Constant Region Research Articles

A Modified Mild-Slope Model for the Hydrodynamic Analysis of Arrays of Heaving WECs in Variable Bathymetry Regions

A simplified model based on the Modified Mild-Slope Equation with inclusions is developed for modelling the scattering of waves from multiple heaving point absorbers arranged in an array in general bottom topography. The model is used, in conjunction with a 3D BEM, in order to estimate the parameters modelling the energy extraction of the devices using data obtained from the hydrodynamic responses and performance of the single floating WEC. Subsequently, the present model is used for specific examples to calculate the wave field and the hydrodynamic performance of arrays of heaving WECs in constant depth and variable bathymetry regions and illustrate the effect of bottom slope and variation on the calculated wave field in the domain and in the vicinity of the devices. The present simplified model provides a low-cost first estimation of the wave conditions in the domain, which could be exploited as a supporting tool for best arrangement and park design purposes.

Intravenous immunoglobulin induces IgG internalization by tolerogenic myeloid dendritic cells that secrete IL-10 and expand Fc-specific regulatory T cells

Intravenous immunoglobulin (IVIG) is used as an immunomodulatory agent in many inflammatory conditions including Multisystem Inflammatory Syndrome-Children (MIS-C) and Kawasaki disease (KD). However, the exact mechanisms underlying its anti-inflammatory action are incompletely characterized. Here, we show that in KD, a pediatric acute vasculitis that affects the coronary arteries, IVIG induces a repertoire of natural Treg that recognize immunodominant peptides in the Fc heavy chain constant region. To address which antigen-presenting cell (APC) populations present Fc peptides to Treg, we studied the uptake of IgG by innate cells in subacute KD patients 2 weeks after IVIG and in children 1.6–14 years after KD. Healthy adults served as controls. IgG at high concentrations was internalized predominantly by two myeloid dendritic cell (DC) lineages, CD14+ cDC2 and ILT-4+ CD4+ tmDC mostly through Fcγ receptor (R) II and to a lesser extent FcγRIII. Following IgG internalization, these two DC lineages secreted IL-10 and presented processed Fc peptides to Treg. The validation of IVIG function in expanding Fc-specific Treg presented by CD14+ cDC2 and ILT-4+ CD4+ tmDC was addressed in a small cohort of patients with MIS-C. Taken together, these results suggest a novel immune regulatory function of IgG in activating tolerogenic innate cells and expanding Treg, which reveals an important anti-inflammatory mechanism of action of IVIG.

Optimized quantitative PCR analysis of random DNA aptamer libraries

The quantitative polymerase chain reaction (qPCR) with detection of duplex DNA yield by intercalator fluorescence is a common and essential technique in nucleic acid analysis. We encountered unexpected results when applying standard qPCR methods to the quantitation of random DNA libraries flanked by regions of fixed sequence, a configuration essential for in vitro selection experiments. Here we describe the results of experiments revealing why conventional qPCR methods can fail to allow automated analysis in such cases, and simple solutions to this problem. In particular we show that renaturation of PCR products containing random regions is incomplete in late PCR cycles when extension fails due to reagent depletion. Intercalator fluorescence can then be lost at standard interrogation temperatures. We show that qPCR analysis of random DNA libraries can be achieved simply by adjusting the step at which intercalator fluorescence is monitored so that the yield of annealed constant regions is detected rather than the yield of full duplex DNA products.

Characterization and determination of bovine immunoglobulin G subtypes in milk and dairy products by UPLC-MS

In this study, ultra-high performance liquid chromatography mass spectrometry (UPLC-MS) method was established for the characterization and quantitative determination of immunoglobulin G (IgG) subtypes (IgG1, IgG2, IgG3) in bovine dairy products. High-resolution mass spectrometry (HRMS) was applied to qualitatively confirm the theoretical peptides with specificity, enzymatic hydrolysis curve and stability among in heavy chain constant (CH1, CH2 and CH3) regions. The characteristic peptides VHNEGLPAPIVR, EPSVFIFPPKPK, GLPAPIVR, VVSALR were screened to quantitative analysis bovine IgG1, IgG2, IgG3 and the total amount of bovine IgG1 and IgG3, respectively. Isotope-labeled peptides were obtained by isotope dimethylation reaction, which aimed to correct the matrix effects. The results showed that the recovery was between 98.7% and 103.5%, and the precision of inter-day and intra-day was less than 6.8%. Moreover, this method had good linearity (R2 ≥ 0.999). Therefore, this research provided an effective method for quantitatively detecting bovine IgG subtypes in milk and dairy products.

