During the COVID-19, elective cataract surgery (CS) was significantly curtailed. We investigated whether consequent reduction of micro-surgical skills practice might lead to higher operative complications.st February 2019 to 13th January 2020 (P1) prior to pandemic; 3rd June 2020 to 11th January 2021 after 1st lockdown (P2); and 25th January to 30th July 2021 (P3) after/during second lockdown.Single-centre, electronic note review of consecutive patients undergoing CS during three periods: 1p = 0.54), but lower than P3 . There was no difference in PCR risk percentage scores between routine and PCR cases during P1 , P2 , or P3 . During P2 and P3, there was a higher rate of cystoid macular oedema (CMO) compared with P1 , with no differences in proportion of diabetics or cases with CMO in combination with PCR. There was no difference in surgeons grade experiencing PCR.2276 operations occurred during P1, 999 during P2, 846 during P3. During P1, posterior capsular rupture (PCR) rate was 1.67%, similar to P2 (1.30%, In P3 following 9 months of curtailed elective CS, PCR rates were increased across all surgeon grades, occurring in cases with similar risk percentage scores. CMO rates were increased during COVID and not related to proportion of diabetics or increased PCR rates. The reduction in elective CS during the pandemic was associated with more complications, perhaps due to attenuation of microsurgical skills. Cataracts are a major cause of blindness. With an aging population, population growth, and mounting rates of age-related diseases associated with cataract, the demand for cataract surgery (CS) is increasing . CS is tOn 11th March 2020, The World Health Organization declared the coronavirus (COVID-19) outbreak a global pandemic . In spriIn addition to this backlog, many ophthalmic surgeons, some for the first time, ceased undertaking micro-surgery with all its associated complexities and skill sets for several months, being redeployed to medical and critical care units or remaining within ophthalmic departments to provide emergency out-patient care. Breaks from performing regular surgery have been shown to lead to deterioration in technical skills . From obThe aim of this study was to investigate these issues by examining the electronic medical records (EMR) of all patients in our unit undergoing elective CS, with reference to surgical complications and case complexity, during two approximate 7-month periods following the first lockdown period in 2020 and during and after the second lockdown period in the UK in 2021 and comparing them to an 11-month period prior to the pandemic.This study was conducted at a public university hospital in the UK. It was approved by our institution’s audit and quality improvement project team (audit number 11822). Data collection adhered to the Tenets of the Declaration of Helsinki and UK Data Protection Act.Patient data were collected from our EMR system . As well as providing patient demographics, past medical history, details of consultations and peri-operative data, the software calculates a posterior capsular rupture (PCR) percentage risk rate where clinical characteristics, identified from the National (UK) Ophthalmology Database as risk factors for PCR , such asst lockdown (P 2), and 25th January 2021 to 30th July 2021 (P 3) after and during second lockdown. Documented findings collected included patient age, gender, right or left eye, pre-cataract surgery best-corrected distance visual acuity (BCDVA), final postoperative BCVA, cystoid macular oedema (CMO) rates, and diabetic status. Visual acuities were recorded in LogMAR acuity. Visual acuities of counting fingers, hand movements, light perception, and no perception of light were rounded up to 1.8 (LogMAR), 2.3, 2.8, and 3.0, respectively [All patients who underwent CS within our unit, performed between February 2019 and July 2021 were included. Patients that had cataract surgery combined with glaucoma, corneal or vitreoretinal surgery were excluded. Reference was made to demographic data, surgeon’s seniority, surgical complexity based on the PCR risk score and peri- and post-operative complications. The different time points examined were 1st February 2019 to 13th January 2020 (P 1) (prior to the COVID-19 pandemic), from 3rd June 2020 to 11th January 2021 after 1ectively .Statistical analysis was performed using GraphPad Prism version 8.0.0 for Mac . Data sets was assessed for normality using the Shapiro-Wilk test. For parametric data unpaired T-test was used to compare means between groups. For non-parametric data sets, the Mann-Whitney was used to compare the medians between different groups, and the Wilcoxon test to compare medians between the same groups. Fisher’s exact tests were used to compare the proportion of cases between groups.P < 0.0001)  and when P2 and P3 were compared (p = 0.0017). In terms of PCR with vitreous loss (PCRVL) there were 28 cases in P1 (1.23%), 7 in P2 (0.7%), and 27 in P3 (3.19%). There was an increase in the PCRVL rate when P1 and P3 (p = 0.006) and when P2 and P3 were