Clinical and healthcare utilization outcomes during the 6 months following COVID infection in children.

BACKGROUND: We aimed to identify the impact of COVID infection in children in the US prior to vaccine availability on clinical and healthcare utilization outcomes within 6 months of infection. METHODS: Using claims data from a large national insurer, we identified 223,842 children with a COVID diagnosis in May 2020-March 2021 and matched them to 223,842 children with a COVID test and no diagnosis. We compared the two cohorts' outcomes during the 6 months after infection/test. RESULTS: Uncommon acute adverse events occurring in <0.5% of cases, including MIS-C (relative risk (RR) = 45.2), myocarditis (RR = 10.3), acute heart failure (RR = 2.14), sepsis (RR = 2.02), and viral pneumonia (RR = 2.43) were more frequent in the COVID cohort (all p < 0.001). Development of arrhythmias (RR = 1.24, p < 0.001) and atherosclerotic cardiovascular disease (RR = 1.41, p = 0.007) were more common in the COVID group, while behavioral health disorders were less common (RR = 0.94, p < 0.001). Lab testing and imaging were slightly higher in the COVID group (RR ranging 1.05-1.11 depending on the service and timeframe), though medical costs did not increase. CONCLUSION: Severe disease and diagnoses of new conditions are rare in children following COVID infection. We observed an increase in cardiac complications, though they may not last long term. IMPACT: Few studies have analyzed the association between COVID infection and medium-term outcomes in children. Our study of >447,000 geographically and socioeconomically diverse children in the US found that uncommon acute adverse events, including myocarditis, MIS-C, and acute heart failure, were more frequent in children with COVID than matched controls, and development of arrhythmias and cardiovascular disease were 1.2 and 1.4 times more common, respectively. Six-month healthcare utilization was similar between cohorts. We provide data on the risks of COVID in children, particularly with respect to cardiac complications, that decision makers may find useful when weighing the benefits and harms of preventive measures.

Measurements of bone-conducted sound in the chinchilla external ear.

We measure bone-conduction (BC) induced skull velocity, sound pressure at the tympanic membrane (TM) and inner-ear compound-action potentials (CAP) before and after manipulating the ear canal, ossicles, and the jaw to investigate the generation of BC induced ear-canal sound pressures and their contribution to inner-ear BC response in the ears of chinchillas. These measurements suggest that in chinchilla: i.) Vibrations of the bony ear canal walls contribute significantly to BC-induced ear canal sound pressures, as occluding the ear canal at the bone-cartilaginous border causes a 10 dB increase in sound pressure at the TM (PTM) at frequencies below 2 kHz. ii.) The contributions to PTM of ossicular and TM motions when driven in reverse by BC-induced inner-ear sound pressures are small. iii.) The contribution of relative motions of the jaw and ear canal to PTM is small. iv.) Comparison of the effect of canal occlusion on PTM and CAP thresholds point out that BC-induced ear canal sound pressures contribute significantly to bone-conduction stimulation of the inner ear when the ear canal is occluded.

Patient-Specific Targeting of the T-Cell Receptor Variable Region as a Therapeutic Strategy in Clonal T-Cell Diseases.

PURPOSE: Targeted therapeutics are a goal of medicine. Methods for targeting T-cell lymphoma lack specificity for the malignant cell, leading to elimination of healthy cells. The T-cell receptor (TCR) is designed for antigen recognition. T-cell malignancies expand from a single clone that expresses one of 48 TCR variable beta (Vß) genes, providing a distinct therapeutic target. We hypothesized that a mAb that is exclusive to a specific Vß would eliminate the malignant clone while having minimal effects on healthy T cells. EXPERIMENTAL DESIGN: We identified a patient with large granular T-cell leukemia and sequenced his circulating T-cell population, 95% of which expressed Vß13.3. We developed a panel of anti-Vß13.3 antibodies to test for binding and elimination of the malignant T-cell clone. RESULTS: Therapeutic antibody candidates bound the malignant clone with high affinity. Antibodies killed engineered cell lines expressing the patient TCR Vß13.3 by antibody-dependent cellular cytotoxicity and TCR-mediated activation-induced cell death, and exhibited specific killing of patient malignant T cells in combination with exogenous natural killer cells. EL4 cells expressing the patient's TCR Vß13.3 were also killed by antibody administration in an in vivo murine model. CONCLUSIONS: This approach serves as an outline for development of therapeutics that can treat clonal T-cell-based malignancies and potentially other T-cell-mediated diseases. See related commentary by Varma and Diefenbach, p. 4024.

