Lymphoma Driver Mutations in the Pathogenic Evolution of an Iconic Human Autoantibody.

Pathogenic autoantibodies arise in many autoimmune diseases, but it is not understood how the cells making them evade immune checkpoints. Here, single-cell multi-omics analysis demonstrates a shared mechanism with lymphoid malignancy in the formation of public rheumatoid factor autoantibodies responsible for mixed cryoglobulinemic vasculitis. By combining single-cell DNA and RNA sequencing with serum antibody peptide sequencing and antibody synthesis, rare circulating B lymphocytes making pathogenic autoantibodies were found to comprise clonal trees accumulating mutations. Lymphoma driver mutations in genes regulating B cell proliferation and V(D)J mutation (CARD11, TNFAIP3, CCND3, ID3, BTG2, and KLHL6) were present in rogue B cells producing the pathogenic autoantibody. Antibody V(D)J mutations conferred pathogenicity by causing the antigen-bound autoantibodies to undergo phase transition to insoluble aggregates at lower temperatures. These results reveal a pre-neoplastic stage in human lymphomagenesis and a cascade of somatic mutations leading to an iconic pathogenic autoantibody.

Embryo mortality in a captive-bred, Critically Endangered amphibian.

The Critically Endangered southern corroboree frog Pseudophryne corroboree is dependent upon captive assurance colonies for its continued survival. Although the captive breeding programme for this species has largely been successful, embryonic mortality remains high (40-90% per year). This study aimed to investigate the causes of mortality in P. corroboree embryos in the captive collection at Melbourne Zoo. During the 2021 breeding season, we investigated 108 abnormal embryos to determine the impact of infections and anatomical deformities on survival and used culture and molecular methods to identify microbes. Overall, 100% of abnormal embryos had fungal infections, and of these, 41.6% also had anatomical deformities. The mortality rate in abnormal embryos was 89.8%; however, we detected no difference in survival in any of the 3 observed fungal growth patterns or between deformed and non-deformed embryos. Sanger sequencing of the ITS region identified fungal isolates belonging to the genus Ilyonectria, the first record in a vertebrate host, and another as a Plectosphaerella sp., which is the first record of infection in an embryo. Dominant bacteria identified were of the genera Herbaspirillum and Flavobacterium; however, their role in the mortality is unknown. Fungal infection and deformities have a significant impact on embryo survival in captive-bred P. corroboree. In a species which relies on captive breeding, identifying and reducing the impacts of embryonic mortality can inform conservation efforts and improve reintroduction outcomes.

Exploring conceptualizations of knowledge translation, transfer and exchange across public health in one UK region: a qualitative mapping study.

OBJECTIVES: Knowledge translation (KT) is becoming common vocabulary, but as a concept it is not clearly defined. Many related terms exist; these are often used interchangeably and given multiple interpretations. While there is a growing body of literature exploring these concepts, using it to inform public health practice, strategy, research and education is challenging given the range of sources and need for local 'contextual fit'. This study explores how various public health stakeholders make sense of, and experience, KT and related concepts. STUDY DESIGN: A qualitative mapping study using a phenomenographic approach. METHODS: Thirty-four academics, students and practitioners working in public health across the north east of England participated in six focus groups and five one-to-one interviews. Discussions were audio-recorded, transcribed and analysed using a thematic framework approach. The framework drew on findings from reviews of the existing literature, whilst allowing unanticipated issues to emerge. RESULTS: Three main themes were identified from the stakeholder discussions: CONCLUSIONS: This study has enabled further development of theoretical understandings of the KT discourses at play in public health, and identified the ways in which these may be bound by discipline and context. Ironically, the findings suggest that terms such as knowledge translation, transfer and exchange are seen as themselves requiring translation, or at least debate and discussion.

Changing characteristics of post-COVID-19 syndrome: Cross-sectional findings from 458 consultations using the Stanford Hall remote rehabilitation assessment tool.

