed that participants' anthropometry and sex could indirectly affect kinematic outcomes., (© 2022. The Author(s).)

Objective: The purpose of this study was to evaluate the imaging and pathologic features and upgrade rate of non-calcified ductal carcinoma in situ (NCDCIS). The study tested the hypothesis that lesions with sonographic findings have higher upgrade rate compared to lesions seen on mammography or MRI only., Methods: This retrospective study included patients with ductal carcinoma in situ (DCIS) diagnosed by image-guided core breast biopsy from December 2009 to April 2018. Patients with microcalcifications on mammography or concurrent ipsilateral cancer on core biopsy were excluded. An upgrade was defined as surgical pathology showing microinvasive or invasive cancer., Results: A total of 71 lesions constituted the study cohort. 62% of cases (44/71) had a mammographic finding, and 38% (27/71) of mammographically occult lesions had findings on either ultrasound, MRI, or both. Of the 67 cases that underwent sonography, a mass was noted in 56/67 (83.6%) cases and no sonographic correlate was identified in 11/67 (16.4%) cases. 21% (15/71) of lesions were upgraded on final surgical pathology. The upgrade rate of patients with sonographic correlate was 27% (15/56) vs with mammographic findings only was 0% (0/11)., Conclusion: DCIS should be considered in the differential diagnosis of architectural distortion, asymmetries, focal asymmetries, and masses, even in the absence of microcalcifications. NCDCIS diagnosed by ultrasound may be an independent risk factor for upgrade., Advances in Knowledge: Radiologists must be aware of imaging features of DCIS and consider increased upgrade rate when NCDCIS is diagnosed by ultrasound.

Purpose: Facebook (Facebook, Inc, Menlo Park, California, USA) is the most popular social networking platform worldwide. Facebook groups are virtual communities of people who share a common interest. Breast Imaging Radiologists is a Facebook group for radiologists with an interest in breast imaging. The purpose of this study was to analyze the membership and activity of the Breast Imaging Radiologists Facebook group (BIRFG) for 2 years since its inception., Methods: Using both the Grytics (www.grytics.com) and Sociograph (www.sociograph.io) analytic engines, the activity of the BIRFG was analyzed retrospectively from its inception on February 11, 2015, through February 12, 2017. Activity data were exported for further qualitative and quantitative analysis using Excel (Microsoft, Redmond, Washington, USA). Member demographic data were obtained by querying public Facebook profiles, US News Doctor Finder (US News & World Report, Washington, DC, USA), Doximity (Doximity, San Francisco, California, USA), and Google (Google Inc, Mountain View, California, USA)., Results: Membership grew from 1 to 774 over the study period, and 84% of the members were female. There were 493 posts, 3,253 comments, and 1,732 reactions; 92% of posts received either comments or reactions. Each post received an average of 6.6 comments, and 55% of members were active over the study period. There was an increase in all measures of activity from year 1 to year 2., Conclusions: Our findings indicate that radiologists find value in using Facebook groups as a forum to network and exchange information about breast imaging. This may be generalizable to other radiology subspecialties. Given the popularity and accessibility of Facebook for personal use, it may prove a more comfortable social medium for radiologists to interact professionally., (Copyright © 2017 American College of Radiology. Published by Elsevier Inc. All rights reserved.)

The purpose of our study was to assess data reproducibility from 2 consecutive front squat workouts, spaced 1 week apart, performed by American college football players (n = 18) as they prepared for their competitive season. For each workout, our methods entailed the performance of 3-6 front squat repetitions per set at 55, 65, and 75% of subject's 1 repetition maximum (1RM) load. In addition, a fourth set was done at a heavier load, with a resistance equal to 80 and 83% of their 1RM values, for the first and second workouts, respectively. A triple-axis accelerometer was affixed to a barbell to quantify exercise performance. Per load, the accelerometer measures peak values for the following indices: force, velocity, and power. To assess data reproducibility, inter-workout comparisons were made for 12 performance indices with 4 statistical test-retest measures: intraclass correlation coefficients, coefficients of variation (CVs), and the SEM expressed in both absolute and relative terms. Current results show that the majority of performance indices exceeded intraclass correlation (0.75-0.80) and CV (10-15%) values previously deemed as acceptable levels of data reproducibility. The 2 indices with the greatest variability were power and velocity values obtained at 55% of the 1RM load; thus, it was concluded that higher movement rates at the lightest load were the most difficult aspect of front squat performance to repeat successfully over time. Our practical applications imply lighter loads, with inherently higher rates of barbell movement, yield lower data reproducibility values.