A promising new tool for literacy instruction: The morphological matrix.

There is growing interest in the role that morphological knowledge plays in literacy acquisition, but there is no research directly comparing the efficacy of different forms of morphological instruction. Here we compare two methods of teaching English morphology in the context of a memory experiment when words were organized by affix during study (e.g., a list of words was presented that all share an affix, such as , , , , etc.) or by base during study (e.g., a list of words was presented that all share a base, such as , , , ). We show that memory for morphologically complex words is better in both conditions compared to a control condition that does not highlight the morphological composition of words, and most importantly, show that studying words in a base-centric format improves memory further still. We argue that the morphological matrix that organizes words around a common base may provide an important new tool for literacy instruction.

Genomic analysis of diet composition finds novel loci and associations with health and lifestyle.

We conducted genome-wide association studies (GWAS) of relative intake from the macronutrients fat, protein, carbohydrates, and sugar in over 235,000 individuals of European ancestries. We identified 21 unique, approximately independent lead SNPs. Fourteen lead SNPs are uniquely associated with one macronutrient at genome-wide significance (P < 5 × 10-8), while five of the 21 lead SNPs reach suggestive significance (P < 1 × 10-5) for at least one other macronutrient. While the phenotypes are genetically correlated, each phenotype carries a partially unique genetic architecture. Relative protein intake exhibits the strongest relationships with poor health, including positive genetic associations with obesity, type 2 diabetes, and heart disease (rg ≈ 0.15-0.5). In contrast, relative carbohydrate and sugar intake have negative genetic correlations with waist circumference, waist-hip ratio, and neighborhood deprivation (|rg| ≈ 0.1-0.3) and positive genetic correlations with physical activity (rg ≈ 0.1 and 0.2). Relative fat intake has no consistent pattern of genetic correlations with poor health but has a negative genetic correlation with educational attainment (rg ≈-0.1). Although our analyses do not allow us to draw causal conclusions, we find no evidence of negative health consequences associated with relative carbohydrate, sugar, or fat intake. However, our results are consistent with the hypothesis that relative protein intake plays a role in the etiology of metabolic dysfunction.

Chronic myeloid leukemia stem cells require cell-autonomous pleiotrophin signaling.

Tyrosine kinase inhibitors (TKIs) induce molecular remission in the majority of patients with chronic myelogenous leukemia (CML), but the persistence of CML stem cells hinders cure and necessitates indefinite TKI therapy. We report that CML stem cells upregulate the expression of pleiotrophin (PTN) and require cell-autonomous PTN signaling for CML pathogenesis in BCR/ABL+ mice. Constitutive PTN deletion substantially reduced the numbers of CML stem cells capable of initiating CML in vivo. Hematopoietic cell-specific deletion of PTN suppressed CML development in BCR/ABL+ mice, suggesting that cell-autonomous PTN signaling was necessary for CML disease evolution. Mechanistically, PTN promoted CML stem cell survival and TKI resistance via induction of Jun and the unfolded protein response. Human CML cells were also dependent on cell-autonomous PTN signaling, and anti-PTN antibody suppressed human CML colony formation and CML repopulation in vivo. Our results suggest that targeted inhibition of PTN has therapeutic potential to eradicate CML stem cells.

A lumped-element model of the chinchilla middle ear.

