Prolonged airway explant culture enables study of health, disease, and viral pathogenesis (preprint)

In vitro models play a major role in studying airway physiology and disease. However, the native lungs complex tissue architecture and non-epithelial cell lineages are not preserved in these models. Ex vivo tissue models could overcome in vitro limitations, but methods for long-term maintenance of ex vivo tissue has not been established. We describe methods to culture human large airway explants, small airway explants, and precision-cut lung slices for at least 14 days. Human airway explants recapitulate genotype-specific electrophysiology, characteristic epithelial, endothelial, stromal and immune cell populations, and model viral infection after 14 days in culture. These methods also maintain mouse, rabbit, and pig tracheal explants. Notably, intact airway tissue can be cryopreserved, thawed, and used to generate explants with recovery of function 14 days post-thaw. These studies highlight the broad applications of airway tissue explants and their use as translational intermediates between in vitro and in vivo studies.

Dynamic Serial Cytokine Measurements During Intravenous Ca-DTPA Chelation in Gadolinium Deposition Disease and Gadolinium Storage Condition: A Pilot Study.

PURPOSE: The aim of this study was to investigate the feasibility of measuring early changes in serum cytokine levels after intravenous diethylenetriaminepentaacetic acid (Ca-DTPA) chelation in patients manifesting either gadolinium deposition disease (GDD) or gadolinium storage condition (GSC) and the possible usefulness of this method in further research. METHODS: Four patients with recent-onset GDD (≤1 year) and 2 patients with long-standing GSC (4 and 9 years) underwent chelation with intravenous bolus administration of Ca-DTPA. Multiple blood draws were performed to measure serum cytokines: at T = 0 (before Ca-DTPA injection) and 1, 5, 10, 30, 60 minutes, and 24 hours after Ca-DTPA injection. Patients rated the severity of GDD symptom flare at 24 hours. The 24-hour urine Gd amounts were measured prechelation and for the 24 hours after chelation. Serum samples were analyzed blind to whether patients had GDD or GSC but with knowledge of the time points characterizing each sample. RESULTS: Urine samples for both GDD and GSC patients showed increases in Gd postchelation. All GDD patients experienced flare reactions postchelation; the 2 GSC patients did not. Two cytokines, EGF and sCD40L, peaked at 30 minutes postchelation in at least 4 of the 6 participants. Three cytokines, ENA78/CXCL5, EOTAXIN/CCL11, and LEPTIN, peaked at 24 hours in at least 4 of the 6 participants. Two participants were high outliers for a large number of cytokines across time points. No clear distinction between GDD and GSC was apparent from the cytokine patterns, although differences were present. CONCLUSIONS: This pilot study describes precise temporal resolution (in the range of minutes) after a cytokine-inciting event. Select cytokines exhibited peak values at different time points. At this preliminary stage of investigation, peak cytokine release seems to reflect the amount of Gd mobilized rather than the severity of the patient symptomatic reaction. Too few subjects were studied to support statistical analysis between GDD and GSC groups, although differences were observed through visual data analysis.

Chronic airway epithelial hypoxia exacerbates injury in muco-obstructive lung disease through mucus hyperconcentration.

Unlike solid organs, human airway epithelia derive their oxygen from inspired air rather than the vasculature. Many pulmonary diseases are associated with intraluminal airway obstruction caused by aspirated foreign bodies, virus infection, tumors, or mucus plugs intrinsic to airway disease, including cystic fibrosis (CF). Consistent with requirements for luminal O2, airway epithelia surrounding mucus plugs in chronic obstructive pulmonary disease (COPD) lungs are hypoxic. Despite these observations, the effects of chronic hypoxia (CH) on airway epithelial host defense functions relevant to pulmonary disease have not been investigated. Molecular characterization of resected human lungs from individuals with a spectrum of muco-obstructive lung diseases (MOLDs) or COVID-19 identified molecular features of chronic hypoxia, including increased EGLN3 expression, in epithelia lining mucus-obstructed airways. In vitro experiments using cultured chronically hypoxic airway epithelia revealed conversion to a glycolytic metabolic state with maintenance of cellular architecture. Chronically hypoxic airway epithelia unexpectedly exhibited increased MUC5B mucin production and increased transepithelial Na+ and fluid absorption mediated by HIF1α/HIF2α-dependent up-regulation of ß and γENaC (epithelial Na+ channel) subunit expression. The combination of increased Na+ absorption and MUC5B production generated hyperconcentrated mucus predicted to perpetuate obstruction. Single-cell and bulk RNA sequencing analyses of chronically hypoxic cultured airway epithelia revealed transcriptional changes involved in airway wall remodeling, destruction, and angiogenesis. These results were confirmed by RNA-in situ hybridization studies of lungs from individuals with MOLD. Our data suggest that chronic airway epithelial hypoxia may be central to the pathogenesis of persistent mucus accumulation in MOLDs and associated airway wall damage.