A CURE for Cloning: Implementing a Modular Approach to Cloning Recombinant Antibodies in an Undergraduate Teaching Lab

An undergraduate course‐based research experience (CURE) is a high impact practice which provides genuine research experiences to a large and diverse student population. We have implemented a six‐week research module involving the cloning and expression of therapeutic antibodies as the culmination of a senior level Biochemistry lab course. We have designed a cloning system that takes advantage of the modular domain structure of antibodies to facilitate changes to an antibody’s affinity, isotype, or allotype. Specifically, our system was developed for readily swapping the antigen‐binding N‐terminal sequences of different antibodies into a set of vectors encoding the conserved regions of the heavy and light chains, all in the background of a strong mammalian expression vector. Gene fragments corresponding to the constant regions of each immunoglobulin chain, as well as the antigen‐binding regions of unique antibodies were designed, and produced by gene synthesis. Various iterations of the module were tested over four different semesters with high levels of success in terms of student engagement and research goals. The resulting plasmid constructs are now being used by our research lab as part of the investigation of the nature of antibody recognition of cancer‐specific Mucin antigens. This project allowed our senior undergraduates to contribute to an active research program, while earning credit toward a required course. It exposed them to modern cloning methodologies, bioinformatics tools, cell culture techniques and participation in a local research conference. It is also a readily scalable project as only the variable regions of new antibodies of interest need be synthesized.

AID function in somatic hypermutation and class switch recombination.

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation of immunoglobulin (Ig) gene variable regions and class switch recombination (CSR) of Ig heavy chain constant regions. Two decades of intensive research has greatly expanded our knowledge of how AID functions in peripheral B cells to optimize antibody responses against infections, while maintaining tight regulation of AID to restrain its activity to protect B cell genomic integrity. The many exciting recent advances in the field include: 1) the first description of AID's molecular structure, 2) remarkable advances in high throughput approaches that precisely track AID targeting genome-wide, and 3) the discovery that the cohesion-mediate loop extrusion mechanism [initially discovered in V(D)J recombination studies] also governs AID-medicated CSR. These advances have significantly advanced our understanding of AID's biochemical properties in vitro and AID's function and regulation in vivo. This mini review will discuss these recent discoveries and outline the challenges and questions that remain to be addressed.

Characterization of the DH475 cooperating antibody and its interaction with the HIV‐1 spike

HIV evolves very quickly, so approaches to design an effective vaccine that elicits protective antibodies have thus far been unsuccessful. Current HIV vaccine design efforts seek to elicit broadly neutralizing antibodies, unique antibodies that target many viral variants, by first eliciting their precursors through prime‐boost regimens. This requires an understanding of the co‐evolution between viruses and antibodies. Towards this goal, we are analyzing a cooperating antibody, called DH475, which exerted pressure on HIV to evolve in such a way that it became sensitive to the DH270 broadly neutralizing antibody lineage. This study aims to elucidate how DH475 binds to the HIV viral spike and identify how DH475 facilitated the development of DH270 broadly neutralizing antibodies. We obtained a 2.90Å crystal structure of DH475 in complex with Man9 glycan, and used site‐directed mutagenesis coupled with biolayer interferometry (BLI) and protein‐protein docking to characterize how DH475 interacts with Env. These investigations revealed a glycan‐dependent epitope, and docking analyses identified an unorthodox binding mode in which the DH475 framework and constant regions participate in binding. While further confirmation of DH475’s binding mode is required, our findings indicate an overlapping epitope between the DH270 lineage and DH475, consistent with its cooperative neutralization ability.