An air-conduction circuit model was developed for the chinchilla middle ear and cochlea. The lumped-element model is based on the classic Zwislocki model of the same structures in human. Model parameters were fit to various measurements of chinchilla middle-ear transfer functions and impedances, using a combination of error-minimization-driven computer-automated and manual fitting methods. The measurements used to fit the model comprise a newer, more-extensive data set than previously used, and include measurements of stapes velocity and inner-ear sound pressure within the vestibule and the scala tympani near the round window. The model is in agreement with studies of the effects of middle-ear cavity holes in experiments that require access to the middle-ear air space. The structure of the model allows easy addition of other sources of auditory stimulation, e.g., the multiple sources of bone-conducted sound-the long-term goal for the model's development-and mechanical stimulation of the ossicles and round window.

The Use of Somatic Hypermutation for the Affinity Maturation of Therapeutic Antibodies.

The engineering of antibodies and antibody fragments for affinity maturation, stability, and other biophysical characteristics is a common aspect of therapeutic development. Maturation of antibodies in B cells during the adaptive immune response is the result of a process called somatic hypermutation (SHM), in which the activation-induced cytidine deaminase (AID) acts to introduce mutations into immunoglobulin (Ig) genes. Iterative selection and clonal expansion of B cells containing affinity-enhancing mutations drive an increase in the overall affinity of antibodies. Here we describe the use of SHM coupled with mammalian cell surface display for the maturation of antibodies in vitro and the complementarity of these methods with the mining of immune lineages using next-generation sequencing (NGS).

Gene discovery and polygenic prediction from a genome-wide association study of educational attainment in 1.1 million individuals.

Here we conducted a large-scale genetic association analysis of educational attainment in a sample of approximately 1.1 million individuals and identify 1,271 independent genome-wide-significant SNPs. For the SNPs taken together, we found evidence of heterogeneous effects across environments. The SNPs implicate genes involved in brain-development processes and neuron-to-neuron communication. In a separate analysis of the X chromosome, we identify 10 independent genome-wide-significant SNPs and estimate a SNP heritability of around 0.3% in both men and women, consistent with partial dosage compensation. A joint (multi-phenotype) analysis of educational attainment and three related cognitive phenotypes generates polygenic scores that explain 11-13% of the variance in educational attainment and 7-10% of the variance in cognitive performance. This prediction accuracy substantially increases the utility of polygenic scores as tools in research.

The importance of correctly characterising the English spelling system when devising and evaluating methods of reading instruction: Comment on Taylor, Davis, and Rastle (2017).

Taylor, Davis, and Rastle employed an artificial language learning paradigm to compare phonics and meaning-based approaches to reading instruction. Adults were taught consonant, vowel, and consonant (CVC) words composed of novel letters when the mappings between letters and sounds were completely systematic and the mappings between letters and meaning were completely arbitrary. At test, performance on naming tasks was better following training that emphasised the phonological rather than the semantic mappings, whereas performance on semantic tasks was similar in the two conditions. The authors concluded that these findings support phonics for early reading instruction in English. However, in our view, these conclusions are not justified given that the artificial language mischaracterised both the phonological and semantic mappings in English. Furthermore, the way participants studied the arbitrary letter-meaning correspondences bears little relation to meaning-based strategies used in schools. To compare phonics with meaning-based instruction it must be determined whether phonics is better than alternative forms of instruction that fully exploit the regularities within the semantic route. This is rarely assessed because of a widespread and mistaken assumption that underpins so much basic and applied research, namely, that the main function of spellings is to represent sounds.

Limits on normal cochlear 'third' windows provided by previous investigations of additional sound paths into and out of the cat inner ear.