Bladder cancer is a common problem facing urologists worldwide. The gold standard for its diagnosis and follow-up is the direct visualization of the tumor using conventional cystoscopy. Despite having high sensitivity and specificity for detecting bladder cancer, conventional cystoscopy is regarded as an invasive procedure which is associated with several complications. In addition, regular follow-up of patients with bladder cancer is a financial burden on the health system. With the progressive development in diagnostic imaging and medical computer software technologies, it was possible to generate virtual reality images to aid the clinician to inspect the interior of the bladder in real time. This technology is considered as a safe test for bladder cancer diagnosis and follow-up, and it is associated with cancer detection rates comparable with conventional cystoscopy. However, it is associated with some drawbacks that limit its use in routine clinical practice at the current time. In this paper, we review the development and clinical applications of this technology.

Prevention programs are a valuable component of the comprehensive services offered at school-based health centers (SBHC). Reducing risky adolescent behaviors is an effective way to reduce the morbidity and mortality burden among the school-age population. Programs using peer educators and youth-initiated websites can increase knowledge and self-esteem and help reduce risky sexual behaviors. Because many SBHCs provide services beyond traditional primary care, there is great need to support and increase the number of SBHC prevention programs targeted at communities at risk.

During plant growth and development, the phytohormone auxin induces a wide array of changes that include cell division, cell expansion, cell differentiation, and organ initiation. It has been suggested that the actin cytoskeleton plays an active role in the elaboration of these responses by directing specific changes in cell morphology and cytoarchitecture. Here we demonstrate that the promoter and the protein product of one of the Arabidopsis vegetative actin genes, ACT7, are rapidly and strongly induced in response to exogenous auxin in the cultured tissues of Arabidopsis. Homozygous act7-1 mutant plants were slow to produce callus tissue in response to hormones, and the mutant callus contained at least two to three times lower levels of ACT7 protein than did the wild-type callus. On the other hand, a null mutation in ACT2, another vegetative actin gene, did not significantly affect callus formation from leaf or root tissue. Complementation of the act7-1 mutants with the ACT7 genomic sequence restored their ability to produce callus at rates similar to those of wild-type plants, confirming that the ACT7 gene is required for callus formation. Immunolabeling of callus tissue with actin subclass-specific antibodies revealed that the predominant ACT7 is coexpressed with the other actin proteins. We suggest that the coexpression, and probably the copolymerization, of the abundant ACT7 with the other actin isovariants in cultured cells may facilitate isovariant dynamics well suited for cellular responses to external stimuli such as hormones.

The immunogenicity of therapeutic Abs limits their long-term use. The processes of complementarity-determining region grafting, resurfacing, and hyperchimerization diminish mAb immunogenicity by reducing the number of foreign residues. However, this does not prevent anti-idiotypic and anti-allotypic responses following repeated administration of cell-binding Abs. Classical studies have demonstrated that monomeric human IgG is profoundly tolerogenic in a number of species. If cell-binding Abs could be converted into monomeric non-cell-binding tolerogens, then it should be possible to pretolerize patients to the therapeutic cell-binding form. We demonstrate that non-cell-binding minimal mutants of the anti-CD52 Ab CAMPATH-1H lose immunogenicity and can tolerize to the "wild-type" Ab in CD52-expressing transgenic mice. This finding could have utility in the long-term administration of therapeutic proteins to humans.