compared (p = 0.001) Table There were 38 cases of PCR in P1 (1.67%), 13 in P2 (1.30%), and 30 in P3 (3.55%) (Table p = 0.18). In P3 the median risk score was 1.87% (0.27–33.80) in routine and 2.71% (0.42–7.89) in PCR cases (p = 0.08) (Table p = 0.28) and P3 (p = 0.32)  in routine and 2.03% (0.84–52.26) in PCR cases (0.83). The median risk score during P2 was 2% (0.27–37.12) in routine and 2.18% (1.36–10.97) in PCR cases (0–53.34 ip = 0.0001) increase in the CMO rate when P1 is compared with P2 (p < 0.0001), P3 (p < 0.0001) and P2 + P3 (p < 0.001). Amongst patients who developed CMO, the proportion of diabetics was similar between P1 and P2 , and P1 and P3  (Table p = 0.16), although rates of combined PCR and CMO were significantly higher in P1 compared with P2 (0%) (p = 0.047) and P2 and P3 combined (p = 0.026). There were no differences in pre-operative BCDVA measurements between eyes that experienced CMO and eyes that did not for P1 (0.3 LogMAR (0.0–2.8) vs. 0.3 LogMAR (−0.2 to 3.00) p = 0.62), P2 (0.36 LogMAR (0.02.8) vs. 0.3 LogMAR (−0.2 to 3.0) p = 0.36) and P3 (0.41 LogMar (0.0–2.8) vs. 0.4 LogMAR (−0.1 to 2.8) p = 0.76).There were 44 cases of post-operative CMO in P1 (1.93%), 49 in P2 (4.9%) and 58 in P3 (6.86%) (Table p = 0.30) and P3 (p = 0.24)   Table .Table 5PIn the UK, during the national COVID-19 lockdown periods, elective CS surgical ceased. In addition, large numbers of ophthalmic staff were redeployed, and it would have been challenging to deliver safe standards of elective care. Following the lockdowns, when elective activity resumed, to safeguard patients and health care professionals, several measures were introduced, including social distancing within clinical settings, asking patients to self-isolate at home and have proof of a negative COVID polymerase chain reaction 3 days prior to surgery. As such, the number of patients that could be operated per list was limited and our case numbers were reduced  (Table After and during the second lockdown (P3), when PCR and PCRVL rates significantly increased Table , patient1) Table , althougp < 0.0001) during COVID with 49 episodes (4.9%) in P2 and 58 (6.85%) during P3. This rise is likely to be multifactorial. It could be postulated that the longer waiting times for surgery in P2 and P3 may have led to more mature cataracts presenting on the day of surgery. One of the limitations of our study is that patients waiting for surgery because of lockdown restrictions were not recalled for re-examination prior to surgery and that PCR risk percentage scores are not routinely re-calculated on the day of surgery even in patients who have had an extended waiting time for CS. Given our currently high PCR and PCRVL rates this may be something that needs to be reconsidered. Potentially denser cataracts may result in the need for higher phacoemulsification cumulative dissipated energy (CDE) and operations taking longer than usual, resulting in increased post-operative inflammation. It has been postulated increased post-operative inflammation is a risk factor for CMO [During P2 and P3, there was an increased rate of CMO compared to P1 (Table  for CMO . UnfortuThere was no difference in the proportions of consultant surgeons operating on cases that experienced PCR in P1, compared to P2 and P3 Table . Whilst BCDVA appeared to be significantly improved in all eyes following surgery and in all periods in eyes both with and without PCR Table . This hiCovid 19 has had a significant impact on ophthalmology, and we continue to adjust our services in response to the evolving nature of the pandemic. Our findings of increased PCR and PCRVL rates in the presence of similar PCR percentage risk scores in P3, suggest that a hiatus of 9 months from regular routine high volume elective CS, perhaps due to micro-surgical skills degradation, is responsible for such operative complications. How issues such as the length of time away from undertaking micro-surgery, personal circumstances, and prior surgical experience can impact ophthalmologists’ performances on returning to CS and subsequent outcomes and complications is unknown and there is little research in this area . GeneralBreaks from performing surgery lead to deterioration in technical skills, especially fine motor skills after 6 months.During the COVID-19 pandemic lockdown periods all elective surgery, including cataract surgery which necessitates fine motor skills, was cancelled.After 9 months of curtailed elective cataract surgery rates of posterior capsular rupture increased across all surgeon grades and was not related to case complexity.Post-operative cystoid macula oedema rates increased during COVID and were not related to the proportion of diabetics or increased PCR rates.The reduction in elective cataract surgery during the pandemic was associated with more complications, perhaps due to attenuation of microsurgical skills.There appears to be a need for more support and guidelines for ophthalmic surgeons of all grades when they return to surgery after an extended hiatus, to improve patient safety.


