PASC (Post Acute Sequelae of COVID-19) is associated with decreased neutralizing antibody titers in both biological sexes and increased ANG-2 and GM-CSF in females.

Post-acute sequelae of COVID-19 (PASC) or the continuation of COVID-19 (Coronavirus disease 2019) symptoms past 12 weeks may affect as many as 30% of people recovering from a SARS-CoV-2 (severe acute respiratory coronavirus 2) infection. The mechanisms regulating the development of PASC are currently not known; however, hypotheses include virus reservoirs, pre-existing conditions, microblood clots, immune dysregulation, as well as poor antibody responses. Importantly, virus neutralizing antibodies are essential for COVID-19 recovery and protection from reinfection but there is currently limited information on these immune regulators and associated cytokines in PASC patients. Understanding the key drivers of general and specific symptoms associated with Long COVID and the presence of virus neutralizing antibodies in PASC will aid in the development of therapeutics, diagnostics, and vaccines which currently do not exist. We designed a cross-sectional study to investigate systemic antibody and cytokine responses during COVID-19 recovery and PASC. In total, 195 participants were recruited in one of four groups: (1) Those who never had COVID-19 (No COVID); (2) Those in acute COVID-19 recovery (Acute Recovery) (4-12 weeks post infection); (3) Those who recovered from COVID-19 (Recovered) (+ 12 weeks from infection); and (4) those who had PASC (PASC) (+ 12 weeks from infection). Participants completed a questionnaire on health history, sex, gender, demographics, experiences with COVID-19 acute and COVID-19 recovery/continuing symptoms. Serum samples collected were evaluated for antibody binding to viral proteins, virus neutralizing antibody titers, and serum cytokine levels using Ella SimplePlex Immunoassay™ panels. We found participants with PASC reported more pre-existing conditions (e.g. such as hypertension, asthma, and obesity), and PASC symptoms (e.g. fatigue, brain fog, headaches, and shortness of breath) following COVID-19 than COVID-19 Recovered individuals. Importantly, we found PASC individuals to have significantly decreased levels of neutralizing antibodies toward both SARS-CoV-2 and the Omicron BA.1 variant. Sex analysis indicated that female PASC study participants had sustained antibody levels as well as levels of the inflammatory cytokines GM-CSF and ANG-2 over time following COVID-19. Our study reports people experiencing PASC had lower levels of virus neutralizing antibodies; however, the results are limited by the collection time post-COVID-19 and post-vaccination. Moreover, we found females experiencing PASC had sustained levels of GM-CSF and ANG-2. With lower levels of virus neutralizing antibodies, this data suggests that PASC individuals not only have had a suboptimal antibody response during acute SARS-CoV-2 infection but may also have increased susceptibility to subsequent infections which may exacerbate or prolong current PASC illnesses. We also provide evidence suggesting GM-CSF and ANG-2 to play a role in the sex-bias of PASC. Taken together, our findings maybe important for understanding immune molecular drivers of PASC and PASC subgroups.

Aptamer-Functionalized Microbubbles Targeted to P-selectin for Ultrasound Molecular Imaging of Murine Bowel Inflammation.

PURPOSE: Our objectives were to develop a targeted microbubble with an anti-P-selectin aptamer and assess its ability to detect bowel inflammation in two murine models of acute colitis. PROCEDURES: Lipid-shelled microbubbles were prepared using mechanical agitation. A rapid copper-free click chemistry approach (azide-DBCO) was used to conjugate the fluorescent anti-P-selectin aptamer (Fluor-P-Ap) to the microbubble surface. Bowel inflammation was chemically induced using 2,4,6-trinitrobenzenesulfonic acid (TNBS) in both Balb/C and interleukin-10-deficient (IL-10 KO) mice. Mouse bowels were imaged using non-linear contrast mode following an i.v. bolus of 1 × 108 microbubbles. Each mouse received a bolus of aptamer-functionalized and non-targeted microbubbles. Mouse phenotypes and the presence of P-selectin were validated using histology and immunostaining, respectively. RESULTS: Microbubble labelling of Fluor-P-Ap was complete after 20 min at 37 ̊C. We estimate approximately 300,000 Fluor-P-Ap per microbubble and confirmed fluorescence using confocal microscopy. There was a significant increase in ultrasound molecular imaging signal from both Balb/C (p = 0.003) and IL-10 KO (p = 0.02) mice with inflamed bowels using aptamer-functionalized microbubbles in comparison to non-targeted microbubbles. There was no signal in healthy mice (p = 0.4051) using either microbubble. CONCLUSIONS: We constructed an aptamer-functionalized microbubble specific for P-selectin using a clinically relevant azide-DBCO click reaction, which could detect bowel inflammation in vivo. Aptamers have potential as a next generation targeting agent for developing cost-efficient and clinically translatable targeted microbubbles.