While most models of cochlear function assume the presence of only two windows into the mammalian cochlea (the oval and round windows), a position that is generally supported by several lines of data, there is evidence for additional sound paths into and out of the inner ear in normal mammals. In this report we review the existing evidence for and against the 'two-window' hypothesis. We then determine how existing data and inner-ear anatomy restrict transmission of sound through these additional sound pathways in cat by utilizing a well-tested model of the cat inner ear, together with anatomical descriptions of the cat cochlear and vestibular aqueducts (potential additional windows to the cochlea). We conclude: (1) The existing data place limits on the size of the cochlear and vestibular aqueducts in cat and are consistent with small volume-velocities through these ducts during ossicular stimulation of the cochlea, (2) the predicted volume velocities produced by aqueducts with diameters half the size of the bony diameters match the functional data within ±10 dB, and (3) these additional volume velocity paths contribute to the inner ear's response to non-acoustic stimulation and conductive pathology.

Energy-efficient waveform for electrical stimulation of the cochlear nerve.

The cochlear implant (CI) is the most successful neural prosthesis, restoring the sensation of sound in people with severe-to-profound hearing loss by electrically stimulating the cochlear nerve. Existing CIs have an external, visible unit, and an internal, surgically-placed unit. There are significant challenges associated with the external unit, as it has limited utility and CI users often report a social stigma associated with prosthesis visibility. A fully-implantable CI (FICI) would address these issues. However, the volume constraint imposed on the FICI requires less power consumption compared to today's CI. Because neural stimulation by CI electrodes accounts for up to 90% of power consumption, reduction in stimulation power will result directly in CI power savings. To determine an energy-efficient waveform for cochlear nerve stimulation, we used a genetic algorithm approach, incorporating a computational model of a single mammalian myelinated cochlear nerve fiber coupled to a stimulator-electrode-tissue interface. The algorithm's prediction was tested in vivo in human CI subjects. We find that implementation of a non-rectangular biphasic neural stimulation waveform may result in up to 25% charge savings and energy savings within the comfortable range of hearing for CI users. The alternative waveform may enable future development of a FICI.

Middle-ear and inner-ear contribution to bone conduction in chinchilla: The development of Carhart's notch.

While the cochlea is considered the primary site of the auditory response to bone conduction (BC) stimulation, the paths by which vibratory energy applied to the skull (or other structures) reaches the inner ear are a matter of continued investigation. We present acoustical measurements of sound in the inner ear that separate out the components of BC stimulation that excite the inner ear via ossicular motion (compression of the walls of the ear canal or ossicular inertia) from the components that act directly on the cochlea (cochlear compression or inertia, and extra-cochlear 'third-window' pathways). The results are consistent with our earlier suggestion that the inner-ear mechanisms play a large role in bone-conduction stimulation in the chinchilla at all frequencies. However, the data also suggest the pathways that conduct vibration to the inner ear via ossicular-motion make a significant contribution to the response to BC stimulation in the 1-3 kHz range, such that interruption of these path leads to a 5 dB reduction in total stimulation in that frequency range. The mid-frequency reduction produced by ossicular manipulations is similar to the 'Carhart's notch' phenomenon observed in otology and audiology clinics in cases of human ossicular disorders. We also present data consistent with much of the ossicular-conducted sound in chinchilla depending on occlusion of the ear canal.

A Randomized, Open-Label, Multicenter, Phase III Study of Epoetin Alfa Versus Best Standard of Care in Anemic Patients With Metastatic Breast Cancer Receiving Standard Chemotherapy.