BACKGROUND: In the UK, there have been multiple waves of COVID-19, with a five-tier alert system created to describe the transmission rate and appropriate restrictions. While acute mortality decreased, there continued to be a significant morbidity, with individuals suffering from persistent, life-restricting symptoms for months to years afterwards. A remote rehabilitation tool was created at the Defence Medical Rehabilitation Centre (DMRC) Stanford Hall to assess post-COVID-19 symptoms and their impact on the UK military.This study aims to understand changes in post-COVID-19 syndrome between wave 1 and wave 2, identify interactions between alert level and symptoms and investigate any predictive nature of acute symptoms for postacute symptomology in a young, physically active population. METHODS: Cross-sectional study of 458 consecutive remote rehabilitation assessments performed at DMRC Stanford Hall between 2 April 2020 and 29 July 2021. Consultations were coded, anonymised, and statistical analysis was performed to determine associations between acute and postacute symptoms, and between symptoms, alert levels and waves. RESULTS: 435 assessments were eligible; 174 in wave 1 and 261 in wave 2. Post-COVID-19 syndrome prevalence reduced from 43% to 2% between the waves. Acutely, widespread pain was more prevalent in wave 2 (p<0.001). Postacutely, there was increased anxiety (p=0.10) in wave 1 and increased sleep disturbance (p<0.001), memory/concentration issues (p<0.001) and shortness of breath/cough (p=0.017) in wave 2. Increasing alert level was associated with increased postacute symptom prevalence (p=0.046), with sleep disturbance increasing at higher alert level (p=0.016). Acute symptoms, including fatigue, sleep disturbance and myalgia, were associated with multiple postacute symptoms. CONCLUSIONS: This study reports the overall prevalence and symptom burden in the UK military in the first two waves of COVID-19. By reporting differences in COVID-19 in different waves and alert level, this study highlights the importance of careful assessment and contextual understanding of acute and postacute illnesses for individual management plans.

Differential glycosylation of polyclonal IgG, IgG-Fc and IgG-Fab isolated from the sera of patients with ANCA-associated systemic vasculitis.

Post-translational modifications (PTMs) of proteins produced in vivo may be tissue, developmentally and/or disease specific. PTMs impact on the stability and function of proteins and offer a challenge to the commercial production of protein biotherapeutics. We have previously reported a marked deficit in galactosylation of oligosaccharides released from polyclonal IgG isolated from sera of patients with the anti-neutrophil cytoplasmic antibodies (ANCA) associated vasculitides; Wegener's granulomatosis (WG) and microscopic polyangiitis (MPA). Whilst normal polyclonal IgG molecules are glycosylated within the IgG-Fc region, approximately 20% of molecules also bear oligosaccharides attached to the variable regions of the light or heavy chain IgG-Fab. It is of interest, therefore to compare profiles of oligosaccharides released from the IgG-Fc and IgG-Fab of normal IgG with that isolated from the sera of patients with WG or MPA. This study shows that whilst the oligosaccharides released from ANCA IgG-Fc are hypogalactosylated those released from IgG-Fab are galactosylated and sialylated. These results show that hypogalactosylation of IgG-Fc is not due to a defect in the glycosylation or processing machinery. It rather suggests a subtle change in IgG-Fc conformation that influences the addition of galactose. Remarkably, this influence is exerted on all plasma cells. Interestingly, a licensed monoclonal antibody therapeutic, produced in Sp2/0 cells, is also shown to be hypogalactosylated in its IgG-Fc but fully galactosylated in its IgG-Fab.

Mechanism and dynamics of conformational ordering in xanthan polysaccharide.

The thermally induced order-disorder transition of xanthan (extracellular bacterial polysaccharide from Xanthomonas campestris) has been investigated by optical rotation, differential scanning calorimetry, stopped-flow reaction kinetics and low-angle laser light scattering, and the results have been analysed in terms of Zimm -Bragg helix-coil transition theory. The reciprocal of the transition midpoint temperature (Tm) varies linearly with the logarithm of cation (K+) the salt dependence of Tm, is in agreement with Manning polyelectrolyte theory the ordered structure. The associated increase in cation binding, calculated from the salt dependence of tm, is in agreement with the Manning polyelectrolyte theory for one of the candidate structures from X-ray diffraction, a 5(1) single helix stabilized by packing of side-chains along the polymer backbone, but not for the alternative double-helix structure that has also been proposed. At each salt concentration, the two fundamental parameters of the Zimm -Bragg theory, s and sigma, were calculated. The equilibrium constant for growth of the ordered structure (s) is derived directly from calorimetric measurement of transition enthalpy (delta Hcal ), and sigma, which quantifies the relative instability of the helix nucleus, is derived from the ratio of delta Hcal to the apparent transition enthalpy (delta Happ ) obtained by van't Hoff analysis of the optical rotation data. The temperature course of conformational ordering calculated theoretically is in good quantitative agreement with experimental results from both optical rotation and scanning calorimetry. The calculated average length of stable, ordered chain-sequences increases with decreasing temperature, but equals or exceeds the total chain length from light scattering only at temperatures more than approximately equal to 70 K below Tm, suggesting that ordered and disordered regions may co-exist within the same xanthan molecule. Consistent with this interpretation, the observed rate of conformational ordering increases sharply under conditions where the starting solution for dynamic measurements is partially ordered, suggesting that ordered sequences within each chain may act as helix nuclei for adjacent disordered regions, so that helix growth, rather than the slower nucleation process, becomes rate limiting.