Plant actins are involved in numerous cytoskeletal processes effecting plant development, including cell division plane determination, cell elongation, and cell wall deposition. Arabidopsis thaliana has five ancient subclasses of actin with distinct patterns of spatial and temporal expression. To test their functional roles, we identified insertion mutants in three Arabidopsis actin genes, ACT2, ACT4, and ACT7, representing three subclasses. Adult plants homozygous for the act2-1, act4-1, and act7-1 mutant alleles appear to be robust, morphologically normal, and fully fertile. However, when grown as populations descended from a single heterozygous parent, all three mutant alleles were found at extremely low frequencies relative to the wild-type in the F2 generation. Thus, all three mutant alleles appear to be deleterious. The act2-1 mutant allele was found at normal frequencies in the F1, but at significantly lower frequencies than expected in the F2 and F3 generations. These data suggest that the homozygous act2-1/act2-1 mutant adult plants have a reduced fitness in the 2N sporophytic portion of the life cycle, consistent with the vegetative expression of ACT2. These data are interpreted in light of the extreme conservation of plant actin subclasses and genetic redundancy.

A mathematical model was developed to help interpret genotype and allele frequency dynamics in selfing populations, with or without apomixis. Our analysis provided explicit time-dependent solutions for the frequencies at diallelic loci in diploid populations under any combination of fertility, viability, and gametic selection through meiotic drive. With no outcrossing, allelic variation is always maintained under gametic selection alone, but with any fertility or viability differences, variation will ordinarily be maintained if and only if the net fitness (fertility x viability) of heterozygotes exceeds that of both homozygotes by a substantial margin. Under pure selfing and Mendelian segregation, heterozygotes must have a twofold fitness advantage; the level of overdominance necessary to preserve genetic diversity declines with apomixis, and increases with segregation distortion if this occurs equally and independently in male and female gametes. A case study was made of the Arabidopsis act2-1 actin mutant over multiple generations initiated from a heterozygous plant. The observed genotypic frequency dynamics were consistent with those predicted by our model for a deleterious, incompletely recessive mutant in either fertility or viability. The theoretical framework developed here should be very useful in dissecting the form(s) and strength of selection on diploid genotypes in populations with negligible levels of outcrossing.

The development of recombinant techniques for the rapid cloning, expression, and characterization of cDNAs encoding antibody (Ab) subunits has revolutionized the field of antibody engineering. By fusion to heterologous protein domains, chain shuffling, and inclusion of self-assembly motifs, novel molecules such as bispecific Abs can now be generated which possess the subset of functional properties designed to fit the intended application. Rapid technological developments in phage display of peptides and proteins have led to a plethora of applications directed towards immunology and antibody engineering. Many of the problems associated with the therapeutic use of Abs are being addressed by the application of these new techniques.

A single-chain Fv (sFv) was expressed from the variable regions of the CD40-specific mAb G28-5. The molecule bound CD40 with a high affinity (2.2 nM) and was a monomer in solution. Surprisingly, G28-5 sFv was a potent CD40 agonist that rapidly crosslinked CD40 on the cell surface but did not crosslink CD40-Ig in solution. G28-5 sFv was a more potent agonist than G28-5 IgG and was able to stimulate CD40 responses by B cells and monocytes. G28-5 IgG partially blocked, whereas G28-5 sFv augmented CD40 responses during stimulation with natural ligand (gp39-CD8 fusion protein). These results indicate that the functional activity of ligands built from the binding site of G28-5 is highly dependent upon the size and physical properties of the molecule both in solution and on the cell surfaces.

Neural network analysis of the response of an array of vapor-sensitive detectors has been used to identify six different types of aviation fuel. The data set included 96 samples of JP-4, JP-5, JP-7, JP-8, JetA, and aviation gasoline (AvGas). A sample of each neat fuel was injected into a continuous stream of breathing air through an injection port from a gas chromatograph. The aspirated sample was then swept from the injection port to the chamber without separation. In the chamber, the sample was exposed to an array of eight vapor-sensitive detectors. The analog output of each detector was digitized and stored while the sample was swept into and through the chamber. The response of each detector was then averaged and stored as the final response or pattern of each sample. It was clear from a visual inspection of each of the radar plots that there was a characteristic pattern in the response of the array to five of the six different fuel types. This was confirmed using neural network analysis to study the entire data set. A two-step procedure was developed to separate the patterns of all six fuel tyes into their respective classes. In the first step, fuels were separated into one of five groups: JP-4, JP-5, JP-7, AvGas, or a combined JP-8/JetA group. In the second step, the fuels in the combined group were separated into either JP-8 or JetA groups.

