Distinct antibody responses to SARS-CoV-2 in children and adults across the COVID-19 clinical spectrum.

Clinical manifestations of COVID-19 caused by the new coronavirus SARS-CoV-2 are associated with age1,2. Adults develop respiratory symptoms, which can progress to acute respiratory distress syndrome (ARDS) in the most severe form, while children are largely spared from respiratory illness but can develop a life-threatening multisystem inflammatory syndrome (MIS-C)3-5. Here, we show distinct antibody responses in children and adults after SARS-CoV-2 infection. Adult COVID-19 cohorts had anti-spike (S) IgG, IgM and IgA antibodies, as well as anti-nucleocapsid (N) IgG antibody, while children with and without MIS-C had reduced breadth of anti-SARS-CoV-2-specific antibodies, predominantly generating IgG antibodies specific for the S protein but not the N protein. Moreover, children with and without MIS-C had reduced neutralizing activity as compared to both adult COVID-19 cohorts, indicating a reduced protective serological response. These results suggest a distinct infection course and immune response in children independent of whether they develop MIS-C, with implications for developing age-targeted strategies for testing and protecting the population.

Acute mechanical hemolysis as a complication of extracorporeal photopheresis in a low-weight child.

Graft-versus-host disease (GVHD) is a complication of allogeneic hematopoietic stem cell transplantation with high morbidity and mortality. Extracorporeal photopheresis (ECP) is an effective therapy for treating medication-refractory GVHD, however, there is scant evidence of whether ECP can be safely performed in patients weighing less than 15 kg. Here, we report the implementation of a successful protocol to perform ECP in a 21-month-old, 10.6 kg female with medication-refractory GVHD. Our initial ECP treatment resulted in significant hemolysis that was most likely mechanical. After procedural adjustments that included modifying the anticoagulation dose and whole blood:anticoagulant ratio, as well as whole blood processing time, the patient tolerated future procedures safely without hemolysis or other adverse events. With appropriate technical modifications, we provide a framework for safely performing ECP in children less than 15 kg.

Linker histone H1 is essential for Drosophila development, the establishment of pericentric heterochromatin, and a normal polytene chromosome structure.

We generated mutant alleles of Drosophila melanogaster in which expression of the linker histone H1 can be down-regulated over a wide range by RNAi. When the H1 protein level is reduced to approximately 20% of the level in wild-type larvae, lethality occurs in the late larval - pupal stages of development. Here we show that H1 has an important function in gene regulation within or near heterochromatin. It is a strong dominant suppressor of position effect variegation (PEV). Similar to other suppressors of PEV, H1 is simultaneously involved in both the repression of euchromatic genes brought to the vicinity of pericentric heterochromatin and the activation of heterochromatic genes that depend on their pericentric localization for maximal transcriptional activity. Studies of H1-depleted salivary gland polytene chromosomes show that H1 participates in several fundamental aspects of chromosome structure and function. First, H1 is required for heterochromatin structural integrity and the deposition or maintenance of major pericentric heterochromatin-associated histone marks, including H3K9Me(2) and H4K20Me(2). Second, H1 also plays an unexpected role in the alignment of endoreplicated sister chromatids. Finally, H1 is essential for organization of pericentric regions of all polytene chromosomes into a single chromocenter. Thus, linker histone H1 is essential in Drosophila and plays a fundamental role in the architecture and activity of chromosomes in vivo.

A core erythroid transcriptional network is repressed by a master regulator of myelo-lymphoid differentiation.

Two mechanisms that play important roles in cell fate decisions are control of a "core transcriptional network" and repression of alternative transcriptional programs by antagonizing transcription factors. Whether these two mechanisms operate together is not known. Here we report that GATA-1, SCL, and Klf1 form an erythroid core transcriptional network by co-occupying >300 genes. Importantly, we find that PU.1, a negative regulator of terminal erythroid differentiation, is a highly integrated component of this network. GATA-1, SCL, and Klf1 act to promote, whereas PU.1 represses expression of many of the core network genes. PU.1 also represses the genes encoding GATA-1, SCL, Klf1, and important GATA-1 cofactors. Conversely, in addition to repressing PU.1 expression, GATA-1 also binds to and represses >100 PU.1 myelo-lymphoid gene targets in erythroid progenitors. Mathematical modeling further supports that this dual mechanism of repressing both the opposing upstream activator and its downstream targets provides a synergistic, robust mechanism for lineage specification. Taken together, these results amalgamate two key developmental principles, namely, regulation of a core transcriptional network and repression of an alternative transcriptional program, thereby enhancing our understanding of the mechanisms that establish cellular identity.