SARS-CoV-2 Variant-Specific Infectivity and Immune Profiles Are Detectable in a Humanized Lung Mouse Model.

Small animal models that accurately model pathogenesis of SARS-CoV-2 variants are required for ongoing research efforts. We modified our human immune system mouse model to support replication of SARS-CoV-2 by implantation of human lung tissue into the mice to create TKO-BLT-Lung (L) mice and compared infection with two different variants in a humanized lung model. Infection of TKO-BLT-L mice with SARS-CoV-2 recapitulated the higher infectivity of the B.1.1.7 variant with more animals becoming infected and higher sustained viral loads compared to mice challenged with an early B lineage (614D) virus. Viral lesions were observed in lung organoids but no differences were detected between the viral variants as expected. Partially overlapping but distinct immune profiles were also observed between the variants with a greater Th1 profile in VIDO-01 and greater Th2 profile in B.1.1.7 infection. Overall, the TKO-BLT-L mouse supported SARS-CoV-2 infection, recapitulated key known similarities and differences in infectivity and pathogenesis as well as revealing previously unreported differences in immune responses between the two viral variants. Thus, the TKO-BLT-L model may serve as a useful animal model to study the immunopathobiology of newly emerging variants in the context of genuine human lung tissue and immune cells.

Rapid Copper-free Click Conjugation to Lipid-Shelled Microbubbles for Ultrasound Molecular Imaging of Murine Bowel Inflammation.

Microbubbles are ultrasound contrast agents that can adhere to disease-related vascular biomarkers when functionalized with binding ligands such as antibodies or peptides. The biotin-streptavidin approach has predominantly been used as the microbubble labeling approach in preclinical imaging. However, due to the immunogenicity of avidin in humans, it is not suitable for clinical translation. What would aid clinical translation is a simple and effective microbubble functionalization approach that could be directly translated from animals to humans. We developed a targeted microbubble to P-selectin, a vascular inflammatory marker, labeled using a strain-promoted [3 + 2] azide-alkyne (azide-DBCO) reaction, comparing its ability to detect bowel inflammation to that of P-selectin targeted microbubbles labeled with a traditional biotin-streptavidin approach. Bowel inflammation was chemically induced using 2,4,6-trinitrobenzenesulfonic acid (TNBS) in Balb/C mice. Each mouse received both non-targeted and P-selectin targeted microbubbles (either biotin-streptavidin or azide-DBCO). Using the biotin-streptavidin reaction, there was a significant increase in the ultrasound molecular imaging signal in inflamed mice using P-selectin targeted (2.30 ± 0.91 a.u.) compared to isotype control microbubbles (1.14 ± 0.7 a.u.) (p = 0.009). Using the azide-DBCO reaction, there was a similar increase in the ultrasound molecular imaging signal in inflamed mice (2.54 ± 0.56 a.u) compared to the isotype control (0.44 ± 0.25 a.u) (p = 0.009). There were no significant differences between the two labeling approaches between non-targeted and P-selectin targeted microbubbles. Mouse inflammatory phenotypes and expression of P-selectin were validated using histology and immunostaining. We constructed P-selectin targeted microbubbles using an azide-DBCO click reaction, which could detect bowel inflammation in vivo. This reaction generated a similar ultrasound molecular imaging signal to biotin-strepavidin-labeled microbubbles. These data show the potential of click chemistry conjugation (azide-DBCO) as a quick, cost-efficient, and clinically translatable approach for developing targeted microbubbles.

Developing a Microbubble-Based Contrast Agent for Synchrotron Multiple-Image Radiography.

