Development of C1q Affinity Chromatography for the Study of C1q-IgG Interactions.

The classical complement system represents a central effector mechanism of Abs initiated by the binding of C1q to target bound IgG. Human C1q contains six heterotrimeric globular head groups that mediate IgG interaction, resulting in an avidity-driven binding event involving multiple IgG molecules binding a single C1q. Accordingly, surface bound IgG molecules are thought to assemble into noncovalent hexameric rings for optimal binding to the six-headed C1q. To study the C1q-Fc interaction of various Abs and screen for altered C1q binding mutants, we developed, to our knowledge, a novel HPLC-based method. Employing a single-chain form of C1q representing one C1q head group, our HPLC methodology was able to detect the interaction between the single-chain monomeric form of C1q and various ligands. We show that, despite a narrow window of specific binding owing to the low affinity of the monomeric C1q-IgG interaction, this approach clearly distinguished between IgG subclasses with established C1q binding properties. IgG3 displayed the strongest binding, followed by IgG1, with IgG2 and IgG4 showing the weakest binding. Fc mutants known to have increased C1q binding through oligomerization or enhanced C1q interaction showed greatly increased column retention, and IgG glycovariants displayed a consistent trend of increasing retention upon increasing galactosylation and sialylation. Furthermore, the column retention of IgG isotypes and glycovariants matches both the cell surface recruitment of C1q and complement-mediated cytotoxicity induced by each variant on an anti-CD20 Ab backbone. This methodology therefore provides a valuable tool for testing IgG Ab (glyco)variants for C1q binding, with clear relevance for therapeutic Ab development.

The Association Between the We Can Do This Campaign and Vaccination Beliefs in the United States, January 2021-March 2022.

Public health campaigns addressing COVID-19 vaccination beliefs may be effective in changing COVID-19 vaccination behaviors, particularly among people who remain vaccine hesitant. The "We Can Do This" COVID-19 public education campaign (the Campaign) was designed to increase COVID-19 vaccine confidence and uptake. This study aims to evaluate whether Campaign dose was associated with changes in vaccination beliefs related to COVID-19 vaccine concerns and perceived risks, the importance of COVID-19 vaccines, the perceived benefits of COVID-19 vaccination, normative beliefs about COVID-19 vaccination, and perceptions about general vaccine safety and effectiveness. The study linked data from four waves of a nationally representative longitudinal panel of U.S. adults (January 2021-March 2022) with Campaign paid digital media data (April 2021-May 2022). We used mixed-effects linear regressions to examine the association between Campaign paid digital impressions and changes in vaccination beliefs. The results provide evidence that Campaign digital impressions were significantly associated with changes in respondent beliefs regarding COVID-19 vaccine concerns and perceived risks, perceived benefits of COVID-19 vaccination, and perceptions about general vaccine safety and effectiveness. Findings suggest that public education campaigns may influence vaccine confidence and uptake by increasing positive vaccination beliefs and reducing vaccine concerns.

Entorhinal vessel density correlates with phosphorylated tau and TDP-43 pathology.

INTRODUCTION: The entorhinal cortex (EC) and perirhinal cortex (PC) are vulnerable to Alzheimer's disease. A triggering factor may be the interaction of vascular dysfunction and tau pathology. METHODS: We imaged post mortem human tissue at 100 µm3 with 7 T magnetic resonance imaging and manually labeled individual blood vessels (mean = 270 slices/case). Vessel density was quantified and compared per EC subfield, between EC and PC, and in relation to tau and TAR DNA-binding protein 43 (TDP-43) semiquantitative scores. RESULTS: PC was more vascularized than EC and vessel densities were higher in posterior EC subfields. Tau and TDP-43 strongly correlated with vasculature density and subregions with severe tau at the preclinical stage had significantly greater vessel density than those with low tau burden. DISCUSSION: These data impact cerebrovascular maps, quantification of subfield vasculature, and correlation of vasculature and pathology at early stages. The ordered association of vessel density, and tau or TDP-43 pathology, may be exploited in a predictive context. HIGHLIGHTS: Vessel density correlates with phosphorylated tau (p-tau) burden in entorhinal and perirhinal cortices. Perirhinal area 35 and posterior entorhinal cortex showed greatest p-tau burden but also the highest vessel density in the preclinical phase of Alzheimer's disease. We combined an ex vivo magnetic resonance imaging model and histopathology to demonstrate the 3D reconstruction of intracortical vessels and its spatial relationship to the pathology.

Vasculopathy and Increased Vascular Congestion in Fatal COVID-19 and Acute Respiratory Distress Syndrome.