PURPOSE: An open-label, noninferiority study to evaluate the impact of epoetin alfa (EPO) on tumor outcomes when used to treat anemia in patients receiving chemotherapy for metastatic breast cancer. METHODS: Women with hemoglobin ≤ 11.0 g/dL, receiving first- or second-line chemotherapy for metastatic breast cancer, were randomly assigned to EPO 40,000 IU subcutaneously once a week or best standard of care. The primary end point was progression-free survival (PFS). Secondary end points included overall survival, time to tumor progression, overall response rate, RBC transfusions, and thrombotic vascular events. RESULTS: In 2,098 patients randomly assigned, median PFS (based on investigator-determined disease progression [PD]) was 7.4 months in both groups (hazard ratio [HR], 1.089; 95% CI, 0.988 to 1.200); upper bound exceeded prespecified noninferiority margin of 1.15. Median PFS per independent review committee-determined PD was 7.6 months in both groups (HR, 1.028; 95% CI, 0.922 to 1.146); upper bound did not exceed prespecified noninferiority margin. Median overall survival at clinical cutoff (1,337 deaths) was 17.2 months in the EPO and 17.4 months in the best standard of care group (HR, 1.057; 95% CI, 0.949 to 1.177), median time to tumor progression was 7.5 months in both groups (HR, 1.094; 95% CI, 0.991 to 1.209), and overall response rate was 50% versus 51% (odds ratio, 0.950; 95% CI, 0.799 to 1.130). RBC transfusions were 5.8% versus 11.4% (P < .001), and thrombotic vascular events were 2.8% versus 1.4% (P = .038), respectively. CONCLUSION: The primary end point, PFS based on investigator-determined PD, did not meet noninferiority criteria. As a consistency assessment with the primary finding, PFS based on independent review committee-determined PD met noninferiority criteria. Overall, this study did not achieve noninferiority objective in ruling out a 15% increased risk in PD/death. RBC transfusion should be the preferred approach for the management of anemia in this population.

A general approach to antibody thermostabilization.

Antibody engineering to enhance thermostability may enable further application and ease of use of antibodies across a number of different areas. A modified human IgG framework has been developed through a combination of engineering approaches, which can be used to stabilize antibodies of diverse specificity. This is achieved through a combination of complementarity-determining region (CDR)-grafting onto the stable framework, mammalian cell display and in vitro somatic hypermutation (SHM). This approach allows both stabilization and maturation to affinities beyond those of the original antibody, as shown by the stabilization of an anti-HA33 antibody by approximately 10°C and affinity maturation of approximately 300-fold over the original antibody. Specificities of 10 antibodies of diverse origin were successfully transferred to the stable framework through CDR-grafting, with 8 of these successfully stabilized, including the therapeutic antibodies adalimumab, stabilized by 9.9°C, denosumab, stabilized by 7°C, cetuximab stabilized by 6.9°C and to a lesser extent trastuzumab stabilized by 0.8°C. This data suggests that this approach may be broadly useful for improving the biophysical characteristics of antibodies across a number of applications.

Nucleotide insertions and deletions complement point mutations to massively expand the diversity created by somatic hypermutation of antibodies.

During somatic hypermutation (SHM), deamination of cytidine by activation-induced cytidine deaminase and subsequent DNA repair generates mutations within immunoglobulin V-regions. Nucleotide insertions and deletions (indels) have recently been shown to be critical for the evolution of antibody binding. Affinity maturation of 53 antibodies using in vitro SHM in a non-B cell context was compared with mutation patterns observed for SHM in vivo. The origin and frequency of indels seen during in vitro maturation were similar to that in vivo. Indels are localized to CDRs, and secondary mutations within insertions further optimize antigen binding. Structural determination of an antibody matured in vitro and comparison with human-derived antibodies containing insertions reveal conserved patterns of antibody maturation. These findings indicate that activation-induced cytidine deaminase acting on V-region sequences is sufficient to initiate authentic formation of indels in vitro and in vivo and that point mutations, indel formation, and clonal selection form a robust tripartite system for antibody evolution.

Mammalian cell display for the discovery and optimization of antibody therapeutics.