Altered abundances of human immunoglobulin M and immunoglobulin G subclasses in Alzheimer\u2019s disease frontal cortex

The immune system has been described to play a role in the development of Alzheimer’s disease (AD), but the distribution of immunoglobulins and their subclasses in brain tissue has not been explored. In this study, examination of pathologically diagnosed frontal cortex gray matter revealed significantly higher levels of IgM and IgG in late-stage AD (Braak and Braak stages V and VI) compared to age-matched controls. While levels of IgG2 and IgG4 constant region fragments were higher in late-stage AD, concentration of native–state IgG4 with free Fc regions was increased in AD III and VI. RNA analysis did not support parenchymal B-cell production of IgG4 in AD III and V, indicating possible peripheral or meningeal B-cell involvement. Changes in the profile of IgM, IgG and IgG subclasses in AD frontal cortex may provide insight into understanding disease pathogenesis and progression.

Functionalisation of Inorganic Material Surfaces with Staphylococcus Protein A: A Molecular Dynamics Study.

Staphylococcus protein A (SpA) is found in the cell wall of Staphylococcus aureus bacteria. Its ability to bind to the constant Fc regions of antibodies means it is useful for antibody extraction, and further integration with inorganic materials can lead to the development of diagnostics and therapeutics. We have investigated the adsorption of SpA on inorganic surface models such as experimentally relevant negatively charged silica, as well as positively charged and neutral surfaces, by use of fully atomistic molecular dynamics simulations. We have found that SpA, which is itself negatively charged at pH7, is able to adsorb on all our surface models. However, adsorption on charged surfaces is more specific in terms of protein orientation compared to a neutral Au (111) surface, while the protein structure is generally well maintained in all cases. The results indicate that SpA adsorption is optimal on the siloxide-rich silica surface, which is negative at pH7 since this keeps the Fc binding regions free to interact with other species in solution. Due to the dominant role of electrostatics, the results are transferable to other inorganic materials and pave the way for new diagnostic and therapeutic designs where SpA might be used to conjugate antibodies to nanoparticles.

Gender dimorphism in IgA subclasses in T2-high asthma

Immunoglobulin A (Chan in J Allergy Clin Immunol 134:1394–14014e4, 2014), the second most abundant immunoglobulin in serum, plays an important role in mucosal homeostasis. In human serum, there are two subclasses of IgA, IgA1 (≅ 90%) and IgA2 (≅ 10%), transcribed from two distinct heavy chain constant regions. This study evaluated the serum concentrations of total IgA, IgA1, and IgA2, and total IgG, IgG1, IgG2, IgG3, and IgG4 in T2-high asthmatics compared to healthy controls and the presence of gender-related variations of immunoglobulins. Total IgA levels were increased in asthmatics compared to controls. Even more marked was the increase in total IgA in male asthmatics compared to healthy male donors. IgA1 were increased only in male, but not in female asthmatics, compared to controls. Concentrations of IgG2, but not IgG1, IgG3, and IgG4, were reduced in asthmatics compared to controls. IgG4 levels were reduced in female compared to male asthmatics. In female asthmatics, IgA and IgA1 levels were increased in postmenopause compared to premenopause. IgA concentrations were augmented in mild, but not severe asthmatics. A positive correlation was found between IgA levels and the age of patients and an inverse correlation between serum concentrations of IgA2 and IgE in asthmatics. A positive correlation between total IgA or IgA2 and IgG2 was found in asthmatics. These results highlight a gender dimorphism in IgA subclasses in male and female T2-high asthmatics. More adequate consideration of immunological gender disparity in asthma may open new opportunities in personalized medicine by optimizing diagnosis and targeted therapy.

Multiscale analysis of some shear layers in a fully developed turbulent channel flow

Multiscale edge detection-wavelet analysis is applied to the velocity field emanating from direct numerical simulations of a fully developed turbulent channel flow. The direct numerical simulations are realized in large computational domains at four different Karman numbers up to 1100. The shear layers (SL) related to the streamwise and spanwise velocity fields involving streamwise and spanwise gradients are analyzed scale-by-scale, over the homogeneous planes. The SL involving the spanwise gradient, in particular the wall vorticity layers surrounding the quasi-streamwise vortices (QSV) are organized into long streamwise streaks, whose spanwise spacing and thickness are determined versus the wavelet-wavenumber. The shear layers related to the streamwise gradient are further analyzed by using the Rice decomposition of the wavelet coefficients into a local phase and a local amplitude. Large zones of constant phase with slowly varying local amplitude have been identified at different scales. It is found that the constant phase-locked regions coincide with the wakes of the buffer layer QSV. The arrival of the quasi-streamwise vortices cause phase jumps and large intermittency in the local amplitude. The analogy between these peculiarities and the imperfect chaos synchronization phenomena together with the consequences in term of wall turbulence control are discussed. These results confirm globally our previous investigation realized through a low Reynolds number channel flow DNS in a small computational box and clarify some points that could not then elucidated.

