The physiological landscape and specificity of antibody repertoires are consolidated by multiple immunizations.

Diverse antibody repertoires spanning multiple lymphoid organs (i.e., bone marrow, spleen, lymph nodes) form the foundation of protective humoral immunity.

Generation of a single-cell B cell atlas of antibody repertoires and transcriptomes to identify signatures associated with antigen specificity.

Although new genomics-based pipelines have potential to augment antibody discovery, these methods remain in their infancy due to an incomplete understanding of the selection process that governs B cell clonal selection, expansion, and antigen specificity. Furthermore, it remains unknown how factors such as aging and reduction of tolerance influence B cell selection. Here we perform single-cell sequencing of antibody repertoires and transcriptomes of murine B cells following immunizations with a model therapeutic antigen target.

Phenotypic determinism and stochasticity in antibody repertoires of clonally expanded plasma cells.

The capacity of humoral B cell-mediated immunity to effectively respond to and protect against pathogenic infections is largely driven by the presence of a diverse repertoire of polyclonal antibodies in the serum, which are produced by plasma cells (PCs). Recent studies have started to reveal the balance between deterministic mechanisms and stochasticity of antibody repertoires on a genotypic level (i.e.

DeepSARS: simultaneous diagnostic detection and genomic surveillance of SARS-CoV-2.

Background: The continued spread of SARS-CoV-2 and emergence of new variants with higher transmission rates and/or partial resistance to vaccines has further highlighted the need for large-scale testing and genomic surveillance. However, current diagnostic testing (e.g.

Profiling the specificity of clonally expanded plasma cells during chronic viral infection by single-cell analysis.

Plasma cells and their secreted antibodies play a central role in the long-term protection against chronic viral infection. However, due to experimental limitations, a comprehensive description of linked genotypic, phenotypic, and antibody repertoire features of plasma cells (gene expression, clonal frequency, virus specificity, and affinity) has been challenging to obtain. To address this, we performed single-cell transcriptome and antibody repertoire sequencing of the murine BM plasma cell population following chronic lymphocytic choriomeningitis virus infection.

Platypus: an open-access software for integrating lymphocyte single-cell immune repertoires with transcriptomes.

High-throughput single-cell sequencing (scSeq) technologies are revolutionizing the ability to molecularly profile B and T lymphocytes by offering the opportunity to simultaneously obtain information on adaptive immune receptor repertoires (VDJ repertoires) and transcriptomes. An integrated quantification of immune repertoire parameters, such as germline gene usage, clonal expansion, somatic hypermutation and transcriptional states opens up new possibilities for the high-resolution analysis of lymphocytes and the inference of antigen-specificity. While multiple tools now exist to investigate gene expression profiles from scSeq of transcriptomes, there is a lack of software dedicated to single-cell immune repertoires.

Applications of Machine and Deep Learning in Adaptive Immunity.

Adaptive immunity is mediated by lymphocyte B and T cells, which respectively express a vast and diverse repertoire of B cell and T cell receptors and, in conjunction with peptide antigen presentation through major histocompatibility complexes (MHCs), can recognize and respond to pathogens and diseased cells. In recent years, advances in deep sequencing have led to a massive increase in the amount of adaptive immune receptor repertoire data; additionally, proteomics techniques have led to a wealth of data on peptide-MHC presentation. These large-scale data sets are now making it possible to train machine and deep learning models, which can be used to identify complex and high-dimensional patterns in immune repertoires.

Single-cell immune repertoire and transcriptome sequencing reveals that clonally expanded and transcriptionally distinct lymphocytes populate the aged central nervous system in mice.

Neuroinflammation plays a crucial role during ageing and various neurological conditions, including Alzheimer's disease, multiple sclerosis and infection. Technical limitations, however, have prevented an integrative analysis of how lymphocyte immune receptor repertoires and their accompanying transcriptional states change with age in the central nervous system. Here, we leveraged single-cell sequencing to simultaneously profile B cell receptor and T cell receptor repertoires and accompanying gene expression profiles in young and old mouse brains.

Antibody discovery and engineering by enhanced CRISPR-Cas9 integration of variable gene cassette libraries in mammalian cells.