PURPOSE: Multiple-image radiography (MIR) is an analyzer-based synchrotron X-ray imaging approach capable of dissociating absorption, refraction, and scattering components of X-ray interaction with the material. It generates additional image contrast mechanisms (besides absorption), especially in the case of soft tissues, while minimizing absorbed radiation dose. Our goal is to develop a contrast agent for MIR using ultrasound microbubbles by carrying out a systematic assessment of size, shell material, and concentration. PROCEDURES: Microbubbles were synthesized with two different shell materials: phospholipid and polyvinyl-alcohol. Polydisperse perfluorobutane-filled lipid microbubbles were divided into five size groups using centrifugation. Two distributions of air-filled polymer microbubbles were generated: 2-3 µm and 3-4 µm. A subset of polymer microbubbles 3-4 µm had iron oxide nanoparticles incorporated into their shell or coated on their surface. Microbubbles were immobilized in agar with different concentrations: 5 × 107, 5 × 106, and 5 × 105 MBs/ml. MIR was conducted on the BioMedical Imaging and Therapy beamline at the Canadian Light Source. Three images were generated: Gaussian amplitude, refraction, and ultra-small-angle X-ray scattering (USAXS). The contrast signal was quantified by measuring mean pixel values and comparing them with agar. RESULTS: No difference was detected in absorption or refraction images of all tested microbubbles. Using USAXS, a significant signal increase was observed with lipid microbubbles 6-10 µm at the highest concentration (p = 0.02), but no signal was observed at lower concentrations. CONCLUSIONS: These data indicate that lipid microbubbles 6-10 µm are candidates as contrast agents for MIR, specifically for USAXS. A minimum concentration of 5 × 107 microbubbles (lipid-shell 6-10 µm) per milliliter was needed to generate a detectable signal.

SARS-CoV-2 infection in the Syrian hamster model causes inflammation as well as type I interferon dysregulation in both respiratory and non-respiratory tissues including the heart and kidney.

COVID-19 (coronavirus disease 2019) caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection is a disease affecting several organ systems. A model that captures all clinical symptoms of COVID-19 as well as long-haulers disease is needed. We investigated the host responses associated with infection in several major organ systems including the respiratory tract, the heart, and the kidneys after SARS-CoV-2 infection in Syrian hamsters. We found significant increases in inflammatory cytokines (IL-6, IL-1beta, and TNF) and type II interferons whereas type I interferons were inhibited. Examination of extrapulmonary tissue indicated inflammation in the kidney, liver, and heart which also lacked type I interferon upregulation. Histologically, the heart had evidence of myocarditis and microthrombi while the kidney had tubular inflammation. These results give insight into the multiorgan disease experienced by people with COVID-19 and possibly the prolonged disease in people with post-acute sequelae of SARS-CoV-2 (PASC).

Sex and age bias viral burden and interferon responses during SARS-CoV-2 infection in ferrets.

SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) hospitalizations and deaths disportionally affect males and older ages. Here we investigated the impact of male sex and age comparing sex-matched or age-matched ferrets infected with SARS-CoV-2. Differences in temperature regulation was identified for male ferrets which was accompanied by prolonged viral replication in the upper respiratory tract after infection. Gene expression analysis of the nasal turbinates indicated that 1-year-old female ferrets had significant increases in interferon response genes post infection which were delayed in males. These results provide insight into COVID-19 and suggests that older males may play a role in viral transmission due to decreased antiviral responses.

Spontaneous hybridization and introgression between walleye (Sander vitreus) and sauger (Sander canadensis) in two large reservoirs: Insights from genotyping by sequencing.

Anthropogenic activities may facilitate undesirable hybridization and genomic introgression between fish species. Walleye (Sander vitreus) and sauger (Sander canadensis) are economically valuable freshwater species that can spontaneously hybridize in areas of sympatry. Levels of genomic introgression between walleye and sauger may be increased by modifications to waterbodies (e.g., reservoir development) and inadvertent propagation of hybrids in stocking programs. We used genotyping by sequencing (GBS) to examine 217 fish from two large reservoirs with mixed populations of walleye and sauger in Saskatchewan, Canada (Lake Diefenbaker, Tobin Lake). Analyses with 20,038 (r90) and 478 (r100) single nucleotide polymorphisms clearly resolved walleye and sauger, and classified hybrids with high confidence. F1, F2, and multigeneration hybrids were detected in Lake Diefenbaker, indicating potentially high levels of genomic introgression. In contrast, only F1 hybrids were detected in Tobin Lake. Field classification of fish was unreliable; 7% of fish were misidentified based on broad species categories. Important for activities such as brood stock selection, 12 of 173 (7%) fish field identified as pure walleye, and one of 24 (4%) identified as pure sauger were actually hybrids. In addition, two of 15 (13%) field-identified hybrids were actually pure walleye or sauger. We conclude that hybridization and introgression are occurring in Saskatchewan reservoirs and that caution is warranted when using these populations in stocking programs. GBS offers a powerful and flexible tool for examining hybridization without preidentification of informative loci, eliminating some of the key challenges associated with other marker types.