Streptococcus pneumoniae serotype 6B sufficient to establish nasopharyngeal carriage in 40% of those nasally inoculated and evaluate nasal mucosal immunity before and after experimental inoculation.Persistent carriage of pneumococcal vaccine serotypes has occurred after introduction of PCV13 vaccination in Africa but the mechanisms are unclear. We tested the feasibility of using a human pneumococcal challenge model in Malawi to understand immune correlates of protection against carriage and to trial alternative vaccine candidates. We aimed to identify a dose of Streptococcus pneumoniae serotype 6B Post inoculation carriage was determined by nasal sampling for bacterial culture and lytA PCR. Immunological responses were measured in serum and nasal mucosal biopsies before and after bacterial inoculation.Healthy student volunteers were recruited and inoculated with saline, 20,000 CFU/naris or 80,000 CFU/naris of Twenty-four subjects completed the feasibility protocol with minimal side effects. pneumococcal carriage was established in 0/6, 3/9 and 4/9 subjects in the saline, 20,000 CFU/naris and 80,000 CFU/naris groups, respectively. Incidental  serotype carriage was common (7/24 participants carried non-6B strains, 29.2%. Experimentally induced type 6B pneumococcal carriage was associated with pro-inflammatory nasal mucosal responses prior to inoculation and altered mucosal recruitment of immune cells post bacterial challenge. There was no association with serum anti-capsular antibody.The serotype 6B experimental human pneumococcal carriage model is feasible in Malawi and can now be used to determine the immunological correlates of protection against carriage and vaccine efficacy in this population. Experimental human pneumococcal carriage has been used to measure humoral and cellular responses in mucosal and systemic compartments and as a method to test new vaccines in high income settings. However, the greatest burden of invasive pneumococcal disease is found in low- and middle- income countries, particularly in sub-Saharan Africa. Despite the roll out of pneumococcal conjugate vaccines in Malawi, vaccine serotype carriage remains high, leading to persistent population transmission and constraining herd effects. This is particularly problematic in the context of prevalent pneumococcal disease vulnerable populations  who are not eligible for vaccination under the WHO expanded programme on immunisation. There is currently poor understanding of the correlates of pneumococcal immunity and vaccine efficacy in these settings. Our group has conducted thirteen individual clinical studies with more than 1500 participants recruited in Liverpool, United Kingdom. A placebo-controlled trial of PCV13 demonstrated a 78% reduced carriage acquisition after nasal challenge in the United Kingdom. Multiple studies have demonstrated the safety of this predominantly asymptomatic colonisation model with mild symptoms of rhinorrhoea, sore throat, earache, fever and malaise infrequently reported.Streptococcus pneumoniae in Malawi, a low-resourced setting with high burden of invasive pneumococcal disease. This model and associated standard operating procedures were transferred from the United Kingdom. We showed that Malawian adult healthy volunteers can be safely challenged with serotype 6B pneumococcus, and that colonisation can be confirmed through classical microbiological culture of nasal wash. These observations were supported by findings of microbiological and immunological investigationsWe report the establishment of a controlled human infection model of S. pneumoniae controlled human infection model to Malawi and this is now ready to be used as a platform to understand host-pathogen interactions, evaluate immune responses to current conjugate vaccines and test new vaccine candidates.Pneumococcal epidemiology in sub-Saharan Africa is characterised by higher rates of carriage and transmission than HIC settings despite introduction of conjugate vaccines. We have safely transferred this Alt-text: Unlabelled box1Streptococcus pneumoniae infection in 2015 Even in the era of conjugate vaccines, 400,000 deaths in children under 5 years were attributed to For ten years, we have used an experimental human pneumococcal carriage model in the United Kingdom to study mucosal immunity against pneumococcal carriage and to test vaccine efficacy Given the knowledge gaps related to VT pneumococcal carriage in high burden countries, we conducted a feasibility study to safely establish experimental pneumococcal carriage in Malawi and used novel nasal microbiopsy techniques to evaluate mucosal immunity before and after experimental inoculation. This work aims to evaluate which vaccines can prevent acquisition of pneumococcal carriage in Malawi, and to determine the critical mechanisms of respiratory tract defence that may be harnessed to develop more effective vaccines suitable for low- and middle-income country use.22.1https://wellcomeopenresearch.org/articles/5–25)10. Briefly, healthy participants aged 18–40 years consented to nasal inoculation of Streptococcus pneumoniae serotype 6B to determine the proportion who subsequently developed colonisation. The majority of participants were undergraduate students from adjacent university campuses. Fluency in written Chichewa or English (to follow safety instructions) were inclusion criteria but no other sampling frame restrictions were imposed This study adhered to a published protocol  in blocks of 12 participants, each block randomised 3:1 with 9 participants receiving pneumococci and 3 receiving saline (0.9%). Rather than using a formal power calculation for this feasibility study, we hypothesised that the infection rate in Malawi would be similar to that in the UK and therefore that a dose of 80,000 colony forming units per naris would be sufficient to establish nasal carriage in 40–50% of participants 2.2The study was ethically approved in Malawi by the National Health Sciences