Hyperferritinemia Screening to Aid Identification and Differentiation of Patients with Hyperinflammatory Disorders.

High ferritin is an important and sensitive biomarker for the various forms of hemophagocytic lymphohistiocytosis (HLH), a diverse and deadly group of cytokine storm syndromes. Early action to prevent immunopathology in HLH often includes empiric immunomodulation, which can complicate etiologic work-up and prevent collection of early/pre-treatment research samples. To address this, we instituted an alert system at UPMC Children's Hospital where serum ferritin > 1000 ng/mL triggered real-time chart review, assessment of whether the value reflected "inflammatory hyperferritnemia (IHF)", and biobanking of remnant samples from consenting IHF patients. We extracted relevant clinical data; periodically measured serum total IL-18, IL-18 binding protein (IL-18BP), and CXCL9; retrospectively classified patients by etiology into infectious, rheumatic, or immune dysregulation; and subjected a subgroup of samples to a 96-analyte biomarker screen. 180 patients were identified, 30.5% of which had IHF. Maximum ferritin levels were significantly higher in patients with IHF than with either hemoglobinopathy or transplant, and highly elevated total IL-18 levels were distinctive to patients with Stills Disease and/or Macrophage Activation Syndrome (MAS). Multi-analyte analysis showed elevation in proteins associated with cytotoxic lymphocytes in all IHF samples when compared to healthy controls and depression of proteins such as ANGPT1 and VEGFR2 in samples from hyperferritinemic sepsis patients relative to non-sepsis controls. This real-time IFH screen proved feasible and efficient, validated prior observations about the specificity of IL-18, enabled early sample collection from a complex population, suggested a unique vascular biomarker signature in hyperferritinemic sepsis, and expanded our understanding of IHF heterogeneity.

Impact of Genetic Ancestry on T-cell Acute Lymphoblastic Leukemia Outcomes.

The influence of genetic ancestry on biology, survival outcomes, and risk stratification in T-cell Acute Lymphoblastic Leukemia (T-ALL) has not been explored. Genetic ancestry was genomically-derived from DNA-based single nucleotide polymorphisms in children and young adults with T-ALL treated on Children's Oncology Group trial AALL0434. We determined associations of genetic ancestry, leukemia genomics and survival outcomes; co-primary outcomes were genomic subtype, pathway alteration, overall survival (OS), and event-free survival (EFS). Among 1309 patients, T-ALL molecular subtypes varied significantly by genetic ancestry, including increased frequency of genomically defined ETP-like, MLLT10, and BCL11B-activated subtypes in patients of African ancestry. In multivariable Cox models adjusting for high-risk subtype and pathways, patients of Admixed American ancestry had superior 5-year EFS/OS compared with European; EFS/OS for patients of African and European ancestry were similar. The prognostic value of five commonly altered T-ALL genes varied by ancestry - including NOTCH1 , which was associated with superior OS for patients of European and Admixed American ancestry but non-prognostic among patients of African ancestry. Furthermore, a published five-gene risk classifier accurately risk stratified patients of European ancestry, but misclassified patients of African ancestry. We developed a penalized Cox model which successfully risk stratified patients across ancestries. Overall, 80% of patients had a genomic alteration in at least one gene with differential prognostic impact by genetic ancestry. T-ALL genomics and prognostic associations of genomic alterations vary by genetic ancestry. These data demonstrate the importance of incorporating genetic ancestry into analyses of tumor biology for risk classification algorithms.

Hyperferritinemia screening to aid identification and differentiation of patients with hyperinflammatory disorders.

High ferritin is an important and sensitive biomarker for hemophagocytic lymphohistiocytosis (HLH), a diverse and deadly group of cytokine storm syndromes. Early action to prevent immunopathology in HLH often includes empiric immunomodulation, which can complicate etiologic work-up and prevent collection of early/pre-treatment research samples. To address this, we instituted an alert system where serum ferritin > 1000ng/mL triggered real-time chart review, assessment of whether the value reflected "inflammatory hyperferritnemia (IHF)", and biobanking of remnant samples from consenting IHF patients. We extracted relevant clinical data; periodically measured serum total IL-18, IL-18 binding protein (IL-18BP), and CXCL9; retrospectively classified patients by etiology into infectious, rheumatic, or immune dysregulation; and subjected a subgroup of samples to a 96-analyte biomarker screen. 180 patients were identified, 30.5% of which had IHF. Maximum ferritin levels were significantly higher in patients with IHF than with either hemoglobinopathy or transplant, and highly elevated total IL-18 levels were distinctive to patients with Stills Disease and/or Macrophage Activation Syndrome (MAS). Multi-analyte analysis showed elevation in proteins associated with cytotoxic lymphocytes in all IHF samples when compared to healthy controls and depression of proteins such as ANGPT1 and VEGFR2 in samples from hyperferritinemic sepsis patients relative to non-sepsis controls. This single-center, real-time IFH screen proved feasible and efficient, validated prior observations about the specificity of IL-18, enabled early sample collection from a complex population, suggested a unique vascular biomarker signature in hyperferritinemic sepsis, and expanded our understanding of IHF heterogeneity.