Assay restriction profiles of three monoclonal antibodies recognizing the G3m(u) allotype. Development of an allotype specific assay.

Three monoclonal antibodies raised against a purified human IgG3 paraprotein were found to exhibit a restriction profile for IgG3/G3m(u) and pan-IgG specificity which was dependent on the assay system. When adapted to an IgG3 subclass capture ELISA, all three McAbs discriminated between paraproteins expressing G3m(u) and antithetical markers G3m(st). One of the antibodies (PNF69C) was selected and conditions were optimised for Gm typing purposes. Using this system G3m(u) could be detected on captured IgG3 derived from human sera. This system may prove useful in the elucidation of Gm allotype profiles.

Evaluation of monoclonal antibodies having specificity for human IgG subclasses: results of the 2nd IUIS/WHO collaborative study.

Following the 1st IUIS/WHO Collaborative Study of monoclonal anti-IgG subclass antibodies, a panel of WHO Specificity Reference Reagents (SRR) was established [Jefferis, R., et al. (1985) Immunol. Lett., 10, 223]. At the time, the hope was expressed that further reagents particularly for IgG2, and other allotypic specificities would become available which could be applied in a wide range of assay protocols. The 2nd study reports the evaluation of nineteen anti-subclass and seven anti-allotype monoclonal antibodies. The anti-IgG1 antibody HP6187 was equivalent in performance to the SRR. Others, that were not of the mouse IgG1 isotype, may be useful for particular applications. The anti-IgG2 antibody HP6200 could be a valuable addition to the WHO SRR; it is specific for an epitope in the Fab region but does not have the light chain bias of HP6014. Antibodies of putative allotype specificity exhibited the claimed specificity when used within protocols similar to those employed by the originating laboratory. It appears to be inherent in the nature of the epitopes (allotopes) recognized that it will take several years before reagents applicable to a wide range of techniques will become available.

Two enzyme linked immunosorbent assays for detecting antibodies against meningococcal capsular polysaccharides A and C.

AIMS: To evaluate two of the recent methods of coating microtitre plates in the enzyme linked immunosorbent assay (ELISA) for detecting human antibodies against meningococcal capsular polysaccharides A and C with a view to validating a specific meningococcal antibody assay for routine clinical use. METHODS: Two four-layer ELISA protocols were standardised: one method utilised meningococcal polysaccharides conjugated to poly-L-lysine polypeptide for coating the microtitre plates; another used polysaccharides mixed with methylated human serum albumin (mHSA). Titration curves were plotted for the ELISAs and the squared Pearson correlation coefficient (R2) was used to determine the degree of accuracy of fit of the curves. Specificity tests were performed by inhibition and adsorption studies. RESULTS: Both methods gave good titration curves with a high R2 of > 0.98, indicating a high degree of accuracy in forming the curves. The titration end point after vaccination, obtained by the mHSA method, was 20 times higher, however, than that obtained by the poly-L-lysine method. Specificity tests showed that in the ELISA using polysaccharide/poly-L-lysine, antibody activity of a pre-vaccination serum sample was inhibited by 37%, and of post-vaccination serum by 50% with 1000-fold excess antigen. Antibody activity (post-vaccination) was reduced by 51% and 59%, respectively, by adsorption with antigen-coated Sepharose beads or adsorption with suspensions of killed meningococci. In contrast, antibody activity of a pre-vaccination serum was inhibited by 60% and a post-vaccination serum by 90% in ELISA employing polysaccharides mixed with mHSA. Reproducibility was better with the use of methylated human serum albumin than with poly-L-lysine; the former showed intrabatch and interbatch coefficients of variation of 4% and 2%, respectively, compared with 43% (intrabatch) and 16% (interbatch) obtained with the poly-L-lysine. CONCLUSION: It is concluded that the antibody assay using meningococcal polysaccharides groups A and C mixed with mHSA is much better than that using polysaccharides coupled with poly-L-lysine.

Trace analysis of gamma-cyclodextrin in a sample of beta-cyclodextrin by capillary electrophoresis.