Research Committee  and in the United Kingdom by the Liverpool School of Tropical Medicine . LSTM also sponsored the study. Informed written consent was recorded for all participants. Deidentified participant data will be made openly available one year after the publication date using the Harvard Dataverse platform2.3lytA by PCR on nasal wash samples. Secondary endpoints were symptoms, mucosal and systemic immune parameters. All subjects completed exit questionnaires to assess study experience.We determined participant symptoms including expected adverse events with open and targeted questions at every clinic visit. Intermittent sampling was performed for immunological analysis at 5 days before intranasal pneumococcal inoculation, and then on days 2, 7, and 14 afterwards: at these time points we collected nasal wash for bacterial detection, peripheral blood, nasal scrapes and nasosorption samples. The primary endpoint was detection of the inoculated pneumococci serotype (6B) by classical culture from nasal wash recovered from the participants at any of days 2, 7 and 14 after pneumococcal challenge. Nasal wash is established as the diagnostic technique from the Liverpool model due to the theoretical risk of nasopharyngeal trauma and pneumococcal invasion from nasal swabbing. In parallel, we confirmed carriage by detection of pneumococcal 2.4S. pneumoniae serotype 6B BHN418 was thawed, centrifuged and washed before being resuspended in 0.9% normal saline at the pre-specified concentration . The prepared inoculum was taken immediately (less than 5 min transit) to the clinical area where 100 µl was instilled into each nostril of the participant Briefly, as described in our study protocol lytA PCR.Nasal wash was collected as described elsewhere 2 for 18–24 h and inspected to identify pneumococcal phenotype. Serotype  was confirmed by latex agglutination using Immulex Pneumotest reagents (Statens serum institute).To quantify colonization density by culture, serial dilutions of the pellet, in 100 µl STGG, were plated on Columbia sheep Blood Agar  containing 4 mg/ml gentamicin (CBG). Plates were incubated at 37 °C with 5% CO2.5lytA qPCR. PCR amplification of the lytA gene was carried out using forward primer (5′-ACGCAATCTAGCAGATGAAGCA-3′) and reverse primer (5′-TCGTGCGTTTTAATTCCAGCT-3′). Master mix was prepared using 12 µl of DEPC-treated H2O; 0.225 µl of each primer (100 µM); 0.125 µl of probe (100 µM); 12.5 µl of Taq. The total volume of PCR reaction used was 25 µl, containing: 22.5 µl of the prepared master mix and 2.5 µl of sample in a set of duplicate wells. Finally, 2.5 µl of lytA plasmid  making up the standard curve was added into each of two wells, beginning with the lowest concentration standard (1 × 10° copies/µl) to the highest concentration (1 × 105 copies/µl). The plate was loaded in a thermocycler  with the following amplification conditions: 95 °C for 10 min; followed by 40 cycles of 95 °C for 15 s and then 60 °C for 1 min.Genomic bacterial DNA was isolated from nasal wash pellets stored in STGG using the Agowa Mag mini-DNA extraction kit  as per manufacturer's instruction using a 300 µl aliquot of the stored nasal wash STGG pellet . The eluted DNA was stored at −20 °C for use in 2.6Nasal microbiopsies were obtained from the inferior turbinate of each participant using microcurettes without local analgesia. Each sample was dropped immediately into transport medium and transferred to the laboratory in less than 10 min. For immunophenotyping, nasal cells were dislodged from curettes by gentle pipetting and suspension. Cells were then live-stained with an antibody cocktail containing anti-human CD3 APC, anti-human CD19 Brilliant Violet 510, TCR Gamma Delta FITC and anti-human CD45 Alexa Fluor 700 , anti-human CD14 PE-Cy7 (BD Biosciences), anti-human CD66b PE (eBiosciences) and MR1 Tetramer Bv421 (NIH Tetramer Core Facility). After fixation, samples were acquired using standard methods on an LSR FORTESSA flow cytometer (BD Biosciences) and analysed using Flowjo v10.5.3 (BD Biosciences).2.7Nasosorption filters were gently applied to the nasal mucosa, transferred in dry collection tubes and stored at −80 °C. Cytokines were eluted from stored nasosorption filters  Ltd., Midhurst, UK) using 200  μl of elution buffer (Millipore) by centrifugation at 1500 g, then the eluate was cleared by further centrifugation at 1595 g. The samples were acquired on a MAGPIX  using a 38-plex magnetic human cytokine kit (Millipore) and analysed with xPONENT software following the manufacturer's instructions. The analytes included sCD40L, EGF, Eotaxin/CCL11, FGF-2, Flt-3 ligand, Fractalkine, G-CSF, GM-CSF, GRO, IFN-α2, IFN-γ, IL-1α, IL-1β, IL-1ra, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IP-10, MCP-1, MCP-3, MDC (CCL22), MIP-1α, MIP-1β, TGF-α, TNF-α, TNF-β and VEGF.2.8Anti-pneumococcal capsular PS antibodies were determined in serum using the World Health Organization internationally standardized method and reagents 2.9p-value of < 0.05 was considered statistically significant, all p-values are two-tailed. All cytokine data were log10-transformed and summarised by medians and IQRs.Statistical analyses were performed using R v.4.0.2  and GraphPad Prism v9.0.0 . Non-normally distributed quantitative measurements are summarised by the median and inter-quartile range (IQR). Exact binomial confidence intervals are reported for estimated proportions. Categorical variables were compared using Fisher's exact test. The nasal cell and serum IgG antibody data were analysed using Friedman test with correction for multiple comparisons using Dunn's multiple comparisons test. A 2.10The funder and sponsor have no role in study design; collection, management, analysis, and interpretation of data; writing of the report; or the decision to submit the report for publication.33.1a priori defined stopping criteria S. pneumoniae serotype 6B strain BHN418 20,000 colony forming units (cfu)/naris or normal saline  and 12 subjects