Quadriparesis and paraparesis following chimeric antigen receptor T-cell therapy in children and adolescents.

ABSTRACT: Immune effector cell-associated neurotoxicity syndrome (ICANS) is a common but potentially severe adverse event associated with chimeric antigen receptor T-cell (CART) therapy, characterized by the development of acute neurologic symptoms following CART infusion. ICANS encompasses a wide clinical spectrum typified by mild to severe encephalopathy, seizures, and/or cerebral edema. As more patients have been treated with CART, new ICANS phenomenology has emerged. We present the clinical course of 5 children who developed acute onset of quadriparesis or paraparesis associated with abnormal brain and/or spine neuroimaging after infusion of CD19- or CD22-directed CART, adverse events not previously reported in children. Orthogonal data from autopsy studies, cerebrospinal fluid (CSF) flow cytometry, and CSF proteomics/cytokine profiling demonstrated chronic white matter destruction, but a notable lack of inflammatory pathologic changes and cell populations. Instead, children with quadriparesis or paraparesis post-CART therapy had lower levels of proinflammatory cytokines, such as interferon gamma, CCL17, CCL23, and CXCL10, than those who did not develop quadriparesis or paraparesis. Taken together, these findings imply a noninflammatory source of this newly described ICANS phenomenon in children. The pathophysiology of some neurologic symptoms following CART may therefore have a more complex etiology than exclusive T-cell activation and excessive cytokine production.

Poly (ADP-ribose) polymerase-1 regulates HIV-1 replication in human CD4+ T cells.

The cellular enzyme poly (ADP-ribose) polymerase-1 (PARP-1) regulates multiple processes that are potentially implicated in HIV-1 infection. However, the role of PARP-1 in HIV-1 infection remains controversial, with reports indicating or excluding that PARP-1 influence early steps of the HIV-1 life cycle. Most of these studies have been conducted with Vesicular Stomatitis virus Glycoprotein G (VSV-G)-pseudotyped, single-round infection HIV-1; limiting our understanding of the role of PARP-1 in HIV-1 replication. Therefore, we evaluated the effect of PARP-1 deficiency or inhibition in HIV-1 replication in human CD4+ T cells. Our data showed that PARP-1 knockout increased viral replication in SUP-T1 cells. Similarly, a PARP-1 inhibitor that targets PARP-1 DNA-binding activity enhanced HIV-1 replication. In contrast, inhibitors affecting the catalytic activity of the enzyme were inactive. In correspondence with the pharmacological studies, mutagenesis analysis indicated that the DNA-binding domain was required for the PARP-1 anti-HIV-1 activity, but the poly-ADP-ribosylation activity was dispensable. Our results also demonstrated that PARP-1 acts at the production phase of the viral life cycle since HIV-1 produced in cells lacking PARP-1 was more infectious than control viruses. The effect of PARP-1 on HIV-1 infectivity required Env, as PARP-1 deficiency or inhibition did not modify the infectivity of Env-deleted, VSV-G-pseudotyped HIV-1. Furthermore, virion-associated Env was more abundant in sucrose cushion-purified virions produced in cells lacking the enzyme. However, PARP-1 did not affect Env expression or processing in the producer cells. In summary, our data indicate that PARP-1 antagonism enhances HIV-1 infectivity and increases levels of virion-associated Env. Importance: Different cellular processes counteract viral replication. A better understanding of these interfering mechanisms will enhance our ability to control viral infections. We have discovered a novel, antagonist effect of the cellular enzyme poly (ADP-ribose) polymerase-1 (PARP-1) in HIV-1 replication. Our data indicate that PARP-1 deficiency or inhibition augment HIV-1 infectivity in human CD4+ T cells, the main HIV-1 target cell in vivo . Analysis of the mechanism of action suggested that PARP-1 antagonism increases in the virus the amounts of the viral protein mediating viral entry to the target cells. These findings identify for the first time PARP-1 as a host factor that regulates HIV-1 infectivity, and could be relevant to better understand HIV-1 transmission and to facilitate vaccine development.

Mapping the cellular biogeography of human bone marrow niches using single-cell transcriptomics and proteomic imaging.

Non-hematopoietic cells are essential contributors to hematopoiesis. However, heterogeneity and spatial organization of these cells in human bone marrow remain largely uncharacterized. We used single-cell RNA sequencing (scRNA-seq) to profile 29,325 non-hematopoietic cells and discovered nine transcriptionally distinct subtypes. We simultaneously profiled 53,417 hematopoietic cells and predicted their interactions with non-hematopoietic subsets. We employed co-detection by indexing (CODEX) to spatially profile over 1.2 million cells. We integrated scRNA-seq and CODEX data to link predicted cellular signaling with spatial proximity. Our analysis revealed a hyperoxygenated arterio-endosteal neighborhood for early myelopoiesis, and an adipocytic localization for early hematopoietic stem and progenitor cells (HSPCs). We used our CODEX atlas to annotate new images and uncovered mesenchymal stromal cell (MSC) expansion and spatial neighborhoods co-enriched for leukemic blasts and MSCs in acute myeloid leukemia (AML) patient samples. This spatially resolved, multiomic atlas of human bone marrow provides a reference for investigation of cellular interactions that drive hematopoiesis.