High resolution methylome analysis reveals widespread functional hypomethylation during adult human erythropoiesis.

Differentiation of hematopoietic stem cells to red cells requires coordinated expression of numerous erythroid genes and is characterized by nuclear condensation and extrusion during terminal development. To understand the regulatory mechanisms governing these widespread phenotypic changes, we conducted a high resolution methylomic and transcriptomic analysis of six major stages of human erythroid differentiation. We observed widespread epigenetic differences between early and late stages of erythropoiesis with progressive loss of methylation being the dominant change during differentiation. Gene bodies, intergenic regions, and CpG shores were preferentially demethylated during erythropoiesis. Epigenetic changes at transcription factor binding sites correlated significantly with changes in gene expression and were enriched for binding motifs for SCL, MYB, GATA, and other factors not previously implicated in erythropoiesis. Demethylation at gene promoters was associated with increased expression of genes, whereas epigenetic changes at gene bodies correlated inversely with gene expression. Important gene networks encoding erythrocyte membrane proteins, surface receptors, and heme synthesis proteins were found to be regulated by DNA methylation. Furthermore, integrative analysis enabled us to identify novel, potential regulatory areas of the genome as evident by epigenetic changes in a predicted PU.1 binding site in intron 1 of the GATA1 gene. This intronic site was found to be conserved across species and was validated to be a novel PU.1 binding site by quantitative ChIP in erythroid cells. Altogether, our study provides a comprehensive analysis of methylomic and transcriptomic changes during erythroid differentiation and demonstrates that human terminal erythropoiesis is surprisingly associated with hypomethylation of the genome.

A large gene network in immature erythroid cells is controlled by the myeloid and B cell transcriptional regulator PU.1.

PU.1 is a hematopoietic transcription factor that is required for the development of myeloid and B cells. PU.1 is also expressed in erythroid progenitors, where it blocks erythroid differentiation by binding to and inhibiting the main erythroid promoting factor, GATA-1. However, other mechanisms by which PU.1 affects the fate of erythroid progenitors have not been thoroughly explored. Here, we used ChIP-Seq analysis for PU.1 and gene expression profiling in erythroid cells to show that PU.1 regulates an extensive network of genes that constitute major pathways for controlling growth and survival of immature erythroid cells. By analyzing fetal liver erythroid progenitors from mice with low PU.1 expression, we also show that the earliest erythroid committed cells are dramatically reduced in vivo. Furthermore, we find that PU.1 also regulates many of the same genes and pathways in other blood cells, leading us to propose that PU.1 is a multifaceted factor with overlapping, as well as distinct, functions in several hematopoietic lineages.

Crosstalk between genetic and epigenetic information through cytosine deamination.

Decades of work have elucidated the existence of two forms of heritable information, namely genetic and epigenetic, which are collectively referred to as the 'dual inheritance'. The underlying mechanisms behind these two modes of inheritance have so far remained distinct. Cytosine deaminases, such as activation-induced cytidine deaminase (AID) and other members of the APOBEC family, have been implicated both in genetic variation of somatic cells and in epigenetic remodeling of germ and pluripotent cells. We hereby synthesize these seemingly dissociated functions into one coherent model, and further suggest that cytosine deaminases, particularly AID, might have a broader influence by modulating epigenetic information in somatic or cancer cells, as well as by triggering genetic variation in germ and pluripotent cells through mutation followed by natural selection. We therefore propose that the AID/APOBEC family of deaminases are likely to have acted as drivers throughout vertebrate evolution.

PU.1 directly regulates cdk6 gene expression, linking the cell proliferation and differentiation programs in erythroid cells.

Cell proliferation and differentiation are highly coordinated processes during normal development. Most leukemia cells are blocked from undergoing terminal differentiation and also exhibit uncontrolled proliferation. Dysregulated expression of transcription factor PU.1 is strongly associated with Friend virus-induced erythroleukemia. PU.1 inhibits erythroid differentiation by binding to and inhibiting GATA-1. PU.1 also may be involved in controlling proliferation of erythroid cells. We reported previously that the G(1) phase-specific cyclin-dependent kinase 6 (CDK6) also blocks erythroid differentiation. We now report that PU.1 directly stimulates transcription of the cdk6 gene in both normal erythroid progenitors and erythroleukemia cells, as well as in macrophages. We propose that PU.1 coordinates proliferation and differentiation in immature erythroid cells by inhibiting the GATA-1-mediated gene expression program and also by regulating expression of genes that control progression through the G(1) phase of the cell cycle, the period during which the decision to differentiate is made.