Rheumatoid Factor and Anti-Modified Protein Antibody Reactivities Converge on IgG Epitopes.

Rheumatoid arthritis (RA) patients often develop rheumatoid factors (RFs), antibodies that bind IgG Fc, and anti-modified protein antibodies (AMPAs), multireactive autoantibodies that commonly bind citrullinated, homocitrullinated, and acetylated antigens. Recently, antibodies that bind citrulline-containing IgG epitopes were discovered in RA, suggesting that additional undiscovered IgG epitopes could exist and that IgG could be a shared antigen for RFs and AMPAs. This study was undertaken to reveal new IgG epitopes in rheumatic disease and to determine if multireactive AMPAs bind IgG. Using sera from patients with RA, systemic lupus erythematosus, Sjögren's disease (SjD), or spondyloarthropathy, IgG binding to native, citrulline-containing, and homocitrulline-containing linear epitopes of the IgG constant region was evaluated by peptide array, with highly bound epitopes further evaluated by enzyme-linked immunosorbent assay (ELISA). Binding of monoclonal AMPAs to IgG-derived peptides and IgG Fc was also evaluated by ELISA. Seropositive RA sera showed high IgG binding to multiple citrulline- and homocitrulline-containing IgG-derived peptides, whereas anti-SSA+ sera from SjD patients showed consistent binding to a single linear native epitope of IgG in the hinge region. Monoclonal AMPAs bound citrulline- and homocitrulline-containing IgG peptides and modified IgG Fc. The repertoire of epitopes bound by AMPAs includes modified IgG epitopes, positioning IgG as a common antigen that connects the otherwise divergent reactivities of RFs and AMPAs.

Sialylation as an Important Regulator of Antibody Function.

Antibodies play a critical role in linking the adaptive immune response to the innate immune system. In humans, antibodies are categorized into five classes, IgG, IgM, IgA, IgE, and IgD, based on constant region sequence, structure, and tropism. In serum, IgG is the most abundant antibody, comprising 75% of antibodies in circulation, followed by IgA at 15%, IgM at 10%, and IgD and IgE are the least abundant. All human antibody classes are post-translationally modified by sugars. The resulting glycans take on many divergent structures and can be attached in an N-linked or O-linked manner, and are distinct by antibody class, and by position on each antibody. Many of these glycan structures on antibodies are capped by sialic acid. It is well established that the composition of the N-linked glycans on IgG exert a profound influence on its effector functions. However, recent studies have described the influence of glycans, particularly sialic acid for other antibody classes. Here, we discuss the role of glycosylation, with a focus on terminal sialylation, in the biology and function across all antibody classes. Sialylation has been shown to influence not only IgG, but IgE, IgM, and IgA biology, making it an important and unappreciated regulator of antibody function.

Stitchr: stitching coding TCR nucleotide sequences from V/J/CDR3 information.

The study and manipulation of T cell receptors (TCRs) is central to multiple fields across basic and translational immunology research. Produced by V(D)J recombination, TCRs are often only recorded in the literature and data repositories as a combination of their V and J gene symbols, plus their hypervariable CDR3 amino acid sequence. However, numerous applications require full-length coding nucleotide sequences. Here we present Stitchr, a software tool developed to specifically address this limitation. Given minimal V/J/CDR3 information, Stitchr produces complete coding sequences representing a fully spliced TCR cDNA. Due to its modular design, Stitchr can be used for TCR engineering using either published germline or novel/modified variable and constant region sequences. Sequences produced by Stitchr were validated by synthesizing and transducing TCR sequences into Jurkat cells, recapitulating the expected antigen specificity of the parental TCR. Using a companion script, Thimble, we demonstrate that Stitchr can process a million TCRs in under ten minutes using a standard desktop personal computer. By systematizing the production and modification of TCR sequences, we propose that Stitchr will increase the speed, repeatability, and reproducibility of TCR research. Stitchr is available on GitHub.