TRILL: ORCHESTRATING MODULAR DEEP-LEARNING WORKFLOWS FOR DEMOCRATIZED, SCALABLE PROTEIN ANALYSIS AND ENGINEERING.

Deep-learning models have been rapidly adopted by many fields, partly due to the deluge of data humanity has amassed. In particular, the petabases of biological sequencing data enable the unsupervised training of protein language models that learn the "language of life." However, due to their prohibitive size and complexity, contemporary deep-learning models are often unwieldy, especially for scientists with limited machine learning backgrounds. TRILL (TRaining and Inference using the Language of Life) is a platform for creative protein design and discovery. Leveraging several state-of-the-art models such as ESM-2, DiffDock, and RFDiffusion, TRILL allows researchers to generate novel proteins, predict 3-D structures, extract high-dimensional representations of proteins, functionally classify proteins and more. What sets TRILL apart is its ability to enable complex pipelines by chaining together models and effectively merging the capabilities of different models to achieve a sum greater than its individual parts. Whether using Google Colab with one GPU or a supercomputer with hundreds, TRILL allows scientists to effectively utilize models with millions to billions of parameters by using optimized training strategies such as ZeRO-Offload and distributed data parallel. Therefore, TRILL not only bridges the gap between complex deep-learning models and their practical application in the field of biology, but also simplifies the orchestration of these models into comprehensive workflows, democratizing access to powerful methods. Documentation: https://trill.readthedocs.io/en/latest/home.html.

Correction: Magnetic coupling between Fe(NO) spin probe ligands through diamagnetic NiII, PdII and PtII tetrathiolate bridges.

[This corrects the article DOI: 10.1039/D3SC01546G.].

Evaluation of strategies to modify Anti-SARS-CoV-2 monoclonal antibodies for optimal functionality as therapeutics.

The current global COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a public health crisis with more than 168 million cases reported globally and more than 4.5 million deaths at the time of writing. In addition to the direct impact of the disease, the economic impact has been significant as public health measures to contain or reduce the spread have led to country wide lockdowns resulting in near closure of many sectors of the economy. Antibodies are a principal determinant of the humoral immune response to COVID-19 infections and may have the potential to reduce disease and spread of the virus. The development of monoclonal antibodies (mAbs) represents a therapeutic option that can be produced at large quantity and high quality. In the present study, a mAb combination mixture therapy was investigated for its capability to specifically neutralize SARS-CoV-2. We demonstrate that each of the antibodies bind the spike protein and neutralize the virus, preventing it from infecting cells in an in vitro cell-based assay, including multiple viral variants that are currently circulating in the human population. In addition, we investigated the effects of two different mutations in the Fc portion (YTE and LALA) of the antibody on Fc effector function and the ability to alleviate potential antibody-dependent enhancement of disease. These data demonstrate the potential of a combination of two mAbs that target two different epitopes on the SARS-CoV2 spike protein to provide protection against SARS-CoV-2 infection in humans while extending serum half-life and preventing antibody-dependent enhancement of disease.

Organizing the Global Diversity of Microviruses.

Microviruses encompass an astonishing array of small, single-stranded DNA phages that, due to the surge in metagenomic surveys, are now known to be prevalent in most environments. Current taxonomy concedes the considerable diversity within this lineage to a single family (the Microviridae), which has rendered it difficult to adequately and accurately assess the amount of variation that actually exists within this group. We amassed and curated the largest collection of microviral genomes to date and, through a combination of protein-sharing networks and phylogenetic analysis, discovered at least three meaningful taxonomic levels between the current ranks of family and genus. When considering more than 13,000 microviral genomes from recognized lineages and as-yet-unclassified microviruses in metagenomic samples, microviral diversity is better understood by elevating microviruses to the level of an order that consists of three suborders and at least 19 putative families, each with their respective subfamilies. These revisions enable fine-scale assessment of microviral dynamics: for example, in the human gut, there are considerable differences in the abundances of microviral families both between urban and rural populations and in individuals over time. In addition, our analysis of genome contents and gene exchange shows that microviral families carry no recognizable accessory metabolic genes and rarely, if ever, engage in horizontal gene transfer across microviral families or with their bacterial hosts. These insights bring microviral taxonomy in line with current developments in the taxonomy of other phages and increase the understanding of microvirus biology. IMPORTANCE Microviruses are the most abundant single-stranded DNA phages on the planet and an important component of the human gut virome. And yet, productive research into their biology is hampered by the inadequacies of current taxonomic ordering: microviruses are lumped into a single family and treated as a monolithic group, thereby obscuring the extent of their diversity and resulting in little comparative research. Our investigations into the diversity of microviruses define numerous groups, most lacking any isolated representatives, and point toward high-value targets for future research. To expedite microvirus discovery and comparison, we developed a pipeline that enables the fast and facile sorting of novel microvirus genomes into well-defined taxonomic groups. These improvements provide new insights into the biology of microviruses and emphasize fundamental differences between these miniature phages and their large, double-stranded DNA phage competitors.