Medical Examiner Surveillance of the COVID-19 Pandemic in King County, Washington, USA.

ABSTRACT: The King County Medical Examiner's Office in Seattle, Wash, initiated a surveillance project with a dedicated team and database tracking the spread of severe acute respiratory syndrome coronavirus 2, deaths due to coronavirus disease 2019 (COVID-19), and deaths occurring within 28 days of COVID-19 vaccination. From January 2020 through July 2022, the results of 13,801 nasal/nasopharyngeal swabs from 7606 decedents tested for the virus were assembled in the surveillance database. Generally, 2 samples were collected and tested separately by 2 different laboratories. Positive rates increased from 5.7% in 2020 to 14.3% in 2022. Of 744 decedents positive for the virus, autopsies were performed on 418 (56%); of these, 106 (25%) died of COVID-19 as either the primary or a contributing cause. Comparison of autopsy findings of those dying of COVID-19 with those positive for the virus but dying of other causes demonstrated increased risk for those with preexisting conditions. Of 1035 deaths reported within 28 days of vaccination, the rates of thrombotic complications and myocarditis were no higher than in other decedents. This study provides evidence of the value to public health surveillance of an adequately resourced medical examiner office in tracking viral spread in the community, understanding disease mortality, and assessing vaccine safety.

Development of an amplicon-based sequencing approach in response to the global emergence of mpox.

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.

COVID-19 Math: You, Me, R0 and Rolling Re-entry

Larson discusses the mathematical equation of the Covid-19 pandemic. He offers a mathematical formula to explain the spread, transmission and infection of the Covid-19. He also explains how measures such as social isolation can help protect the most vulnerable population such as the senior citizens and those with chronic medical conditions and asserts that with the significant number of re-openings, if done with meticulous care, will not increase the current value of infections. The author also emphasizes the importance of masks, social distancing and temperature checks and until vaccine is available, everything will be back to normal.

Facilitating Real-Time, Multidirectional Learning for Clinicians in a Low-Evidence Pandemic Response.

As COVID-19 was declared a health emergency in March 2020, there was immense demand for information about the novel pathogen. This paper examines the clinician-reported impact of Project ECHO COVID-19 Clinical Rounds on clinician learning. Primary sources of study data were Continuing Medical Education (CME) Surveys for each session from the dates of March 24, 2020 to July 30, 2020 and impact surveys conducted in November 2020, which sought to understand participants' overall assessment of sessions. Quantitative analyses included descriptive statistics and Mann-Whitney testing. Qualitative data were analyzed through inductive thematic analysis. Clinicians rated their knowledge after each session as significantly higher than before that session. 75.8% of clinicians reported they would 'definitely' or 'probably' use content gleaned from each attended session and clinicians reported specific clinical and operational changes made as a direct result of sessions. 94.6% of respondents reported that COVID-19 Clinical Rounds helped them provide better care to patients. 89% of respondents indicated they 'strongly agree' that they would join ECHO calls again.COVID-19 Clinical Rounds offers a promising model for the establishment of dynamic peer-to-peer tele-mentoring communities for low or no-notice response where scientifically tested or clinically verified practice evidence is limited.

Cardiopulmonary Phenotypes of Post Acute Sequelae of Severe Acute Respiratory Syndrome Coronavirus 2: A Narrative Review.

The acute effects of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are well known; however, the long-term cardiopulmonary effects are less well characterized. The phenotypic expression of acute infection is heterogeneous, ranging from a complete absence of symptoms to shock, multisystem organ failure, and death. Patients with severe or critical coronavirus disease (COVID-19) who survive their initial illness can require a prolonged period of recovery lasting weeks to months. This specific patient group is part of a larger and even more heterogeneous group of patients who initially experience mild-to-moderate symptoms that fail to resolve over time. Collectively, patients recovering from severe or critical COVID-19 and those who continue to experience symptoms following a lower acuity infection are considered to have Post Acute Sequalae of SARS-CoV-2 infection (PASC). Using prognostic factors like myocardial infarction, myocarditis, pulmonary embolism, acute respiratory distress syndrome, need for mechanical ventilation or extracorporeal membrane oxygenation, and advanced pharmaceutical therapies that primarily occur or are instituted in the acute phase of illness one can begin to develop a taxonomy or corpus of PASC in its varied forms.