A reliability and survival model for one and two failure modes system with applications to complete and censored datasets

Lifetime distributions that have bathtub-shaped failure rates with low and long constant region at the mid-time period are more realistic for modelling failure time datasets with similar failure rates. In this paper, we introduce a new model named flexible additive Chen–Gompertz distribution. The model has increasing and bathtub-shaped failure rate with or without low and long constant region, and can be adequately used in reliability context. Some distributional properties of the model are obtained. We present a complete Bayesian analysis of the proposed model for censored and non-censored datasets and provide the Bayes estimators of the model parameters using the Metropolis–Hastings algorithm by utilizing the square error loss function. We have also given the maximum likelihood estimators of the model parameters for complete and right-censored datasets and performed simulations on the two methods for different settings of the parameters and sample sizes. The applicability of the proposed distribution is demonstrated by fitting three different failure time datasets each with bathtub failure rate from reliability and survival analysis. The results of the applications illustrate that the model provides a good fit to all the three datasets compared to the other well-known Chen and Weibull extended distributions as evidenced by some goodness-of-fit tests.

Reliable Detection of T-Cell Clonality by Flow Cytometry in Mature T-Cell Neoplasms Using TRBC1: Implementation as a Reflex Test and Comparison with PCR-Based Clonality Testing.

The T-cell receptor β constant region 1 (TRBC1) antibody can identify T-cell clonality and distinguish pathological from normal T cells. This study aims to establish optimal cutpoints for establishing monotypia and validate the diagnostic abilities of the TRBC1 antibody when used as a reflex test in conjunction with an existing T-cell antibody panel. We used 46 normal peripheral blood specimens and examined 8 patients with reactive lymphoproliferations to determine the normal biological range of TRBC1 on CD4+ and CD8+ T cells. We also evaluated 43 patient specimens that were submitted for investigation of a lymphoproliferative disorder for CD2/CD3/CD4/CD5/CD7/CD8/CD16/CD26/CD45/CD56/TCR αβ/TCR γδ, along with TRBC1 expression. The results were compared to TCR gene rearrangement patterns using polymerase chain reaction (PCR) analysis. Statistical analysis established differing cutoff points for establishing monotypia dependent on restricted TRBC1 or TRBC2 usage. Direct comparison with molecular analysis indicated that no specimen identified with the restricted expression of TRBC1 was reported as polyclonal by PCR with a concordance rate of 97% between a clonal PCR result and monotypic TRBC1 expression. Incorporation of the TRBC1 antibody using statistically derived cutoff points in a reflex setting for the evaluation of a suspected T-cell neoplasm improves the identification of clonal T-cell populations by flow cytometry and correlates well with molecular methods.

Antibody-Mediated Inhibition of Insulin-Degrading Enzyme Improves Insulin Activity in a Diabetic Mouse Model.

Diabetes is a metabolic disease that may lead to different life-threatening complications. While insulin constitutes a beneficial treatment, its use may be limited due to increased degradation and an increase in side effects such as weight gain and hypoglycemia. Small molecule inhibitors to insulin-degrading enzyme (IDE) have been previously suggested as a potential treatment for diabetes through their ability to reduce insulin degradation and thus increase insulin activity. Nevertheless, their tendency to bind to the zinc ion in the catalytic site of IDE may affect other important metalloproteases and limit their clinical use. Here, we describe the isolation of an IDE-specific antibody that specifically inhibits insulin degradation by IDE. Using phage display, we generated a human IDE-specific antibody that binds human and mouse IDE with high affinity and specificity and can differentiate between active IDE to a mutated IDE with reduced catalytic activity in the range of 30 nM. We further assessed the ability of that IDE-inhibiting antibody to improve insulin activity in vivo in an STZ-induced diabetes mouse model. Since human antibodies may stimulate the mouse immune response to generate anti-human antibodies, we reformatted our inhibitory antibody to a “reverse chimeric” antibody that maintained the ability to inhibit IDE in vitro, but consisted of mouse constant regions, for reduced immunogenicity. We discovered that one intraperitoneal (IP) administration of the IDE-specific antibody in STZ-induced diabetic mice improved insulin activity in an insulin tolerance test (ITT) assay and reduced blood glucose levels. Our results suggest that antibody-mediated inhibition of IDE may be beneficial on improving insulin activity in a diabetic environment.