Recent advances are described for the isolation and affinity maturation of antibodies that couple in vitro somatic hypermutation (SHM) with mammalian cell display, replicating key aspects of the adaptive immune system. SHM is dependent on the action of the B cell specific enzyme, activation-induced cytidine deaminase (AID). AID-directed SHM in vitro in non-B cells, combined with mammalian display of a library of human antibodies, initially naïve to SHM, can be used to isolate and affinity mature antibodies via iterative cycles of fluorescence-activated cell sorting (FACS) under increasingly stringent sort conditions. SHM observed in vitro closely resembles SHM observed in human antibodies in vivo in both mutation type and positioning in the antibody variable region. In addition, existing antibodies originating from mouse immunization, in vivo based libraries, or alternative display technologies such as phage can also be affinity matured in a similar manner. The display system has been developed to enable simultaneous high-level cell surface expression and secretion of the same protein through alternate splicing, where the displayed protein phenotype remains linked to genotype, allowing soluble secreted antibody to be simultaneously characterized in biophysical and cell-based functional assays. This approach overcomes many of the previous limitations of mammalian cell display, enabling direct selection and maturation of antibodies as full-length, glycosylated IgGs.

Mammalian cell display and somatic hypermutation in vitro for human antibody discovery.

Human therapeutic antibody discovery has utilized a variety of systems, from in vivo immunization of human immunoglobulin-expressing mice, to in vitro display of antibody libraries. Of the in vitro antibody display technologies, mammalian cell display provides a number of advantages with the ability to co-select immunoglobulin molecules for high expression level in mammalian cells, native folding, and biophysical properties appropriate for drug development. Mammalian cell display has been achieved using either transient or stable expression systems, using a number of alternate transmembrane domains to present antibody on the cell surface. The unique capability of mammalian cells to present IgG in its fully post-translationally modified format also allows selection of antibodies for functional properties. One limitation of mammalian cell based systems, however, has been the smaller library size that can be presented compared to phage display approaches. Until recently, this has necessitated the use of libraries biased toward a particular antigen, such as libraries derived from immunized donors, to achieve success. An alternative approach has now been developed which recapitulates key aspects of the in vivo immune system through reproducing somatic hypermutation (SHM) in vitro. Libraries representing a naïve human B lymphocyte antibody repertoire are created by PCR amplification of the rearranged (D)J segments of heavy and light chain variable regions from human donors and incorporating the resulting sequence diversity into panels of human germline VH and VL genes. The resulting antibodies are presented as full length IgG on the surface of HEK293 cells. After isolation of antibodies binding to individual target antigens, subsequent affinity maturation using in vitro SHM is induced by expression of activation-induced cytidine deaminase (AID). Selection of antibodies from naïve fully human libraries using mammalian cell display coupled with in vitro SHM is an efficient methodology for the generation of high affinity human antibodies with excellent properties for drug development.

Simultaneous surface display and secretion of proteins from mammalian cells facilitate efficient in vitro selection and maturation of antibodies.

A mammalian expression system has been developed that permits simultaneous cell surface display and secretion of the same protein through alternate splicing of pre-mRNA. This enables a flexible system for in vitro protein evolution in mammalian cells where the displayed protein phenotype remains linked to genotype, but with the advantage of soluble protein also being produced without the requirement for any further recloning to allow a wide range of assays, including biophysical and cell-based functional assays, to be used during the selection process. This system has been used for the simultaneous surface presentation and secretion of IgG during antibody discovery and maturation. Presentation and secretion of monomeric Fab can also be achieved to minimize avidity effects. Manipulation of the splice donor site sequence enables control of the relative amounts of cell surface and secreted antibody. Multi-domain proteins may be presented and secreted in different formats to enable flexibility in experimental design, and secreted proteins may be produced with epitope tags to facilitate high-throughput testing. This system is particularly useful in the context of in situ mutagenesis, as in the case of in vitro somatic hypermutation.

Humanization of antibodies using heavy chain complementarity-determining region 3 grafting coupled with in vitro somatic hypermutation.

A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine hßNGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with a CDR-grafted light chain, displayed on the surface of HEK293 cells, and matured using in vitro somatic hypermutation. A high affinity humanized antibody was derived that was considerably more potent than the parental antibody, possessed a low pm dissociation constant, and demonstrated potent inhibition of hßNGF activity in vitro. The resulting antibody contained half the heavy chain murine donor sequence compared with the same antibody humanized using traditional methods.