Multiple models for outbreak decision support in the face of uncertainty.

Policymakers must make management decisions despite incomplete knowledge and conflicting model projections. Little guidance exists for the rapid, representative, and unbiased collection of policy-relevant scientific input from independent modeling teams. Integrating approaches from decision analysis, expert judgment, and model aggregation, we convened multiple modeling teams to evaluate COVID-19 reopening strategies for a mid-sized United States county early in the pandemic. Projections from seventeen distinct models were inconsistent in magnitude but highly consistent in ranking interventions. The 6-mo-ahead aggregate projections were well in line with observed outbreaks in mid-sized US counties. The aggregate results showed that up to half the population could be infected with full workplace reopening, while workplace restrictions reduced median cumulative infections by 82%. Rankings of interventions were consistent across public health objectives, but there was a strong trade-off between public health outcomes and duration of workplace closures, and no win-win intermediate reopening strategies were identified. Between-model variation was high; the aggregate results thus provide valuable risk quantification for decision making. This approach can be applied to the evaluation of management interventions in any setting where models are used to inform decision making. This case study demonstrated the utility of our approach and was one of several multimodel efforts that laid the groundwork for the COVID-19 Scenario Modeling Hub, which has provided multiple rounds of real-time scenario projections for situational awareness and decision making to the Centers for Disease Control and Prevention since December 2020.

Evaluating chest pain in patients with post COVID conditions permission to think outside of the box.

Chest pain is among the most common symptoms of post-COVID-19 Conditions (PCC) that prompts medical attention. Because the SARS-CoV-2 virus has proclivity for many organs and organ systems in the chest, ranging from the heart, lungs, great vessels, lymphatics, and peripheral nerves, clinicians evaluating patients with chest pain must consider a broad differential diagnosis and take a comprehensive approach to management.

SARS‐CoV‐2 spike protein capture by peptide functionalized networks

Coronavirus disease 2019 (COVID‐19) has significantly impacted human health, the global economy, and society. Viruses residing on common surfaces represent a potential source of contamination for the general population. Spike binding peptide 1, SBP1 is a 23 amino acid peptide, which has micromolar binding affinity (1.3 μM) towards the spike protein receptor‐binding domain. We hypothesize that if we can covalently immobilize this SBP1 peptide in a covalent crosslinked network system, we can develop a surface that would preferentially bind spike protein and, therefore, which could limit viral spread. A series of covalently crosslinked networks of hydroxy ethyl acrylate (HEA) with different primary chain lengths and crosslinker density was prepared. Later, this network system was functionalized using 2% SBP1 peptide. Our study found that with a shorter chain length and lower crosslinker density, the HEA network system alone could capture almost 80% of the spike protein. We reported that the efficiency could be enhanced almost by 17% with higher crosslinker density.

Heart Transplantation from COVID-19 Positive Donors. A word of caution

Background During the COVID-19 pandemic, efforts to maintain solid-organ transplantation have continued, including the use of SARS-CoV-2 positive heart donors. Methods We present our institution's initial experience with SARS-CoV-2 positive heart donors. All donors met our institution's Transplant Center criteria, including a negative bronchoalveolar lavage (BAL) PCR result. All but one patient received postexposure prophylaxis (PEP) with anti-spike monoclonal antibody therapy, remdesivir, or both. Results A total of 6 patients received a heart transplant from a SARS-CoV-2 positive donor. One heart transplant was complicated by catastrophic secondary graft dysfunction requiring venoarterial extracorporeal membrane oxygenation and retransplant. The remaining five patients did well postoperatively and were discharged home. None of the patients had evidence of COVID-19 infection after surgery. Conclusion Heart transplants from SARS-CoV-2 PCR positive donors are feasible and safe with adequate screening and PEP.

Heatwaves and mortality in Queensland 2010-2019: implications for a homogenous state-wide approach.

Heatwaves are a significant cause of adverse health outcomes and mortality in Australia, worsening with climate change. In Queensland, the northeastern-most state, little is known about the impact of heatwaves outside of the capital city of Brisbane. This study aims to explore the impact of heatwaves on mortality across various demographic and environmental conditions within Queensland from 2010 to 2019. The Excess Heat Factor was used to indicate heatwave periods at the Statistical Area 2 (SA2) level. Registered deaths data from the Australian Bureau of Statistics and heatwave data from the Bureau of Meteorology were matched using a case-crossover approach. Relative risk and 95% confidence intervals were calculated across years, regions, age, sex, rurality, socioeconomic status, and cause of death. Heatwaves were associated with a 5% increase in all-cause mortality compared to deaths on non-heatwave days, with variability across the state. The risk of death on a heatwave day versus a non-heatwave day varied by heatwave severity. Individuals living in urban centers, the elderly, and those living in regions of lower socioeconomic status were most impacted by heatwave mortality. The relative risk of dying from neoplasms, nervous system conditions, respiratory conditions, and mental and behavioral conditions increased during heatwaves. As heatwaves increase in Queensland due to climate change, understanding the impact of heatwaves on mortality across Queensland is important to tailor public health messages. There is considerable variability across communities, demographic groups, and medical conditions, and as such messages need to be tailored to risk.