A new capillary electrophoretic method for trace analysis of gamma-cyclodextrin, gamma-CD, in a sample of beta-CD has been developed, building on our recent work in which the tetraphenylborate ion, Ph4B-, was found to bind to gamma-CD three orders of magnitude more strongly than to beta-CD. The method involves measurement of the change of net electrophoretic mobility of Ph4B- and its CD complexes in a background electrolyte containing a fixed concentration of beta-CD. Good linearity was found between 1/deltamu and 1/Cgamma where deltamu is the difference in the mobility of Ph4B- in the beta-CD solution at a given and at zero concentration of gamma-CD, and Cgamma the gamma-CD concentration. The limit of detection for gamma-CD in a beta-CD sample was found to be 0.020% (w/w), and high precision and accuracy were obtained.

Improved sensitivity in detection of chlormequat by liquid chromatography-mass spectrometry.

Two published separations, using electrospray mass spectrometry (ES-MS), exhibit significant differences in limits of detection (LODs) for chlormequat cation in pear. Separation on ODS1, confirmed to result from ion-exchange, gives shorter analysis times and calibration over a wider concentration range than on an SCX cation-exchange column. The superior LOD using ODS1 (0.04 ng ml(-1) vs. 1.0 ng ml(-1)) results mainly from better chromatographic peak shape. Separation on ODS1 combined with optimised ES-MS detection allows direct quantification of chlormequat on an ion trap instrument at levels lower than those required for residue analysis in foods and also in drinking water.

Self-consistent framework for standardising mobilities in free solution capillary electrophoresis: applications to oligoglycines and oligoalanines.

A theoretical analysis of deviations from ideality in ionic transport is presented to correct mobilities, mu, measured in free solution capillary electrophoresis (CE) to mobility at infinite dilution, mu degree (limiting mobility). Non-ideality is treated at the same level of approximation as in equilibrium, using a correction factor for the sum of the analyte and counter-ion radius originally suggested by Robinson and Stokes (Electrolyte Solutions, 1961). Unlike previous corrections using Debye-Hückel-Onsager theory, which are strictly applicable only at very low ionic strengths, this treatment is expected to be valid for univalent ions migrating in a uni-univalent background electrolyte for ionic strengths up to 0.075 mol kg-1, a range typical of CE experiments. The analysis is applied to the determination of mu degree in acidic and basic buffers for oligoalanines and oligoglycines with degree of polymerisation 2 to 6. Limiting mobilities for the fully protonated and deprotonated peptides are found to be numerically equal but opposite in sign, consistent with a change in charge from +1 to -1. In all uni-univalent buffers studied (borate, citrate, low pH lithium phosphate and sodium phosphate) mu degree values established using data over a range of pH and ionic strength are found to be identical and in excellent agreement with previous values from isotachophoresis. Values of mu degree in high pH sodium phosphate buffer are systematically 0.2.10(-8) m2 V-1 s-1 higher than those in other buffers; this may be attributed to limitations of the model for a buffer with 1+:2- and 1+:3- ions. This self-consistent framework for standardising mobilities in free solution CE is expected to be widely applicable to univalent analytes migrating in a 1:1 background electrolyte.

High-performance liquid chromatography applications of optical rotation detection with compensation for scattering and absorbance at the laser wavelength.

Use of instrumentation developed to enable simultaneous monitoring of optical rotation (OR) and transmittance allows OR measurements to be made in the presence of high levels of absorbance, scattering or other effects that change the intensity of the plane-polarised light at the photodiode detector. This extends the application of OR detection to areas where it was previously difficult. Examples of the application of high-performance liquid chromatography (HPLC) with the improved OR detector include (i) the analytical scale separation of fructose and sucrose and (ii) the semi-preparative separation of enantiomers of warfarin and Trögers base. A signal-to-noise improvement of up to 150% is found when comparing signals with and without correction for transmittance changes. The improved OR detector has been used in series with a UV detector and the system shown to be suitable for on-line measurement of peak purity in separations using a chiral column under overload conditions.

Ultrastructural evidence for intramolecular double stranding in iota-carrageenan.

Kinetic studies of primary processes of conformational ordering in gel-forming biopolymers have suggested that a change in mechanism from intermolecular to intramolecular multistrand formation occurs on lowering the concentration of biopolymer. We report here ultrastructural observations consistent with intramolecular double stranding in a carbohydrate polymer, iota-carrageenan, by arresting this process of primary conformational ordering by an ultra-rapid freeze fixation technique. High-resolution transmission electron microscopy (TEM) revealed isolated iota-carrageenan chains showing a range of morphologies (linear, circular, and hairpin) consistent with intramolecular stranding. Control experiments in which iota-carrageenan was frozen in the disordered form revealed longer and thinner strands.

Characterization of peptides formed during fermentation of cocoa bean.