A novel mutation in TRAC in a patient with abnormal newborn screening for severe combined immunodeficiency

Background: The T cell receptor (TCR)-α chain plays a key role in TCR structure and function. Biallelic mutations in TRAC, encoding the constant region of the TCR-α chain, obliterates TCR expression and results in immunodeficiency. TCR-α chain deficiency presents at infancy or childhood with repeated viral and bacterial infections, enlarged liver, spleen, and lymph nodes as well as autoimmune features and lymphoma (OMIM #615387). Aim: To broaden the genotypic and phenotypic spectrum of TCR-α chain deficiency. Methods: We present a case report of a patient with severe combined immunodeficiency (SCID) due to a novel autosomal recessive mutation in TRAC. Results: Our patient was identified at 13 days of life due to abnormal T cell receptor excision circle levels detected by newborn screening (NBS). Immune evaluation revealed profound lymphopenia, depressed responses to the mitogen PHA and a skewed T cell repertoire, all consistent with SCID. The patient was found to carry a novel homozygous mutation in the TRAC gene. Conclusion: A novel homozygous mutation in the TRAC gene caused profound T cell lymphopenia and aberrant in vitro mitogenic response, the hallmarks of SCID. Statement of Novelty: TCR-α chain deficiency is a rare and relatively new condition and not very well defined. We herein report a novel mutation in TRAC resulting in SCID.

Stereotyped B-cell responses are linked to IgG constant region polymorphisms in multiple sclerosis.

Clonally related B cells infiltrate the brain, meninges, and cerebrospinal fluid of MS patients, but the mechanisms driving the B-cell response and shaping the immunoglobulin repertoires remain unclear. Here, we used single-cell full-length RNA-seq and BCR reconstruction to simultaneously assess the phenotypes, isotypes, constant region polymorphisms, and the paired heavy- and light-chain repertoires in intrathecal B cells. We detected extensive clonal connections between the memory B cell and antibody-secreting cell (ASC) compartments and observed clonally related cells of different isotypes including IgM/IgG1, IgG1/IgA1, IgG1/IgG2, and IgM/IgA1. There was a strong dominance of the G1m1 allotype constant region polymorphisms in ASCs, but not in memory B cells. Tightly linked to the G1m1 allotype, we found a preferential pairing of the immunoglobulin heavy-chain variable (IGHV)4 gene family with the κ variable (IGKV)1 gene family. The IGHV4-39 gene was most used and showed the highest frequency of pairing with IGKV1-5 and IGKV1(D)-33. These results link IgG constant region polymorphisms to stereotyped B-cell responses in MS and indicate that the intrathecal B-cell response in these patients could be directed against structurally similar epitopes.

The role of chromatin loop extrusion in antibody diversification.

Cohesin mediates chromatin loop formation across the genome by extruding chromatin between convergently oriented CTCF-binding elements. Recent studies indicate that cohesin-mediated loop extrusion in developing B cells presents immunoglobulin heavy chain (Igh) variable (V), diversity (D) and joining (J) gene segments to RAG endonuclease through a process referred to as RAG chromatin scanning. RAG initiates V(D)J recombinational joining of these gene segments to generate the large number of different Igh variable region exons that are required for immune responses to diverse pathogens. Antigen-activated mature B cells also use chromatin loop extrusion to mediate the synapsis, breakage and end joining of switch regions flanking Igh constant region exons during class-switch recombination, which allows for the expression of different antibody constant region isotypes that optimize the functions of antigen-specific antibodies to eliminate pathogens. Here, we review recent advances in our understanding of chromatin loop extrusion during V(D)J recombination and class-switch recombination at the Igh locus.