CD19 is a B cell surface protein capable of forming non-covalent molecular complexes with a number of other B cell surface proteins including the CD21/CD81/Leu-13 complex as well as with surface immunoglobulin. CD19 tyrosine phosphorylation increases after B cell activation, and is proposed to play a role in signal transduction through its cytoplasmic domain, which contains nine tyrosine residues. Several second messenger proteins have been shown to immunoprecipitate with CD19, including p59 Fyn (Fyn), p59 Lyn (Lyn) and phosphatidylinositol-3 kinase (PI-3 kinase). These associations are predicted to occur via the src-homology 2 (SH2) domains of the second messenger proteins. Two of the cytoplasmic tyrosines in the CD19 cytoplasmic region contain the consensus binding sequence for the PI-3 kinase SH2 domain (YPO4-X-X-M). However, the reported consensus binding sequence for the Fyn and Lyn SH2 domains (YPO4-X-X-I/L) is not found in CD19. We investigated the capacity of CD19 cytoplasmic tyrosines to bind both Fyn and PI-3 kinase SH2-domain fusion proteins. In activated B cells, both Fyn and PI-3 kinase SH2-domain fusion proteins precipitate CD19. Using synthetic tyrosine-phosphorylated peptides comprising each of the CD19 cytoplasmic tyrosines and surrounding amino acids, we investigated the ability of the Fyn SH2 and PI-3 kinase SH2 fusion proteins to bind to the different CD19 cytoplasmic phosphotyrosine peptides. ELISA revealed that the two CD19 cytoplasmic tyrosine residues contained within the Y-X-X-M sequences (Y484 and Y515) bound preferentially to the PI-3 kinase SH2-domain fusion proteins. Two different tyrosines (Y405 and Y445) bound preferentially to the Fyn SH2-domain fusion protein via a novel sequence, Y-E-N-D/E, different from that previously reported for the Fyn SH2 domain. In precipitation studies, peptide Y484 was able to compete with tyrosine phosphorylated CD19 specifically for binding to the PI-3 kinase SH2 domain fusion proteins, while peptides Y405 and Y445 were able to compete specifically for binding to the Fyn SH2 domain fusion proteins. These results indicate that CD19 may be capable of binding both Fyn and PI-3 kinase concurrently, suggesting a mechanism for CD19 signal transduction, in which binding of PI-3 kinase to the Fyn SH3 domain results in activation of PI-3 kinase.

We have constructed anti-CD40 immunotoxins consisting of the single chain Fv (sFv) region of the anti-human CD40 mAb G28-5 fused to a truncated form of Pseudomonas exotoxin, PE40. CD40 is an integral membrane glycoprotein found on the surface of B-lineage cells, including lymphomas and leukemias, as well as certain carcinomas. Two forms of the immunotoxin were constructed, one with the light chain variable (VL) region of the sFv preceding the heavy chain variable region (VH) [G28-5 sFv(VL-VH)-PE40] and the second with the sFv in the opposite orientation [G28-5 sFv(VH-VL)-PE40]. Although both forms of G28-5 sFv-PE40 specifically bound to CD40 in ELISAs, the binding of G28-5 sFv(VL-VH)-PE40 was > 10-fold higher. A number of malignant B- and T-cell lines were screened for CD40 expression and susceptibility to G28-5 sFv(VL-VH)-PE40. All of the B-lineage cells tested were CD40 positive and sensitive to the anti-CD40 immunotoxin, with EC50s ranging from 2.5-70 ng/ml, whereas none of the T cell leukemias or lymphomas were antigen positive or were affected by the immunotoxins. Consistent with the antigen-binding results, the VL-VH immunotoxin was > 10-fold more cytotoxic than the VH-VL immunotoxin. The anti-CD40 single-chain immunotoxin fusion protein G28-5 sFv(VL-VH)-PE40 represents a potent and specific cytotoxic agent for the elimination of normal and transformed B-lineage cells expressing CD40.