Antibody engineering in mammalian cells offers the important advantage of expression and screening of libraries in their native conformation, increasing the likelihood of generating candidates with more favorable molecular properties. Major advances in cellular engineering enabled by CRISPR-Cas9 genome editing have made it possible to expand the use of mammalian cells in biotechnological applications. Here, we describe an antibody engineering and screening approach where complete variable light (V) and heavy (V) chain cassette libraries are stably integrated into the genome of hybridoma cells by enhanced Cas9-driven homology-directed repair (HDR), resulting in their surface display and secretion.

Genome Engineering of Hybridomas to Generate Stable Cell Lines for Antibody Expression.

From the perspective of academic and small research laboratories, the most common and practical strategy for recombinant expression of full-length monoclonal antibodies is to perform transient plasmid transfection of mammalian cells, resulting in small-scale and limited protein production. The generation of stable antibody producing mammalian cell lines enables larger-scale and consistent expression, however this approach is rarely pursued due to the time-consuming and expensive process of single colony screening and characterization. In order to bridge the gap between the simplicity of transient transfection and consistent production by stable cell lines, we describe a method to stably integrate antibody genes into the endogenous immunogenomic loci of hybridoma cells using CRISPR/Cas9 genome editing.

Integrating high-throughput screening and sequencing for monoclonal antibody discovery and engineering.

Monoclonal antibody discovery and engineering is a field that has traditionally been dominated by high-throughput screening platforms (e.g. hybridomas and surface display).

Ozonized low density lipoprotein (ozLDL) inhibits NF-kappaB and IRAK-1-associated signaling.

Objective: Recent studies have provided strong evidence for the presence of ozone in atherosclerotic lesions. In addition, modification of LDL has been suggested to be involved in atherosclerosis. In the present study we wanted to investigate whether LDL exposed to ozone (ozLDL) is able to modulate the NF-kappaB system, as a paradigm for inflammatory signaling.

C/EBP beta blocks p65 phosphorylation and thereby NF-kappa B-mediated transcription in TNF-tolerant cells.

TNF is a major mediator of inflammation, immunity, and apoptosis. Pre-exposure to TNF reduces sensitivity to restimulation, a phenomenon known as tolerance, considered as protective in sepsis, but also as a paradigm for immunoparalysis. Earlier experiments in TNF-tolerant cells display inhibition of NF-kappaB-dependent IL-8 gene expression at the transcriptional level with potential involvement of C/EBPbeta.

Reference values for cardiac troponins T and I in healthy neonates.

Objectives: Cardiac troponins T (cTnT) and I (cTnI) are the most sensitive biochemical parameters in the detection of myocardial damage. In neonates, in utero exposure to tocolytic therapy results in detectable values of cardiac troponins after delivery. Additionally, some preliminary results suggest that the upper reference limits for healthy newborns are higher than those for adults but definitive reference limits for newborns are not available.

Detection of IKKbeta-IKKgamma subcomplexes in monocytic cells and characterization of associated signaling.

The IkappaB kinase (IKK) complex is one major step in the regulation of the NF-kappaB/Rel system that is involved in inflammatory and immune responses as well as in proliferation and apoptosis. At present it is not clear whether besides the "classical" IKKalpha-IKKbeta-IKKgamma configuration additional complexes exist in vivo that solely contain IKKbeta and IKKgamma (without IKKalpha). In the current study we were able to demonstrate in monocytic cells that endogenous complexes, which only include IKKbeta as the kinase-active molecule do indeed exist in vivo and that these complexes contain IKKgamma as an additional component.

Transcriptional inhibition of interleukin-8 expression in tumor necrosis factor-tolerant cells: evidence for involvement of C/EBP beta.

There is some evidence that the potent cytokine tumor necrosis factor (TNF) is able to induce tolerance after repeated stimulation of cells. To investigate the molecular mechanisms mediating this phenomenon, the expression of interleukin-8 (IL-8), which is regulated by transcription factors NF-kappaB and C/EBPbeta, was monitored under TNF tolerance conditions. Pretreatment of monocytic cells for 72 h with low TNF doses inhibited TNF-induced (restimulation with a high dose) IL-8 promoter-dependent transcription as well as IL-8 production.