were randomised  to the higher inoculum of 80,000 cfu/naris or normal saline . All inoculated participants completed the study follow up as per protocol, and all reported a satisfactory experience. The study commenced November 2019 and was paused (at n = 20 recruited participants) due to the emerging COVID-19 pandemic in March 2020. The study recommenced in October 2020 with additional safety procedures . Participant characteristics are described in Twenty-nine participants were screened and 24 inoculated  before t3.2lytA density (copies per ml) is provided in the supplementary material (Table S1). We found high concordance between classic microbiological culture and lytA PCR positivity for S. pneumoniae . Serotype 6B specific concordance could not be calculated as lytA detection cannot distinguish different serotypes.Inoculation with pneumococcal serotype 6B, resulted in 3/9 (33% [CI 7–70]) and 4/9 (44% [CI 14–79]) of participants developing nasal colonisation, in the 20,000 CFU and 80,000 CFU groups, respectively. The inoculation doses (median (IQR) of serotype 6B were 21,834  and 91,667  for the 20,000 and 80,000 CFU groups, respectively. The prevalence of incidental carriage was relatively common with non-6B serotypes identified in 7/24 (29.2% [13–51]) participants  These in3.3p = 0.012, Friedman test with Dunn correction), but this was not observed in the experimental carriage positive individuals or those that received normal saline , but was similar with those that received normal saline     a. Neutrorection) b–c, likerection) d, suggesrection) d. In add3.4Nasal inflammation has been associated with increased propensity for experimental pneumococcal carriage 3.5p > 0.05, Friedman test with Dunn correction). These findings suggest that experimental nasal pneumococcal challenge or experimental human pneumococcal carriage did not induce serum anti-6B polysaccharide IgG levels beyond natural exposure in this setting (Serum anti-6B polysaccharide IgG antibody levels have been shown to increase in subjects that establish pneumococcal carriage following nasal experimental challenge with serotype 6B  setting . Further4This study has shown for the first time that experimental human pneumococcal carriage can be safely carried out in Malawi. Malawi has very different pneumococcal epidemiology and biology compared to Europe likely underpinned by differences in human-pathogen interaction as well as the physical environment, and socio-economic circumstances including the effects of crowding. Our primary objective was achieved in that our study showed that recruitment, screening of volunteers and safe completion of the study per protocol was feasible with local adaptation of the protocol initially developed in the UK. Observed carriage rates were consistent with that observed in the UK and the model behaved similarly. There were no specific local difficulties with recruitment, inoculum was delivered accurately and safely and the rates of experimental pneumococcal carriage mirrored those from our Europe-based studies.Our secondary objectives were to measure carriage symptoms and to determine if immunological sampling would be possible in sufficient yield to study key differences in carriage epidemiology observed between the UK and Malawi This study in Malawi shows some similarity with work previously published from Liverpool, UK. The predominantly student population had no difficulty in completing the protocol lytA PCR as a confirmatory method of determining carriage was much lower than in the UK. The frequency of naturally acquired carriage episodes in Malawi is therefore a factor that must be considered in experimental design using the human challenge model in future studies. Specifically, it may be important to use molecular methods to characterise the several serotypes present in the nasopharynx. It was surprising, however, that we saw no evidence of increased experimental carriage in those participants already experiencing natural carriage as our previous studies of the nasal microbiome suggested a “carriage tolerant” phenotype in participants with pre-existing pneumococcal colonisation There were, however, some important differences in our study from the UK experience and from our expected results. The baseline and incidental naturally acquired pneumococcal carriage rate were higher than that in Liverpool, and so the usefulness of The main strength of this study is that it shows experimental human pneumococcal carriage to be feasible in Africa, a continent with a residual burden of pneumococcal disease that may not be controlled by current conjugate vaccination strategy. A further strength of the study is that we piloted the use of sophisticated mucosal analyses to unpick immunological differences between Europe and Africa. This feasibility study has generated a number of hypotheses that should now be tested in larger cohorts, most important being the association of nasal mucosal inflammation with frequent pneumococcal carriage.The main limitations in this study are related to the small numbers of participants. Larger studies will confirm that the rate of reported symptoms remain low. Further, we achieved accurate challenge dosing of controlled shipment from Liverpool ,11. It rAll authors read and approved the final version of the manuscript • Conceptualization BM, KJ, SB, EN, LMT, MYRH, NPB, JR, DMF, SBG • Data Curation and Verification BM, KJ, SB, TC, EN, LMT, MYRH, SBG • Funding Acquisition BM, KJ, JR, DMF, SBG • Investigation BM, KJ, SB, TC, EN, LMT, MYRH, NPB, JR, DMF, SBG • Methodology BM, KJ, SB, TC, EN, LMT, MYRH, NPB, JR, DMF, SBG • Project Administration BM, KJ, SB, EN, LMT, SBG • Resources BM, KJ, JR, DMF, SBG • Supervision BM, KJ, SB, TC, LMT, SBG • Validation BM, KJ, SB, EN, TC • Visualization BM, KJ, SB, TC, MYRH, SBG • Writing – Original Draft BM, KJ, SBG • Writing – Review & Editing BM, KJ, SB, TC, EN, LMT, MYRH, NPB, JR, DMF, SBG.The authors declare no competing interests.