Modeling identifies variability in SARS-CoV-2 uptake and eclipse phase by infected cells as principal drivers of extreme variability in nasal viral load in the 48 h post infection.

The SARS-CoV-2 coronavirus continues to evolve with scores of mutations of the spike, membrane, envelope, and nucleocapsid structural proteins that impact pathogenesis. Infection data from nasal swabs, nasal PCR assays, upper respiratory samples, ex vivo cell cultures and nasal epithelial organoids reveal extreme variabilities in SARS-CoV-2 RNA titers within and between the variants. Some variabilities are naturally prone to clinical testing protocols and experimental controls. Here we focus on nasal viral load sensitivity arising from the timing of sample collection relative to onset of infection and from heterogeneity in the kinetics of cellular infection, uptake, replication, and shedding of viral RNA copies. The sources of between-variant variability are likely due to SARS-CoV-2 structural protein mutations, whereas within-variant population variability is likely due to heterogeneity in cellular response to that particular variant. With the physiologically faithful, agent-based mechanistic model of inhaled exposure and infection from (Chen et al., 2022), we perform statistical sensitivity analyses of the progression of nasal viral titers in the first 0-48 h post infection, focusing on three kinetic mechanisms. Model simulations reveal shorter latency times of infected cells (including cellular uptake, viral RNA replication, until the onset of viral RNA shedding) exponentially accelerate nasal viral load. Further, the rate of infectious RNA copies shed per day has a proportional influence on nasal viral load. Finally, there is a very weak, negative correlation of viral load with the probability of infection per virus-cell encounter, the model proxy for spike-receptor binding affinity.

Neoadjuvant-Adjuvant or Adjuvant-Only Pembrolizumab in Advanced Melanoma.

BACKGROUND: Whether pembrolizumab given both before surgery (neoadjuvant therapy) and after surgery (adjuvant therapy), as compared with pembrolizumab given as adjuvant therapy alone, would increase event-free survival among patients with resectable stage III or IV melanoma is unknown. METHODS: In a phase 2 trial, we randomly assigned patients with clinically detectable, measurable stage IIIB to IVC melanoma that was amenable to surgical resection to three doses of neoadjuvant pembrolizumab, surgery, and 15 doses of adjuvant pembrolizumab (neoadjuvant-adjuvant group) or to surgery followed by pembrolizumab (200 mg intravenously every 3 weeks for a total of 18 doses) for approximately 1 year or until disease recurred or unacceptable toxic effects developed (adjuvant-only group). The primary end point was event-free survival in the intention-to-treat population. Events were defined as disease progression or toxic effects that precluded surgery; the inability to resect all gross disease; disease progression, surgical complications, or toxic effects of treatment that precluded the initiation of adjuvant therapy within 84 days after surgery; recurrence of melanoma after surgery; or death from any cause. Safety was also evaluated. RESULTS: At a median follow-up of 14.7 months, the neoadjuvant-adjuvant group (154 patients) had significantly longer event-free survival than the adjuvant-only group (159 patients) (P = 0.004 by the log-rank test). In a landmark analysis, event-free survival at 2 years was 72% (95% confidence interval [CI], 64 to 80) in the neoadjuvant-adjuvant group and 49% (95% CI, 41 to 59) in the adjuvant-only group. The percentage of patients with treatment-related adverse events of grades 3 or higher during therapy was 12% in the neoadjuvant-adjuvant group and 14% in the adjuvant-only group. CONCLUSIONS: Among patients with resectable stage III or IV melanoma, event-free survival was significantly longer among those who received pembrolizumab both before and after surgery than among those who received adjuvant pembrolizumab alone. No new toxic effects were identified. (Funded by the National Cancer Institute and Merck Sharp and Dohme; S1801 ClinicalTrials.gov number, NCT03698019.).

Heart Transplantation from COVID-19-Positive Donors: A Word of Caution.