Rationale: The leading cause of death in coronavirus disease 2019 (COVID-19) is severe pneumonia, with many patients developing acute respiratory distress syndrome (ARDS) and diffuse alveolar damage (DAD). Whether DAD in fatal COVID-19 is distinct from other causes of DAD remains unknown. Objective: To compare lung parenchymal and vascular alterations between patients with fatal COVID-19 pneumonia and other DAD-causing etiologies using a multidimensional approach. Methods: This autopsy cohort consisted of consecutive patients with COVID-19 pneumonia (n = 20) and with respiratory failure and histologic DAD (n = 21; non-COVID-19 viral and nonviral etiologies). Premortem chest computed tomography (CT) scans were evaluated for vascular changes. Postmortem lung tissues were compared using histopathological and computational analyses. Machine-learning-derived morphometric analysis of the microvasculature was performed, with a random forest classifier quantifying vascular congestion (CVasc) in different microscopic compartments. Respiratory mechanics and gas-exchange parameters were evaluated longitudinally in patients with ARDS. Measurements and Main Results: In premortem CT, patients with COVID-19 showed more dilated vasculature when all lung segments were evaluated (P = 0.001) compared with controls with DAD. Histopathology revealed vasculopathic changes, including hemangiomatosis-like changes (P = 0.043), thromboemboli (P = 0.0038), pulmonary infarcts (P = 0.047), and perivascular inflammation (P < 0.001). Generalized estimating equations revealed significant regional differences in the lung microarchitecture among all DAD-causing entities. COVID-19 showed a larger overall CVasc range (P = 0.002). Alveolar-septal congestion was associated with a significantly shorter time to death from symptom onset (P = 0.03), length of hospital stay (P = 0.02), and increased ventilatory ratio [an estimate for pulmonary dead space fraction (Vd); p = 0.043] in all cases of ARDS. Conclusions: Severe COVID-19 pneumonia is characterized by significant vasculopathy and aberrant alveolar-septal congestion. Our findings also highlight the role that vascular alterations may play in Vd and clinical outcomes in ARDS in general.

Waning efficacy in a long-term AAV-mediated gene therapy study in the murine model of Krabbe disease.

Neonatal AAV9-gene therapy of the lysosomal enzyme galactosylceramidase (GALC) significantly ameliorates central and peripheral neuropathology, prolongs survival, and largely normalizes motor deficits in Twitcher mice. Despite these therapeutic milestones, new observations identified the presence of multiple small focal demyelinating areas in the brain after 6-8 months. These lesions are in stark contrast to the diffuse, global demyelination that affects the brain of naive Twitcher mice. Late-onset lesions exhibited lysosomal alterations with reduced expression of GALC and increased psychosine levels. Furthermore, we found that lesions were closely associated with the extravasation of plasma fibrinogen and activation of the fibrinogen-BMP-SMAD-GFAP gliotic response. Extravasation of fibrinogen correlated with tight junction disruptions of the vasculature within the lesioned areas. The lesions were surrounded by normal appearing white matter. Our study shows that the dysregulation of therapeutic GALC was likely driven by the exhaustion of therapeutic AAV episomal DNA within the lesions, paralleling the presence of proliferating oligodendrocyte progenitors and glia. We believe that this is the first demonstration of diminishing expression in vivo from an AAV gene therapy vector with detrimental effects in the brain of a lysosomal storage disease animal model. The development of this phenotype linking localized loss of GALC activity with relapsing neuropathology in the adult brain of neonatally AAV-gene therapy-treated Twitcher mice identifies and alerts to possible late-onset reductions of AAV efficacy, with implications to other genetic leukodystrophies.

Factors associated with myocardial SARS-CoV-2 infection, myocarditis, and cardiac inflammation in patients with COVID-19.

COVID-19 has been associated with cardiac injury and dysfunction. While both myocardial inflammatory cell infiltration and myocarditis with myocyte injury have been reported in patients with fatal COVID-19, clinical-pathologic correlations remain limited. The objective was to determine the relationships between cardiac pathological changes in patients dying from COVID-19 and cardiac infection by SARS-CoV-2, laboratory measurements, clinical features, and treatments. In a retrospective study, 41 consecutive autopsies of patients with fatal COVID-19 were analyzed for the associations between cardiac inflammation, myocarditis, cardiac infection by SARS-CoV-2, clinical features, laboratory measurements, and treatments. Cardiac infection was assessed by in situ hybridization and NanoString transcriptomic profiling. Cardiac infection by SARS-CoV-2 was present in 30/41 cases: virus+ with myocarditis (n = 4), virus+ without myocarditis (n = 26), and virus- without myocarditis (n = 11). In the cases with cardiac infection, SARS-CoV-2+ cells in the myocardium were rare, with a median density of 1 cell/cm2. Virus+ cases showed higher densities of myocardial CD68+ macrophages and CD3+ lymphocytes, as well as more electrocardiographic changes (23/27 vs 4/10; P = 0.01). Myocarditis was more prevalent with IL-6 blockade than with nonbiologic immunosuppression, primarily glucocorticoids (2/3 vs 0/14; P = 0.02). Overall, SARS-CoV-2 cardiac infection was less prevalent in patients treated with nonbiologic immunosuppression (7/14 vs 21/24; P = 0.02). Myocardial macrophage and lymphocyte densities overall were positively correlated with the duration of symptoms but not with underlying comorbidities. In summary, cardiac infection with SARS-CoV-2 is common among patients dying from COVID-19 but often with only rare infected cells. Cardiac infection by SARS-CoV-2 is associated with more cardiac inflammation and electrocardiographic changes. Nonbiologic immunosuppression is associated with lower incidences of myocarditis and cardiac infection by SARS-CoV-2.