Here we investigated the effects of in utero or postnatal exposure to cis-9, trans-11  and 10,12 CLA on postnatal development of the mammary gland and its responsiveness to ovarian steroids. In the first experiment we fed dams different CLA prior to and during gestation, then cross fostered female pups onto control fed dams prior to assessing the histomorphology of their mammary glands. Pregnant dams in the second experiment were similarly exposed to CLA, after which their female pups were ovariectomized then treated with 17β-estradiol (E), progesterone (P) or E + P for 5 days. In a third experiment, mature female mice were fed different CLA for 28 days prior to ovariectomy, then treated with E, P or E + P. Our data indicate that 10,12 CLA modifies the responsiveness of the mammary glands to E or E + P when exposure occurs either in utero, or postnatally. These findings underline the sensitivity of the mammary glands to dietary fatty acids and reinforce the potential for maternal nutrition to impact postnatal development of the mammary glands and their risk for developing cancer.We previously showed that dietary The online version contains supplementary material available at 10.1007/s10911-021-09499-y. The in utero environment impacts postnatal development, organ function, and susceptibility to disease , 2 and cDevelopment of the mammary glands begins in utero with patterning of the milk lines, which, in mice, give rise to 5 placode pairs that develop into individual glands along the mammary ridge . Cross-tDietary fats can affect normal development of the mammary glands, and these responses can increase susceptibility of the mammary epithelium to carcinogenesis. Indeed, female offspring of rats fed a high-fat diet (HFD) during pregnancy had more TEB during puberty , which icis and/or trans configuration. These fatty acids occur naturally in meat and milk from ruminants, predominantly as the cis-9, trans-11  isomer [trans-hydrogenation of vegetable oils yields multiple CLA isomers including trans-10, cis-12 . Dietary 10,12 CLA dramatically reduces adiposity in rodents [Conjugated linoleic acids (CLA) are 18:2 fatty acids with conjugated double bonds in a ) isomer , whereas rodents , 15, lea rodents . This cl rodents , increas rodents  in their rodents . Given t rodents , we hypo rodents , we alsoAll experiments were approved by the University of California, Davis Institutional Animal Care and Use Committee. Balb/cJ mice  had ad libitum access to food and water and were housed under a 14 h light:10 h dark cycle. All diet formulations were based on a modified AIN93G control diet . Experimental diets were the control diet with 1% fat (by weight) replaced with 9,11 or 10,12 CLA. The content of 9,11 and 10,12 CLA in the experimental diets was 6.39 and 6.81% of total fatty acids , were assigned to one of the following diet regimens: (1) control diet (n = 2), or that with 1% fat replaced by either (2) 9,11 (n = 1) or (3) 10,12 CLA (n = 1) for the entire gestation. At day 19 of gestation, dams were euthanized by CO2 inhalation, followed by exsanguination .Eight-week old female mice were mated and upon detection of a seminal plug (day 1 of gestation) were assigned to one of the following diet regimens: (1) the control diet, or that with 1% fat replaced by either (2) 9,11 or (3) 10,12 CLA for the entire gestation, (4) the control diet for the first 10 days of gestation followed by the 10,12 CLA diet for the remainder of gestation, (5) or the 10,12 CLA diet for the first 10 days of gestation followed by the control diet until parturition  the control diet, or that with 1% fat replaced by either (2) 9,11 or (3) 10,12 CLA for the entire gestation, or (4) the control diet for the first 10 days of gestation followed by the 10,12 CLA diet for the remainder of gestation  inhalation prior to euthanasia (n = 4–13/hormone/diet), as described above.Sexually-mature female mice (56-70d of age) were fed either the control diet or that containing 9,11 or 10,12 CLA for 28d before they were OVX as described above. After a period of 7d to clear endogenous ovarian hormones, mice were administered daily injections of vehicle , E (1 μg/d 17β-estradiol), P (1 mg/d), or E + P, for 4d. On the final day of hormone treatment mice were administered BrdU  2 h before euthanasia by carbon dioxide  were fixed overnight in 4% paraformaldehyde (Experiment 1) or Tellyesniczky’s Fixative (Experiment 2), dehydrated, and embedded in paraffin. Whole mounts prepared from inguinal mammary glands  were demounted and oriented in paraffin so that the mammary ducts between the teat and the lymph node could be sectioned transversely. Sections (4–5 μm) were stained with either hematoxylin and eosin, or Gomori’s trichrome.Paraffin sections (4–5 μm) of thoracic mammary glands were subjected to immunohistochemistry, either for BrdU using a biotinylated anti-BrdU monoclonal antibody  as described , or a biAnalysis of fatty acids in lipids extracted from diets was performed as described . Lipids P ≤ 0.05.Data were analyzed by one- or two-way ANOVA using the Proc GLM procedure in SAS  or using Prism8 (Graphpad Software), followed by a post-hoc Dunnet or Tukey test while controlling for multiple testing. Data were transformed where appropriate. Least square means comparisons were considered significant at P < 0.05, Table P < 0.05, Table To confirm maternal–fetal transfer of CLA we first analyzed the fatty acid profile of whole fetuses at day 19 of gestation that had been carried by dams fed 9,11 or 10,12 CLA during gestation. The level of 9,11 CLA was increased in fetuses from dams fed the 9,11 CLA diet compared to those from dams fed 10,12 CLA or the control diet during gestation . The highest % of 9,11 CLA in the mammary glands was in 21-day old mice exposed to 9,11 CLA in utero . Amongst OVX mice not treated with hormones, offspring from dams fed 10,12 CLA for the second half of gestation had heavier mammary glands compared to those born to control-fed dams, while offspring from dams fed 10,12 CLA throughout gestation had smaller mammary glands , with a smaller increase induced by E + P  but was reduced in response to dietary 10,12 CLA Supplementary file2 (TIFF 1751 KB)Supplementary file3 (TIFF 12591 KB)Below is the link to the electronic supplementary material.