To examine the effects of ligand engagement and accessory molecule juxtaposition on T cell receptor (TCR) signaling, we prepared LFA-3/ICAM-1 Rg and LFA-3/VCAM-1 Rg bispecific immunoglobulin fusion proteins (Rg, recombinant globulin). These novel fusion proteins allowed us to examine the effects of ligand driven co-engagement of T cell proteins CD2 and LFA-1 or CD2 and VLA-4 on TCR-dependent mobilization of intracellular Ca2+. We observed that preincubation of resting T cells with LFA-3/ICAM-1 Rg or LFA-3/VCAM-1 Rg fusion proteins resulted in significantly enhanced mobilization of intracellular Ca2+ following TCR-accessory molecule cross-linking relative to T cells preincubated with each of the monospecific Rgs alone or with combinations of the monospecific Rg fusion proteins. In addition, such coengagement stimulated TCR-dependent activation and tyrosine phosphorylation of phospholipase C gamma 1 (PLC gamma 1). These results suggest that when T cells interact with antigen presenting cells the engagement of multiple cell adhesion molecules such as CD2, LFA-1, and VLA-4 primes the T cell to respond more effectively to signals delivered through the TCR.

T cell receptor (TCR)-mediated stimulation of T hybridomas leads to cell activation and lymphokine production that is followed by a long-term hyporesponsiveness. To investigate the biochemical events involved in the induction and maintenance of this antigen receptor hyporesponsiveness or anergy, we have expressed a G protein/PLC beta 1-coupled muscarinic subtype 1 acetylcholine receptor in a murine T cell hybrid. Transfected cells were capable of responding to both muscarinic agonists and TCR ligands by inducing interleukin-2 secretion that was sensitive to cyclosporin A and dexamethasone. Both receptors induced tyrosine kinase (TK) activity, but muscarinic stimulation did not affect tyrosine phosphorylation of PLC gamma 1, nor did the TK inhibitor, herbimycin, block muscarinic receptor-mediated calcium mobilization. These data indicate that in T cells, the muscarinic receptor mediates T cell effector functions by regulating a TK-independent proximal pathway which later converges with the TCR pathway. Using these cells, we have explored the long-term consequences of T cell stimulation via antigen or muscarinic receptors. Our results show that hyporesponsiveness specifically follows TCR engagement and appears to result from a defect in the early signal transduction initiated by TCR cross-linking. A study of TCR-mediated signaling supports this model by showing that tyrosine phosphorylation and calcium mobilization are deficient in hyporesponsive T cells.

Single-chain antibody molecules were expressed from modified eukaryotic expression vectors as individual protein domains encoded on interchangeable cDNA cassettes. Two different single-chain antibody derivatives were constructed by linking individual light- and heavy-chain variable domains. The first was specific for the L6 tumour-associated antigen and the second was specific for human CD3. Each single-chain variable domain was genetically fused with an Fc 'tag' and expressed as a fusion protein in a COS cell transient transfection system. These single-chain antibody derivatives demonstrated specific binding to cells expressing appropriate antigen and bound with affinities similar to native antibody. The CD3 single chain molecule mediated stronger activation of PLC gamma 1 and similar levels of T-cell proliferation compared with native antibody. A bispecific Fv single-chain cassette was created by fusing the expression cassettes encoding the binding domains for L6 and CD3 single-chain molecules using oligonucleotide primers encoding a short 27-residue 'helical' peptide linker. The CD3-L6 variable domains were fused to the Fc tag and expressed in COS cells. The CD3-L6FvIg bispecific fusion protein mediated adhesion between T cells and L6-positive tumour cells, and stimulated potent T-cell proliferation and cytotoxicity against tumour cells expressing the L6 antigen.

Stimulation of the human monocytic cell line THP-1 by cross-linking either Fc gamma receptor I (Fc gamma RI) or Fc gamma receptor II (Fc gamma RII) gave rise to the rapid phosphorylation of multiple intracellular proteins. The pattern of proteins that were phosphorylated appeared to be identical. Analysis of these proteins by specific immunoprecipitation indicated that stimulation through either receptor did indeed give rise to phosphorylation of the same set of proteins. These included: Fc gamma RII, phospholipase C (PLC) gamma 1, PLC gamma 2, Vav, GAP, and a protein that co-precipitated with the Fc gamma receptors and migrated with a molecular weight of about 70,000. Co-cross-linking an F(ab')2 anti-CD45 monoclonal antibody together with monoclonal antibodies to either of the Fc gamma receptors inhibited phosphorylation of all these proteins. Analysis of the tyrosine kinases in the cells revealed that both receptors stimulated the phosphorylation and activation of a kinase recognized by antibodies to Syk. Furthermore, the Syk kinase became associated with the Fc gamma RII following receptor cross-linking. These data indicate that although the two Fc gamma receptors have different cytoplasmic tails, they are coupled to the same signal transduction cascade that is regulated by CD45 and involves the activation of Syk.