Improving species status assessments under the U.S. Endangered Species Act and implications for multispecies conservation challenges worldwide.

Despite its successes, the U.S. Endangered Species Act (ESA) has proven challenging to implement due to funding limitations, workload backlog, and other problems. As threats to species survival intensify and as more species come under threat, the need for the ESA and similar conservation laws and policies in other countries to function efficiently has grown. Attempts by the U.S. Fish and Wildlife Service (USFWS) to streamline ESA decisions include multispecies recovery plans and habitat conservation plans. We address species status assessment (SSA), a USFWS process to inform ESA decisions from listing to recovery, within the context of multispecies and ecosystem planning. Although existing SSAs have a single-species focus, ecosystem-based research can efficiently inform multiple SSAs within a region and provide a foundation for transition to multispecies SSAs in the future. We considered at-risk grassland species and ecosystems within the southeastern United States, where a disproportionate number of rare and endemic species are associated with grasslands. To initiate our ecosystem-based approach, we used a combined literature-based and structured World Café workshop format to identify science needs for SSAs. Discussions concentrated on 5 categories of threats to grassland species and ecosystems, consistent with recommendations to make shared threats a focus of planning under the ESA: (1) habitat loss, fragmentation, and disruption of functional connectivity; (2) climate change; (3) altered disturbance regimes; (4) invasive species; and (5) localized impacts. For each threat, workshop participants identified science and information needs, including database availability, research priorities, and modeling and mapping needs. Grouping species by habitat and shared threats can make the SSA process and other planning processes for conservation of at-risk species worldwide more efficient and useful. We found a combination of literature review and structured discussion effective for identifying the scientific information and analysis needed to support the development of multiple SSAs. Article impact statement: Species status assessments can be improved by an ecosystem-based approach that groups imperiled species by shared habitats and threats.

Mejoramiento de la Evaluación del Estado de una Especie bajo el Acta de Especies en Peligro de los Estados Unidos y Sus Consecuencias para los Retos de la Conservación Multiespecie a Nivel Mundial Resumen A pesar de su éxito, el Acta de Especies en Peligro de los E.U.A. (AEP) ha sido un reto de implementación por las limitaciones en su financiamiento, el retraso en la carga de trabajo y otros problemas. Conforme se intensifican las amenazas a la supervivencia de las especies y más especies resultan amenazadas, aumenta la necesidad de que la AEP y las políticas similares de otros países funcionen efectivamente. Los intentos por parte del Servicio Estadounidense de Pesca y Fauna (SEPF) para optimizar las decisiones de la AEP incluyen planes multiespecie de recuperación y planes de conservación de hábitat (PRH). Abordamos la evaluación del estado de las especies (EEE), un proceso del SEPF para orientar las decisiones del AEP desde el listado hasta la recuperación, dentro del contexto de la planeación multiespecie y de ecosistemas. Aunque las EEE existentes tienen un enfoque sobre una única especie, la investigación basada en el ecosistema puede orientar eficientemente a múltiples EEE dentro de una región y proporcionar una base para la transición a las EEE multiespecie en el futuro. Consideramos a las especies y los ecosistemas en riesgo de los pastizales del sureste de los Estados Unidos, en donde un número desproporcionado de especies raras y endémicas está asociado con los pastizales. Para iniciar nuestra estrategia basada en el ecosistema, usamos un formato de taller de World Café estructurado y basado en la literatura para identificar la necesidad de tener EEE. Las discusiones se centraron en cinco categorías de amenazas para las especies y ecosistemas de los pastizales, consistentes con las recomendaciones para volver a las amenazas compartidas un foco de la planeación bajo la AEP: (1) pérdida del hábitat, fragmentación y disrupción de la conectividad funcional; (2) cambio climático; (3) regímenes alterados de perturbación; (4) especies invasoras; y (5) impactos localizados. Para cada amenaza, los participantes del taller identificaron las necesidades científicas y de información, incluyendo la disponibilidad de bases de datos, prioridades de la investigación y necesidades de modelado y mapeado. La agrupación de las especies por hábitat y amenaza compartida puede hacer más eficientes y útiles el proceso de EEE y otros procesos de planeación de la conservación de especies en riesgo a nivel mundial. Encontramos una combinación de revisiones bibliográficas y discusiones estructuradas para identificar la información y el análisis necesarios para respaldar el desarrollo de múltiples EEE.