Increased IkappaB kinase activity is associated with activated NF-kappaB in acute myeloid blasts.

NF-kappaB/Rel transcription factors are modulators of immune and inflammatory processes and are also involved in malignancy. Phosphorylation of the IkappaB inhibitors by the IkappaB kinase (IKK) complex leads to their proteasomal degradation, resulting in activated NF-kappaB. Here, we investigated the activation status of NF-kappaB and the IKK complex in acute myeloid leukemia (AML).

Chlamydia pneumoniae activates IKK/I kappa B-mediated signaling, which is inhibited by 4-HNE and following primary exposure.

Chlamydia pneumoniae may be involved in atherosclerosis by inducing inflammation as well as LDL oxidation. The transcription factor NF-kappa B is found in an active state in atherosclerotic lesions. This study examined the effect of C.

Effects of implantable cardioverter defibrillator implantation and shock application on biochemical markers of myocardial damage.

Background: Implantable cardioverter defibrillator (ICD) implantation is a common approach in patients at high risk of sudden cardiac death. To check for normal function, it is necessary to test the ICD. For this purpose, repetitive induction and termination of ventricular fibrillation by direct current shocks is required.

Expression of messenger RNA of the cardiac isoforms of troponin T and I in myopathic skeletal muscle.

In the absence of clinical signs, elevated values of the cardiac isoforms of troponin T (cTnT) and I (cTnI) can be found in the serum samples of some patients with skeletal muscle myopathies; the cause is unclear. We studied the messenger RNA (mRNA) expression of cTnT and cTnI in the skeletal muscles of 24 patients with histologically proven myopathies and in 18 patients in whom a myopathy could be excluded. For cTnT- and cTnI-mRNA determination, we designed specific primer pairs for nested polymerase chain reaction.

Cardiac troponin T and I in end-stage renal failure.

Background: In patients suffering from end-stage renal failure, cardiac troponin T (cTnT) and I (cTnI) may be increased in serum without other signs of acute myocardial damage. Whether these increases are specific to myocardial injury or nonspecific is not completely clear.

Methods: We investigated time courses of cTnT and cTnI over 1 year and the clinical outcome over 2 years in 59 patients with end-stage renal failure undergoing chronic hemodialysis.

Biotinylated steroid derivatives as ligands for biospecific interaction analysis with monoclonal antibodies using immunosensor devices.

Systematic ligand-binding studies of the biospecific interaction between steroids and antisteroid antibodies can be performed in real time using biosensor techniques. In this study, quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) biosensor systems were applied. Different biotinylated testosterone (T) and 17beta-estradiol (E2) derivatives were preincubated with streptavidin and immobilized on the sensor surfaces.

Clinical value of cystatin C determination.

It has been reported that cystatin C (cys-C) is elevated in patients with malignant disease. In order to investigate whether this phenomenon is linked to or independent of renal function, and at the same time examine the role of this marker in other pathological situations, cys-C concentrations were compared with 24-h creatinine clearance values in three groups of patients; the first group were undergoing treatment for malignant disease, the second group were renal transplant patients and the third randomly taken from patients for whom a routine creatinine clearance had been requested. Several patients with malignant disease had high cys-C levels without any correspondence to creatinine clearance values.

Differential effects of lipopolysaccharide and tumor necrosis factor on monocytic IkappaB kinase signalsome activation and IkappaB proteolysis.

The inflammatory mediators lipopolysaccharide (LPS) and tumor necrosis factor (TNF) are potent activators of NF-kappaB. This study compared the effect of these stimuli on endogenous IkappaB kinase (IKK) signalsome activation and IkappaB phosphorylation/proteolysis in human monocytic cells and investigated the role of the signalsome proteins IKK-alpha, IKK-beta, NF-kappaB-inducing kinase (NIK), IKK-gamma (NF-kappaB essential modulator), and IKK complex-associated protein. Kinase assays showed that TNF elicited a rapid but short-lived induction of IKK activity with a 3-fold greater effect on IKK-alpha than on IKK-beta, peaking at 5 min.