CD20 is a B cell-specific 35/37 kDa integral membrane protein which modulates proliferation and differentiation of normal resting B cells when stimulated by CD20 antibodies. An increase in c-myc mRNA levels occurs within hours after treatment of resting B cells with CD20 mAb; however earlier events in the CD20 signal transduction pathway have not been described. Here we demonstrate that anti-CD20 mediated induction of c-myc mRNA is inhibited by the tyrosine kinase inhibitor herbimycin A, that CD20 is associated with both tyrosine and serine kinase activity, and that tyrosine phosphorylation of multiple substrates is induced within minutes upon ligation of CD20 with mAb. Association of the tyrosine and serine kinases with CD20 was stable in lysis buffer containing 1% NP40 and 0.25% deoxycholate. Under the same conditions, antibodies against several other B cell surface molecules failed to co-precipitate tyrosine kinase activity, however, a serine kinase was precipitated by the anti-CD19 mAb, B43. Both phospholipase C-gamma 1 and -gamma 2 were phosphorylated on tyrosine after cross-linking of CD20-bound mAb, and this correlated with increases in intracellular calcium that were partially resistant to depletion of extracellular calcium with EGTA. The pattern of tyrosine phosphorylated proteins observed in whole cell lysates after anti-CD20 cross-linking appeared to be a subset of those induced by anti-IgM; however, differences in phosphoproteins induced by anti-IgM and anti-CD20 were detected using a fynSH2-fusion protein.

Cross-linking of B cell surface immunoglobulins (sIg) results in activation of mature B cells and stimulates a molecular signaling mechanism for antigen-specific B cell expansion and differentiation. This signaling pathway is dependent on tyrosine (Tyr) phosphorylation and results in the activation of sIg-associated src family kinases and p72SYK. Rapid Tyr phosphorylation occurs on multiple protein substrates. Here we show that activation of B cells by cross-linking sIg results in an increase in Tyr phosphorylation of the lineage-restricted B cell surface antigen CD19, and show that it is a major substrate of activated Tyr kinase following sIg stimulation. Lower levels of constitutive CD19 Tyr phosphorylation occurred in most sIg+ mature B cell lines examined and in normal dense tonsillar B cells. We also find that when CD19 is Tyr-phosphorylated it becomes competent to interact with SH2 domains suggesting a mechanism whereby, following B cell activation, CD19 could be linked to intracellular signaling pathways. In sIg- pre-B cell lines, CD19 was expressed but was not constitutively phosphorylated on tyrosine. Upon CD19 cross-linking, Tyr phosphorylation of CD19 was induced in sIg- pre-B cell lines. CD19 cross-linking also directly induced Tyr phosphorylation of CD19 and other substrates in mature B cells. The ability of CD19 to signal in the absence of sIg expression may provide important stimulation in pre-B cell development.

UV radiation is known to induce lymphocyte nonresponsiveness both in vitro and in vivo. We have found that UV radiation rapidly induced tyrosine phosphorylation and calcium signaling in normal human peripheral blood lymphocytes. In the leukemic T cell line Jurkat and the Burkitt's lymphoma cell line Ramos, UV rapidly induced tyrosine phosphorylation in a wavelength-dependent manner, giving strong signals after UVB and UVC, but not UVA, irradiation. Similarly, in Jurkat cells UV-induced calcium signals were dependent on the dose of UVB or UVC irradiation over a range of 150-1200 J/m2, but only a small signal was observed for UVA at a dose of 1200 J/m2. The UV-induced calcium signals were blocked by the tyrosine kinase inhibitor herbimycin A, indicating that they were dependent on tyrosine phosphorylation. Phospholipase C (PLC) gamma 1 was tyrosine phosphorylated in response to UV irradiation but to a lesser extent than observed after CD3 cross-linking. However, PLC gamma 1-associated proteins demonstrated to bind to the PLC gamma 1 SH2 domain were tyrosine phosphorylated strongly after UV irradiation. A similar dose response was observed for the inhibition by herbimycin A of UV-induced calcium signals and UV-induced tyrosine phosphorylation of PLC gamma 1 and associated proteins. We propose that in contrast to CD3/Ti stimulation, UV aberrantly triggers lymphocyte signal transduction pathways by a mechanism that bypasses normal receptor control.