Type I interferons (IFN-I) exhibit various biological effects during viral infections, and they have been successfully used for clinical treatment of viral diseases. Humans express 12 IFNα subtypes, which strongly differ in their antiviral responses against different viruses. Here we analyzed the antiviral activity of all human IFNα subtypes against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to identify the underlying immune signatures and explore their therapeutic potential. Our data provide a systemic pattern of antiviral host effector responses mediated by high antiviral IFN-I, which could help to identify key cellular effectors targeted in novel therapeutic approaches against SARS-CoV-2 infection. Type I interferons (IFN-I) exert pleiotropic biological effects during viral infections, balancing virus control versus immune-mediated pathologies, and have been successfully employed for the treatment of viral diseases. Humans express 12 IFN-alpha (α) subtypes, which activate downstream signaling cascades and result in distinct patterns of immune responses and differential antiviral responses. Inborn errors in IFN-I immunity and the presence of anti-IFN autoantibodies account for very severe courses of COVID-19; therefore, early administration of IFN-I may be protective against life-threatening disease. Here we comprehensively analyzed the antiviral activity of all IFNα subtypes against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to identify the underlying immune signatures and explore their therapeutic potential. Prophylaxis of primary human airway epithelial cells (hAEC) with different IFNα subtypes during SARS-CoV-2 infection uncovered distinct functional classes with high, intermediate, and low antiviral IFNs. In particular, IFNα5 showed superior antiviral activity against SARS-CoV-2 infection in vitro and in SARS-CoV-2–infected mice in vivo. Dose dependency studies further displayed additive effects upon coadministration with the broad antiviral drug remdesivir in cell culture. Transcriptomic analysis of IFN-treated hAEC revealed different transcriptional signatures, uncovering distinct, intersecting, and prototypical genes of individual IFNα subtypes. Global proteomic analyses systematically assessed the abundance of specific antiviral key effector molecules which are involved in IFN-I signaling pathways, negative regulation of viral processes, and immune effector processes for the potent antiviral IFNα5. Taken together, our data provide a systemic, multimodular definition of antiviral host responses mediated by defined IFN-I. This knowledge will support the development of novel therapeutic approaches against SARS-CoV-2. Without the capacity to produce or recognize interferons (IFN), mammalian hosts rapidly succumb in the case of viral infections. Accordingly, humans with loss-of-function mutations in the IFN signaling pathway even fail to control attenuated viruses. Therefore, IFNs are indispensable mediators of the first immediate intrinsic cellular defenses against invading pathogens, such as viruses. So far, three different types of IFNs, types I (IFN-I), IFN-II, and IFN-III, have been identified and classified based on their genetic, structural, and functional characteristics as well as receptor usages –3. IFN-I5The outbreak of novel viruses, as exemplified by the recent emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the disease COVID-19, has emphasized the urgent need for fast and effective therapeutic strategies. Indeed, IFN-I treatment is currently being explored as an emergency treatment against COVID-19 in various clinical trials –23, and 2650 per milliliter) and genome amplification. Interestingly, we observed a differential antiviral pattern for the individual subtypes, with IFNα5, α4, α8, α14, and IFNλ3 exhibiting the strongest antiviral effects with up to 105-fold reduction in viral titers . Immunofluorescence analysis of VeroE6 cells pretreated with IFNα5, IFNα7, and IFNα16 confirmed their different antiviral activities against SARS-CoV-2 . To determine the inhibitory concentration 50 (IC50), we performed dose–response analyses covering concentrations from 19 U/mL to 80,000 U/mL for the pretreatment. SARS-CoV-2 replication was assessed by quantification of viral titers (Tissue Culture Infection Dose 50 [TCID50] per milliliter) and viral antigens applying a previously described in-cell enzyme-linked immunosorbent assay (icELISA) . Corroborating previous results, a striking clustering of the antiviral subtypes according to their antiviral potency was observed, which allowed their separation into classes of low , intermediate  and high  antiviral activities against SARS-CoV-2  were determined. Again, a distinct antiviral pattern became evident . Prototypical ISG expression patterns, as analyzed by qRT-PCR, revealed subtype-specific gene expression signatures . In order to address whether the observed antiviral activities were SARS-CoV-2 specific, we additionally tested IAV (IAV/PR8) in hAECs. Interestingly, pretreatment of hAECs with the IFN subtypes revealed differences compared to SARS-CoV-2. In general, antiviral responses could be clustered into strong antiviral activities for IFNα2, IFNα4, IFNα5, IFNα8, IFNα14, and IFNλ3  for 16 h. This time point was chosen to include early and late ISGs into our analyses. We included IFNλ3  as a positive control, since its potent antiviral activity against SARS-CoV-2 and other respiratory pathogens has been documented , 32. FolRS-CoV-2 B. AnalysicELISA)   compared to mock-treated cells. Similar to the observed antiviral effects, a general clustering was apparent which showed similar expression patterns for low to intermediate antiviral subtypes  and intermediate to high antiviral subtypes . Interestingly, we observed a clear difference in the numbers of significantly up- and down-regulated genes after treatment with IFNα subtypes compared to mock-treated cells, which positively correlated with antiviral activity . Gene ontology (GO) pathway analysis revealed higher expression of genes mostly involved in antiviral immune response among the medium and high antiviral subtypes, as well as pathways which can be associated with protein localization, translation, oxidative phosphorylation, RNA metabolism, endoplasmic reticulum (ER) stress, signaling pathways, and lymphocyte activation . We identified several DEGs for each subtype, with IFNα5 expressing the most unique genes , followed by IFNλ3 (670 DEGs) . A comparison