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Background COVID‐19 can present with lymphopenia and extraordinary complex multiorgan pathologies that can trigger long‐term sequela. Aims Given that inflammasome products, like caspase‐1, play a role in the pathophysiology of a number of co‐morbid conditions, we investigated caspases across the spectrum of COVID‐19 disease. Materials & Methods We assessed transcriptional states of multiple caspases and using flow cytometry, the expression of active caspase‐1 in blood cells from COVID‐19 patients in acute and convalescent stages of disease. Non‐COVID‐19 subject presenting with various comorbid conditions served as controls. Results Single‐cell RNA‐seq data of immune cells from COVID‐19 patients showed a distinct caspase expression pattern in T cells, neutrophils, dendritic cells, and eosinophils compared with controls. Caspase‐1 was upregulated in CD4+ T‐cells from hospitalized COVID‐19 patients compared with unexposed controls. Post‐COVID‐19 patients with lingering symptoms (long‐haulers) also showed upregulated caspase‐1activity in CD4+ T‐cells that ex vivo was attenuated with a select pan‐caspase inhibitor. We observed elevated caspase‐3/7levels in red blood cells from COVID‐19 patients compared with controls that was reduced following caspase inhibition. Discussion Our preliminary results suggest an exuberant caspase response in COVID‐19 that may facilitate immune‐related pathological processes leading to severe outcomes. Further clinical correlations of caspase expression in different stages of COVID‐19 will be needed. Conclusion Pan‐caspase inhibition could emerge as a therapeutic strategy to ameliorate or prevent severe COVID‐19., This study assesses transcriptional states of multiple caspases and the expression of active caspase‐1 in blood cells from COVID‐19 patients in acute and convalescent stages of disease. Elevated caspase‐3/7 levels in red blood cells is observed in COVID‐19 patients compared to controls. Post‐COVID‐19 patients with lingering symptoms show up‐regulated caspase‐1 activity in CD4+ T‐cells that is attenuated ex vivo with a select pan‐caspase inhibitor. An exuberant caspase response in COVID‐19 that may facilitate immune‐related pathological processes leading to severe outcomes.

COVID-19 can present with lymphopenia and extraordinary complex multi-organ pathologies that can trigger long-term sequela. Given that inflammasome products, like caspase-1, play a role in the pathophysiology of a number of co-morbid conditions, we investigated caspases across the spectrum of COVID-19 disease. We assessed transcriptional states of multiple caspases and using flow cytometry, the expression of active caspase-1 in blood cells from COVID-19 patients in acute and convalescent stages of disease. Non-COVID-19 subjects presenting with various co-morbid conditions served as controls. Single-cell RNA-seq data of immune cells from COVID-19 patients showed a distinct caspase expression pattern in T cells, neutrophils, dendritic cells and eosinophils compared to controls. Caspase-1 was upregulated in CD4+ T-cells from hospitalized COVID-19 patients compared to unexposed controls. Post-COVID-19 patients with lingering symptoms (long-haulers) also showed up-regulated caspase-1 activity in CD4+ T-cells that ex vivo was attenuated with a select pan-caspase inhibitor. We observed elevated caspase-3/7 levels in red blood cells from COVID-19 patients compared to controls that was reduced following caspase inhibition. Our preliminary results suggest an exuberant caspase response in COVID-19 that may facilitate immune-related pathological processes leading to severe outcomes. Further clinical correlations of caspase expression in different stages of COVID-19 will be needed. Pan-caspase inhibition could emerge as a therapeutic strategy to ameliorate or prevent severe COVID-19.