Cell-cell interactions induced between T cells and monocytes by certain soluble anti-CD3 monoclonal antibodies (MAbs) were previously shown to be required for high-level production of HIV-1 by peripheral blood mononuclear cells (PBMCs) from infected donors. Staphylococcal enterotoxin or superantigen (SAg) is another mitogen inducing monocytes-T cell interactions that exhibit potent induction of HIV-1 production. Antibodies to several adhesion molecules were used to test the requirements for T cell- and monocyte-associated adhesion molecules in HIV-1 production following activation with anti-CD3 or SAg. Blocking of either CD2-LFA-3, or CD18-ICAM-1, inhibited anti-CD3- or SAg-induced HIV-1 production by more than 90% without inhibiting CD4+ T cell proliferation. Inhibition of HIV production was observed when either the T cell or monocyte coreceptor was bound by MAbs to these adhesion molecules. Blocking of CD28-B7 interactions by soluble CTLA-4 fusion protein, a CD28 homolog, inhibited both HIV-1 production and CD4+ T cell proliferation. Fc binding was not required for HIV-1 inhibition by MAbs to CD2 and CD18, because Fab or F(ab')2 fragments of these MAbs inhibited HIV-1 production by more than 80%. A chimeric single-chain MAb to CD2 was produced, containing heavy and light chain variable regions from MAb 35.1 to CD2 linked to the constant regions of human IgG1 (CD2 SFv-Ig). This humanized CD2 SFv-Ig inhibited HIV-1 production by 30% to > 98%. These results thus indicate that simultaneous engagement of multiple adhesion pathways between T cells and monocytes are required for HIV production by patients PBMCs and may have implications for therapy of HIV infections.

Epidermal growth factor (EGF) or platelet-derived growth factor binding to their receptor on fibroblasts induces tyrosine phosphorylation of PLC gamma 1 and stable association of PLC gamma 1 with the receptor protein tyrosine kinase. Similarly in lymphocytes, cross-linking of antigen receptors induces the formation of molecular complexes incorporating PLC gamma 1; however, associated kinase activity is thought to be mediated through cytoplasmic protein tyrosine kinase(s). In this report, we generated a fusion protein containing the SH2 domains of human PLC gamma 1 and human IgG1 heavy chain constant region to identify lymphocyte phosphoprotein-binding PLC gamma 1 SH2 domains following cellular activation. As in EGF- or platelet-derived growth factor-stimulated fibroblasts, PLC gamma 1 is coprecipitated in activated lymphocytes, complexed with associated tyrosine-phosphorylated proteins. One of these, a 35/36-kDa protein found prominently in T cells and at lower levels in B cells, bound to the fusion protein in immunoprecipitation experiments. The fusion protein showed lineage restricted association with a 74-kDa phosphoprotein in T cells and a 93-kDa phosphoprotein in B cells. It bound to activated EGF receptor in fibroblasts as expected, and protein tyrosine kinase activity was precipitated from EGF-stimulated cells. However, PLC gamma 1-associated protein tyrosine kinase activity was not detected in activated lymphocytes. These data suggest that lymphocyte PLC gamma 1 SH2-binding proteins are cell lineage specific and may be transiently associated with activated PLC gamma 1.