Analysis by SDS-PAGE and GPC-MS of fermented cocoa extracts shows changes in the amount and composition of the major proteins, accompanied by formation of complex distributions of peptides. MS/MS studies and application of SEQUEST sequencing software have allowed identification of two related peptides, a hexapeptide and a nonapeptide, formed from vicilin, one of the cocoa storage proteins. Time course studies of the two peptides show different abundance profiles and indicate, in part, production of the hexapeptide from the nonapeptide.

Determination of enantiomeric purity of ephedrine and pseudoephedrine by high-performance liquid chromatography with dual optical rotation/UV absorbance detection.

A reversed-phase high-performance liquid chromatography (HPLC) method with dual optical rotation/UV absorbance detection has been developed for the determination of enantiomeric purity of ephedrine hydrochloride and pseudoephedrine hydrochloride using an achiral column. The method gave a correlation coefficient of 0.9997 for the plot of log(optical rotation response) versus log (concentration) over the range of 0.06-10 mg ml(-1) of (+)-ephedrine hydrochloride (20 microliters injection). The limit of detection was 1.0 micrograms. Enantiomeric purity is shown to be most readily determined by measuring optical rotation, alpha, and absorbance, A, responses for standard and unknown samples, and using the equation (alpha/A)u/(alpha/A)s = (2xu - 1)/(2xs - 1), where x is the mole fraction of one of the enantiomers and subscripts s and u refer to standard and unknown, respectively. In blind trials using unknown mixtures of (+)- and (+/-)-ephedrine hydrochloride and a (+)-ephedrine hydrochloride standard, enantiomeric purities were determined to +/- 0.4% (95% confidence level) with five or six replicate 50 micrograms injections. The method has also been applied to the determination of the enantiomer mole fraction of (+)-pseudoephedrine hydrochloride in a cough linctus, giving xu = 0.99 +/- 0.01 with seven replicate injections of 20-fold diluted linctus samples containing 7.5 micrograms of the chiral compound being assayed. Unlike conventional polarimetry, the method does not require chemically-pure samples and can be orders of magnitude more economical in material.

Improved visualization of folded collagen alpha-chains by ultra-rapid freezing.

Transmission electron microscopy techniques are commonly employed to examine the folded polymer structure of collagen polypeptides. These techniques include deposition of a sample by spraying, slow freeze-fixation, air drying and vacuum drying the specimen at room temperature, and using additives such as glycerol in the collagen preparation. Here we report preliminary observations of type I collagen alpha-chains, folded in water, at a concentration of 35 micrograms ml-1 and 10 micrograms ml-1, visualized by an ultra-rapid, freeze-fixation technique designed to minimize structural deformation caused by spraying, additives and poor freeze-fixation. The technique also allows the use of submicrolitre sample volumes of known concentrations with negligible loss and shearing, while at the same time providing excellent contrast to the collagen polymer for electron microscopy. This technique can be employed to study the structure of a wide range of macromolecules (proteins and carbohydrates).

Apoptotic cell-derived ICAM-3 promotes both macrophage chemoattraction to and tethering of apoptotic cells.

A wide range of molecules acting as apoptotic cell-associated ligands, phagocyte-associated receptors or soluble bridging molecules have been implicated within the complex sequential processes that result in phagocytosis and degradation of apoptotic cells. Intercellular adhesion molecule 3 (ICAM-3, also known as CD50), a human leukocyte-restricted immunoglobulin super-family (IgSF) member, has previously been implicated in apoptotic cell clearance, although its precise role in the clearance process is ill defined. The main objective of this work is to further characterise the function of ICAM-3 in the removal of apoptotic cells. Using a range of novel anti-ICAM-3 monoclonal antibodies (mAbs), including one (MA4) that blocks apoptotic cell clearance by macrophages, alongside apoptotic human leukocytes that are normal or deficient for ICAM-3, we demonstrate that ICAM-3 promotes a domain 1-2-dependent tethering interaction with phagocytes. Furthermore, we demonstrate an apoptosis-associated reduction in ICAM-3 that results from release of ICAM-3 within microparticles that potently attract macrophages to apoptotic cells. Taken together, these data suggest that apoptotic cell-derived microparticles bearing ICAM-3 promote macrophage chemoattraction to sites of leukocyte cell death and that ICAM-3 mediates subsequent cell corpse tethering to macrophages. The defined function of ICAM-3 in these processes and profound defect in chemotaxis noted to ICAM-3-deficient microparticles suggest that ICAM-3 may be an important adhesion molecule involved in chemotaxis to apoptotic human leukocytes.