Background The short stability window of several hours from blood collection to measuring basophil activation has limited the use of flow cytometry-based basophil activation assays in clinical settings. We examine if it is possible to extend this window to 1 day allowing for shipment of samples between laboratories. Several options exist for reporting the results including reporting all the measured values directly, calculating ratios and reporting a single value covering all measured results. Each of these options have different stability and value to the physician. Methods Whole blood samples from peanut allergic patients were stimulated with four different peanut concentrations at Day 0, Day 1, and Day 2. Samples were stored under temperature-controlled conditions. Flow cytometry was used to analyze the samples. The basophil activation and degranulation were measured as percentage of positive CD63 basophils and CD203c MFI fold change. Shipped samples were transported under ambient conditions. Results The results show that CD63 is a stable marker at Day 1. The CD203c ratio decreases significantly at Day 1. Calculating the CD63/IgE ratio proves to be more stable than CD63 alone. The most stable readouts are the semi-quantitative results and the trajectory of the dose response curve. Finally, we confirmed that the stability can be extended to samples shipped overnight to the laboratory. Conclusions It is possible to extend the stability of the basophil activation assay to 1 day for samples stored at 18-25°C as well as samples shipped under ambient conditions as long as the temperature is within the 2-37°C range.

Currently, there is no effective vaccine and only one FDA approved early-stage therapy against SARS-CoV-2 infection as an indication to prevent disease progression. Cellular caspases play a role in the pathophysiology of a number of disorders that the co-morbid conditions seen in severe COVID-19 disease. In this study, we assessed transcriptional states of caspases in blood cells from COVID-19 patients. Gene expression levels of select caspases were increased inin vitroSARS-CoV-2 infection models and single cell RNA-Seq data of blood from COVID-19 patients showed a distinct caspase expression in T cells, neutrophils, and dendritic cells. Flow cytometric evaluation of CD4 T cells showed up-regulation of caspase-1 in hospitalized COVID-19 patients compared to unexposed controls. Convalescent COVID-19 patients with lingering symptoms (“long haulers”) showed persistent up-regulation of caspase-1 in CD4 T cells that was attenuatedex vivofollowing co-culture with a select pan-caspase inhibitor. Further, we observed elevated caspase-3 levels in red blood cells from COVID-19 patients compared to controls that were responsive to caspase inhibition. Our results expose an exuberant caspase response in COVID-19 that may facilitate immune-related pathological processes leading to severe outcomes. Pan-caspase inhibition could emerge as a therapeutic strategy to ameliorate or prevent severe COVID-19 outcomes.

Eosinophilic disorders of the gastrointestinal tract are an emerging subset of immune pathologies within the spectrum of allergic inflammation. Eosinophilic Esophagitis (EoE), once considered a rare disease, is increasing in incidence, with a rate of over 1 in 10,000 in the US, for unknown reasons. The clinical management of EoE is challenging, thus there is an urgent need for understanding the etiology and pathophysiology of this eosinophilic disease to develop better therapeutic approaches. In this open label, single arm, unblinded study, we evaluated the effects of an anti-IgE treatment, omalizumab, on local inflammation in the esophagus and clinical correlates in patients with EoE. Omalizumab was administered for 12 weeks to 15 subjects with long standing EoE. There were no serious side effects from the treatment. Esophageal tissue inflammation was assessed both before and after therapy. After 3 months on omalizumab, although tissue Immunoglobulin E (IgE) levels were significantly reduced in all but two of the subjects, we found that full remission of EoE, which is defined as histologic and clinical improvement only in 33% of the patients. The decrease in tryptase-positive cells and eosinophils correlated significantly with the clinical outcome as measured by improvement in endoscopy and symptom scores, respectively. Omalizumab-induced remission of EoE was limited to subjects with low peripheral blood absolute eosinophil counts. These findings demonstrate that in a subset of EoE patients, IgE plays a role in the pathophysiology of the disease and that anti-IgE therapy with omalizumab may result in disease remission. Since this study is open label there is the potential for bias, hence the need for a larger double blind placebo controlled study. The data presented in this pilot study provides a foundation for proper patient selection to maximize clinical efficacy. Trial Registration ClinicalTrials.gov NCT01040598