Although cortical (CD4+CD8+) thymocytes mobilize intracellular calcium poorly when CD3/TCR is ligated, we have found that murine cortical thymocytes can transduce strong biochemical signals in response to ligation of the CD3/Ti TCR complex (CD3/TCR) and that the signals are regulated by CD4 and CD8 interactions with CD3/TCR. Striking increases in intracellular calcium were observed in cortical thymocytes from transgenic mice containing productively rearranged alpha and beta TCR genes, when CD3 or TCR was cross-linked with CD4 or CD8 using heteroconjugated mAb. However, in mature T cells derived from lymph nodes of these mice, identical stimuli elicited calcium responses that were significantly smaller in magnitude. A thymocyte cell line that expresses a low level of the transgenic TCR and has a phenotype characteristic of cortical thymocytes (CD4+CD8+J11d+Thy-1+) was established from a female alpha beta TCR transgenic mouse. Cross-linking of CD4 or CD8 molecules to CD3/TCR induced strong calcium responses in these cells. Responses were weak or absent when CD3 or TCR were aggregated alone. Heteroconjugates of Thy-1xCD3 did not increase the intracellular calcium concentration in transgenic thymocytes or in the thymocyte cell line, although Thy-1 is highly expressed on immature cells. Enhanced tyrosine phosphorylation was observed when CD3 or TCR was cross-linked with CD4 or CD8 on transgenic thymocytes or on the thymocyte cell line, in comparison with aggregation of CD3/TCR alone. Taken together, these data show that CD4 and CD8 molecules allow the weakly expressed CD3/TCR of cortical thymocytes to transduce strong intracellular signals upon receptor ligation. These signals may be involved in selection processes at the CD4+CD8+ stage of differentiation.

Phosphorylation of proteins on tyrosine residues during activation was studied in CD3+ CEM T cells. Crosslinking of either CD4 alone or CD3/Ti alone induced weak and transient responses, but the patterns of induced tyrosine-phosphorylated proteins were different. A synergistic but still transient response occurred by the specific interaction of CD4 with CD3/Ti, whereas simultaneous but separate ligation of CD3/Ti and CD4 decreased rather than increased tyrosine phosphorylation of proteins in comparison to CD3/Ti stimulation alone. Stimulation of T cells with immobilized anti-CD3 induced strong and prolonged tyrosine phosphorylation of distinct substrates. CD4 therefore regulates protein tyrosine kinase activation by specific interaction with CD3/Ti, whereas immobilized anti-CD3 may differ from anti-CD3 in solution in the activation of protein tyrosine phosphatase(s) such as CD45.

We evaluated the effects of recombinant human GH (rhGH) in 16 men and women more than 60 yr of age. After 10 days of dietary equilibration and control collections, subjects were randomly assigned to receive 0.03, 0.06, or 0.12 mg/kg rhGH by daily injection for 7 days. A brisk rise in circulating somatomedin-C (insulin-like growth factor-I) occurred in all subjects, and this rise was dose dependent. rhGH produced striking changes in nitrogen retention, sodium excretion, and the parathyroid-vitamin D axis. Twenty-four-hour urinary nitrogen excretion decreased from 8.00 +/- 0.33 to 5.01 +/- 0.33 g (P less than 0.001), and sodium excretion decreased from 45.9 +/- 2.96 to 21.2 +/- 3.48 mmol/day (P less than 0.001). Serum calcium concentrations did not change, but serum inorganic phosphorus levels of 1.08 +/- 0.04 mmol/L at baseline increased significantly after rhGH treatment to 1.33 +/- 0.04 mmol/L (P less than 0.001). Increases were also observed in circulating PTH (53.2 +/- 6 vs. 39.5 +/- 4.2 ng/L; P less than 0.01) and calcitriol (82.8 vs. 65.8 pmol/L; P less than 0.05). A rise in serum osteocalcin (10.3 +/- .86 vs. 8.0 +/- 0.5 micrograms/L; P less than 0.05) was accompanied by increased urinary excretion of hydroxyproline (628 +/- 63 vs. 406 +/- 44 mumol/day; P less than 0.01). Despite the reduction in sodium excretion, marked increases were observed in urinary calcium (6.04 +/- 0.97 vs. 3.27 +/- 0.40 mmol/day; P less than 0.01). rhGH significantly impaired oral glucose tolerance and reduced insulin sensitivity, but was otherwise well tolerated and produced no systematic changes in weight or blood pressure. The results of this study indicate that rhGH requires further study as a potential agent for attenuating or reversing the loss of muscle and bone in elderly people.