Developing a Microbubble-Based Contrast Agent for Synchrotron In-Line Phase Contrast Imaging.

OBJECTIVE: X-ray phase contrast imaging generates contrast from refraction of X-rays, enhancing soft tissue contrast compared to conventional absorption-based imaging. Our goal is to develop a contrast agent for X-ray in-line phase contrast imaging (PCI) based on ultrasound microbubbles (MBs), by assessing size, shell material, and concentration. METHODS: Polydisperse perfluorobutane-core lipid-shelled MBs were synthesized and size separated into five groups between 1 and 10 µm. We generated two size populations of polyvinyl-alcohol (PVA)-MBs, 2-3 µm and 3-4 µm, whose shells were either coated or integrated with iron oxide nanoparticles (SPIONs). Microbubbles were then embedded in agar at three concentrations: 5 × 107, 5 × 106 and 5 × 105 MBs/ml. In-line phase contrast imaging was performed at the Canadian Light Source with filtered white beam micro-computed tomography. Phase contrast intensity was measured by both counting detectable MBs, and comparing mean pixel values (MPV) in minimum and maximum intensity projections of the overall samples. RESULTS: Individual lipid-MBs 6-10 µm, lipid-MBs 4-6 µm and PVA-MBs coated with SPIONs were detectable at each concentration. At the highest concentration, lipid-MBs 6-10 µm and 4-6 µm showed an overall increase in positive contrast, whereas at a moderate concentration, only lipid-MBs 6-10 µm displayed an increase. Negative contrast was also observed from two largest lipid-MBs at high concentration. CONCLUSION: These data indicate that lipid-MBs larger than 4 µm are candidates for PCI, and 5 × 106 MBs/ml may be the lowest concentration suitable for generating visible phase contrast in vivo. SIGNIFICANCE: Identifying a suitable MB for PCI may facilitate future clinical translation.

Contrast-enhanced ultrasound imaging for assessing organ perfusion in rainbow trout (Oncorhynchus mykiss).

Contrast-enhanced ultrasound (CEUS) imaging has great potential as a non-lethal, inexpensive monitoring tool in aquatic toxicology. It is a well-established clinical imaging approach that combines real-time, quantitative assessment of organ blood flow, with morphological data. In humans, it has been extensively used to measure changes in blood flow that can be attributed to cancer, inflammation, and other biological abnormalities. However, it has yet to be explored as a tool for fish physiology or environmental toxicology. In this study, our goal was to determine if CEUS could be used to visualize and measure blood flow in the liver of a rainbow trout. All rainbow trout received two injections of an ultrasound contrast agent, microbubbles. A subset received a third injection after administration of propranolol, a non-specific beta1 & 2-blocker, to determine if changes in blood flow could be detected. Ultrasound contrast time-intensity curves (TIC) were obtained, fit to a lognormal model, and different perfusion parameters were calculated. Contrast enhancement was observed in all rainbow trout livers, with high percentage between repeated measurements, including blood flow (80.6 ± 27.3%), area under the curve (73.2 ± 14%), blood volume (84 ± 14.2%) and peak enhancement (86.7 ± 7.5%). After administration of propranolol, we detected a non-significant (p > 0.05) increase in area under the curve (102.6 ± 44.2%), peak enhancement (77.3 ± 106.4), blood volume (48.2 ± 74.5%), and decrease in hepatic blood flow (-17.3 ± 37.1%). These data suggest that CEUS imaging is suitable to measure organ blood flow in fish, and demonstrates tremendous potential for exploring different organs, fish species, and effects of chemical contaminants in future studies.