BACKGROUND: During the COVID-19 pandemic, efforts to maintain solid-organ transplantation have continued, including the use of SARS-CoV-2-positive heart donors. METHODS: We present our institution's initial experience with SARS-CoV-2-positive heart donors. All donors met our institution's Transplant Center criteria, including a negative bronchoalveolar lavage polymerase chain reaction result. All but 1 patient received postexposure prophylaxis with anti-spike monoclonal antibody therapy, remdesivir, or both. RESULTS: A total of 6 patients received a heart transplant from a SARS-CoV-2-positive donor. One heart transplant was complicated by catastrophic secondary graft dysfunction requiring venoarterial extracorporeal membrane oxygenation and retransplant. The remaining 5 patients did well postoperatively and were discharged from the hospital. None of the patients had evidence of COVID-19 infection after surgery. CONCLUSION: Heart transplants from SARS-CoV-2 polymerase chain reaction-positive donors are feasible and safe with adequate screening and postexposure prophylaxis.

An ex vivo human precision-cut lung slice platform provides insight into SARS-CoV-2 pathogenesis and antiviral drug efficacy (preprint)

COVID-19 has claimed millions of lives since the emergence of SARS-CoV-2, and lung disease appears the primary cause of the death in COVID-19 patients. However, the underlying mechanisms of COVID-19 pathogenesis remain elusive, and there is no existing model where the human disease can be faithfully recapitulated and conditions for the infection process can be experimentally controlled. Herein we report the establishment of an ex vivo human precision-cut lung slice (hPCLS) platform for studying SARS-CoV-2 pathogenicity and innate immune responses, and for evaluating the efficacy of antiviral drugs against SARS-CoV-2. We show that while SARS-CoV-2 continued to replicate during the course of infection of hPCLS, infectious virus production peaked within 2 days, and rapidly declined thereafter. Although most proinflammatory cytokines examined were induced by SARS-CoV-2 infection, the degree of induction and types of cytokines varied significantly among hPCLS from individual donors, reflecting the heterogeneity of human populations. In particular, two cytokines (IP-10 and IL-8) were highly and consistently induced, suggesting a role in the pathogenesis of COVID-19. Histopathological examination revealed focal cytopathic effects late in the infection. Transcriptomic and proteomic analyses identified molecular signatures and cellular pathways that are largely consistent with the progression of COVID-19 in patients. Furthermore, we show that homoharringtonine, a natural plant alkaloid derived from Cephalotoxus fortunei, not only inhibited virus replication but also production of pro-inflammatory cytokines, and ameliorated the histopathological changes of the lungs caused by SARS-CoV-2 infection, demonstrating the usefulness of the hPCLS platform for evaluating antiviral drugs.

Transient loss and recovery of oral chemesthesis, taste and smell with COVID-19 (preprint)

Anosmia is common with respiratory virus infections, but loss of taste or chemesthesis is rare. Reports of true taste loss with COVID-19 were viewed skeptically until confirmed by multiple studies. Nasal menthol thresholds are elevated in some with prior COVID-19 infections, but data on oral chemesthesis are lacking. Many patients recover quickly, but precise timing and synchrony of recovery are unclear. Here, we collected broad sensory measures over 28 days, recruiting adults (18-45 years) who were COVID-19 positive or recently exposed (close contacts per U.S. CDC criteria at the time of the study) in the first half of 2021. Participants received nose clips, red commercial jellybeans (Sour Cherry and Cinnamon), and scratch-n-sniff cards (ScentCheckPro). Among COVID-19 cases who entered the study on or before Day 10 of infection, Gaussian Process Regression showed odor identification and odor intensity (two distinct measures of function) each declined relative to controls (close contacts who never developed COVID-19), but effects were larger for intensity than identification. To assess changes during early onset, we identified four COVID-19 cases who enrolled on or prior to Day 1 of their illness: this allowed for visualization of baseline ratings, loss, and recovery of function over time. Four controls were matched for age, gender, and race. Variables included sourness and sweetness (Sour Cherry jellybeans), oral burn (Cinnamon jellybeans), mean orthonasal intensity of four odors (ScentCheckPro), and perceived nasal blockage. Data were plotted over 28 days, creating panel plots for the eight cases and controls. Controls exhibited stable ratings over time. By contrast, COVID-19 cases showed sharp deviations over time. No single pattern of taste loss or recovery was apparent, implying different taste qualities might recover at different rates. Oral burn was transiently reduced for some before recovering quickly, suggesting acute loss may be missed in data collected after acute illness ends. Changes in odor intensity or odor identification were not explained by nasal blockage. Collectively, intensive daily testing shows orthonasal smell, oral chemesthesis and taste were each altered by acute COVID-19 infection, and this disruption was dyssynchronous for different modalities, with variable loss and recovery rates across modalities and individuals.