Approaching the Interpretation of Discordances in SARS-CoV-2 Testing.

The coronavirus disease 2019 pandemic has upended life throughout the globe. Appropriate emphasis has been placed on developing effective therapies and vaccines to curb the pandemic. While awaiting such countermeasures, mitigation efforts coupled with robust testing remain essential to controlling spread of the disease. In particular, serological testing plays a critical role in providing important diagnostic, prognostic, and therapeutic information. However, this information is only useful if the results can be accurately interpreted. This pandemic placed clinical testing laboratories and requesting physicians in a precarious position because we are actively learning about the disease and how to interpret serological results. Having developed robust assays to detect antibodies generated against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and serving the hardest-hit areas within the New York City epicenter, we found 3 types of discordances in SARS-CoV-2 test results that challenge interpretation. Using representative clinical vignettes, these interpretation dilemmas are highlighted, along with suggested approaches to resolve such cases.

A randomized double-blind controlled trial of convalescent plasma in adults with severe COVID-19.

BACKGROUNDAlthough convalescent plasma has been widely used to treat severe coronavirus disease 2019 (COVID-19), data from randomized controlled trials that support its efficacy are limited.METHODSWe conducted a randomized, double-blind, controlled trial among adults hospitalized with severe and critical COVID-19 at 5 sites in New York City (USA) and Rio de Janeiro (Brazil). Patients were randomized 2:1 to receive a single transfusion of either convalescent plasma or normal control plasma. The primary outcome was clinical status at 28 days following randomization, measured using an ordinal scale and analyzed using a proportional odds model in the intention-to-treat population.RESULTSOf 223 participants enrolled, 150 were randomized to receive convalescent plasma and 73 to receive normal control plasma. At 28 days, no significant improvement in the clinical scale was observed in participants randomized to convalescent plasma (OR 1.50, 95% confidence interval [CI] 0.83-2.68, P = 0.180). However, 28-day mortality was significantly lower in participants randomized to convalescent plasma versus control plasma (19/150 [12.6%] versus 18/73 [24.6%], OR 0.44, 95% CI 0.22-0.91, P = 0.034). The median titer of anti-SARS-CoV-2 neutralizing antibody in infused convalescent plasma units was 1:160 (IQR 1:80-1:320). In a subset of nasopharyngeal swab samples from Brazil that underwent genomic sequencing, no evidence of neutralization-escape mutants was detected.CONCLUSIONIn adults hospitalized with severe COVID-19, use of convalescent plasma was not associated with significant improvement in day 28 clinical status. However, convalescent plasma was associated with significantly improved survival. A possible explanation is that survivors remained hospitalized at their baseline clinical status.TRIAL REGISTRATIONClinicalTrials.gov, NCT04359810.FUNDINGAmazon Foundation, Skoll Foundation.

Antibody responses to SARS-CoV2 are distinct in children with MIS-C compared to adults with COVID-19.

Clinical manifestations of COVID-19 caused by the novel coronavirus SARS-CoV-2 are associated with age. While children are largely spared from severe respiratory disease, they can present with a SARS-CoV-2-associated multisystem inflammatory syndrome (MIS-C) similar to Kawasaki's disease. Here, we show distinct antibody (Ab) responses in children with MIS-C compared to adults with severe COVID-19 causing acute respiratory distress syndrome (ARDS), and those who recovered from mild disease. There was a reduced breadth and specificity of anti-SARS-CoV-2-specific antibodies in MIS-C patients compared to the COVID patient groups; MIS-C predominantly generated IgG Abs specific for the Spike (S) protein but not for the nucleocapsid (N) protein, while both COVID-19 cohorts had anti-S IgG, IgM and IgA Abs, as well as anti-N IgG Abs. Moreover, MIS-C patients had reduced neutralizing activity compared to COVID-19 cohorts, indicating a reduced protective serological response. These results suggest a distinct infection course and immune response in children and adults who develop severe disease, with implications for optimizing treatments based on symptom and age.

Diagnosis of a rare fetal haemoglobinopathy in the age of next-generation sequencing.

Neonatal cyanosis resulting from a fetal methaemoglobin variant is rare. Most such variants are only described in a few published case reports. We present the case of a newborn with unexplained persistent cyanosis, ultimately determined to have a γ-chain mutation causing Hb FM-Fort Ripley. This neonatal haemoglobinopathy can be challenging to diagnose, as significant oxygen desaturation may result from barely detectable levels of the mutant haemoglobin and co-oximetry studies may show a falsely normal methaemoglobin level. Our analysis of the infant's haemoglobin included high-performance liquid chromatography, cellulose acetate electrophoresis and citrate agar electrophoresis, which showed trace amounts of a suspected variant. Ultimately, the diagnosis was made through a novel application of next-generation sequencing (NGS). NGS-based diagnostic approaches are becoming increasingly available to clinicians, and our case provides a framework and evidence for the utilisation of such testing paradigms in the diagnosis of a rare cause of neonatal cyanosis.

Drosophila H1 regulates the genetic activity of heterochromatin by recruitment of Su(var)3-9.

Eukaryotic genomes harbor transposable elements and other repetitive sequences that must be silenced. Small RNA interference pathways play a major role in their repression. Here, we reveal another mechanism for silencing these sequences in Drosophila. Depleting the linker histone H1 in vivo leads to strong activation of these elements. H1-mediated silencing occurs in combination with the heterochromatin-specific histone H3 lysine 9 methyltransferase Su(var)3-9. H1 physically interacts with Su(var)3-9 and recruits it to chromatin in vitro, which promotes H3 methylation. We propose that H1 plays a key role in silencing by tethering Su(var)3-9 to heterochromatin. The tethering function of H1 adds to its established role as a regulator of chromatin compaction and accessibility.

The multidimensional nature of epigenetic information and its role in disease.

This year marks the 10th anniversary of the publications that reported the initial human genome sequence. In the historic press conference that announced this landmark accomplishment, it was proclaimed that the genome sequence would "revolutionize the diagnosis, prevention, and treatment of most, if not all, human diseases." However, subsequent work over the past decade has revealed that "complex diseases" are much more intricate than originally thought. Even with the advent of several new powerful technologies, our understanding of the underlying genetic etiologies of most complex and non-Mendelian diseases is far from complete. These results have raised the possibility that the DNA sequence, i.e., genetic information, may not be the only relevant source of information in order to understand the molecular basis of disease. In this review, we assemble evidence that information encoded beyond the DNA sequence, i.e., epigenetic information, may hold the key to a better understanding of various pathological conditions. Unlike the genetic information encoded within the DNA sequence, epigenetic information can be stored in multiple dimensions, such as in the form of DNA modifications, RNA, or protein. Ideas presented here support the view that to better understand the molecular etiology of diseases, we need to gain a better understanding of both the genetic and epigenetic components of biological information. We hence believe that the fast development of genome-wide technologies will facilitate a better understanding of both genetic and epigenetic dimensions of disease.

GATA-1 directly regulates p21 gene expression during erythroid differentiation.

Lineage-determination transcription factors coordinate cell differentiation and proliferation by controlling the synthesis of lineage-specific gene products as well as cell cycle regulators. GATA-1 is a master regulator of erythropoiesis. Its role in regulating erythroid-specific genes has been extensively studied, whereas its role in controlling genes that regulate cell proliferation is less understood. Ectopic expression of GATA-1 in erythroleukemia cells releases the block to their differentiation and leads to terminal cell division. An early event in reprogramming the erythroleukemia cells is induction of the cyclin-dependent kinase inhibitor p21. Remarkably, ectopic expression of p21 also induces the erythroleukemia cells to differentiate. We now report that GATA-1 directly regulates transcription of the p21 gene in both erythroleukemia cells and normal erythroid progenitors. Using reporter, electrophoretic mobility shift, and chromatin immunoprecipitation assays, we show that GATA-1 stimulates p21 gene transcription by binding to consensus binding sites in the upstream region of the p21 gene promoter. This activity is also dependent on a binding site for Sp1/KLF-like factors near the transcription start site. Our findings indicate that p21 is a crucial downstream gene target and effector of GATA-1 during red blood cell terminal differentiation.