between high, medium, and low antiviral subtypes revealed that 19 genes were commonly differentially expressed among all subtypes, including MX1 and OAS2  RNAs  were sent to Ingenuity Pathway Analysis  for biological analysis. In order to investigate cellular responses following viral infection, we included SARS-CoV-2–infected hAECs (18 h p.i.) in our analysis. Global transcriptomic analysis revealed unique DEGs, both up- and down-regulated upon IFN treatment , 39 for tivation A. Strikitivation A. We nextivation B. A sepativation B. We obsand OAS2 D. The mosubtypes D. Interenc) RNAs E. In conSI Appendix, Fig. S5A). Principal component analysis revealed a clustering according to donor and/or infection and time points . In addition to host cell proteins, various viral peptides were identified, which correlate to viral titers depending on the respective donor . For all donors, no SARS-CoV-2 peptides could be detected following treatment with IFNα5 and IFNλ3. Pretreatment of cells with IFN subtypes resulted in up- or down-regulation of a variety of proteins compared to untreated hAECs, depending on the IFN stimulation . In order to perform statistical analysis, we considered proteins that were measured in a minimum of three of four donors; however, on/off analysis (defined as full absence of a protein in one group of a pairwise comparison) revealed additional proteins which might be of interest . GO analysis of proteins differentially abundant between untreated and IFN-treated samples at each time point (untreated vs. IFN) identified enrichment of antiviral immune responses for all IFNs except IFNα16 . For IFNα16, only proteins associated with lymphocyte regulation were induced, which likely do not contribute to SARS-CoV-2 restriction in cell culture but may be very important in vivo. At 72 h, pathways belonging to proteolysis, metabolism, and protein localization were additionally enriched after treatment with IFNα5 and IFNλ3. The most prominent up-regulated proteins, associated with IFN signaling , were found to be on–off regulated and present only upon treatment with IFNα5, IFNα7, and IFNλ3. Additional ISGs, including IFIT3, OAS2, and IFITM3, were on–off regulated after 72 h and SARS-CoV-2 infection, except for IFNα16 treatment . Interestingly, the comparison of samples in the presence or absence of SARS-CoV-2 (mock vs. CoV-2) showed a striking trend toward down-regulation of proteins upon SARS-CoV-2 infection. Enrichment of biological processes associated with complement activation and O-glycan processing . In contrast, the strongest biological effects on antiviral immune responses after treatment with IFNα5 and IFNλ3, for example, IFN signaling as well as antigen presentation, nuclear factor κB signaling, or lymphocyte regulation, were not affected by viral infection. Interestingly, proteins belonging to other pathways, for example, antigen presentation by major histocompatibility complex (MHC) class I or proteolysis, seemed to be less abundantly represented under viral infection in the IFNα5-treated samples, a phenomenon which was not as prominent after treatment with IFNλ3 . STRING  analysis , where we aim to identify key cellular factors that are expressed before viral infection, we included a late time point, 72 h posttreatment both in the presence (t = 72 h [CoV-2]) and absence of viral infection (t = 72 h [mock]), to investigate potential antiviral mechanisms and potential intervention by viral effectors  . Given the clear antiviral but incomplete inhibitory effect of both treatment modalities, we next studied a potential beneficial effect of IFNα5 or IFNα2 when coadministered with remdesivir . Likewise, posttreatment in combination with remdesivir resulted in a dose-dependent, additive viral inhibition with over 70% (−/−γc−/−CD47−/− triple-knockout (TKO) mice, which received human fetal lung transplants (humanized lung-only mice [LoM]). Treatment for 4 d with the highly antiviral IFNα5, starting 2 h postchallenge with SARS-CoV-2, significantly reduced viral titers in human lung organoids  , 41 for icELISA) A and B. icELISA) B. IntrigicELISA) B, which over 70% D and F fover 70% G–I. To frganoids J. Taken IFN-I serve as one of the first lines of defense and are induced almost immediately upon viral encounters. IFN-I foster intrinsic immunity, stimulate innate immunity, and recruit and orchestrate adaptive immunity. They can modulate the immune system in several ways, by exerting a wide range of biological activities including antiviral, antiproliferative, immunomodulatory, and regulatory activities. Importantly, impaired IFN-I activity are correlated with severe courses of COVID-19, highlighting their clinical importance . Accordi446 VeroE6 cells were seeded in a T75 flask and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), L-glutamine, penicillin, and streptomycin. The next day, the cells were infected with isolated virus, and, after 3 d of incubation, the supernatant was harvested, and cell debris was removed by centrifugation. Aliquots of the supernatant were prepared and stored at −80 °C. Viral titers were determined by performing an end-point dilution assay or a plaque assay in order to calculate the TCID50 or the plaque-forming units (PFU), respectively. Virus stock was sequenced and assigned to B.1.1.10 according to the Pangolin database  gel. By phase separation of the products with Triton X-114, the remaining endotoxin was removed from the solution. Endotoxin levels were tested using ToxinSensor (GenScript) and are below 0.25 EU/mL. The activity of each subtype was determined using the human ISRE-Luc reporter cell line, a retinal pigment epithelial cell line transfected with a plasmid containing the Firefly Luciferase gene, stably integrated under control of the ISRE. Following stimulation with IFNα, chemiluminescence can be detected and used to calculate the respective activity in units against commercially available IFNα (PBL Assay Science) (IFNα subtypes were produced and purified as previously described . BrieflyScience) . Protein2 atmosphere. Thereafter, the supernatant was aspirated, and the cells were incubated in 100 µL of crystal violet solution  for 5 min. Subsequently, the crystal violet solution was aspirated, cells were washed with PBS, and the number of wells with intact or damaged cell layer was determined. The TCID50 per milliliter was calculated by the Spearman & Kärber algorithm.VeroE6 cells were seeded at a density of 10,000 cells per well in a 96-well plate and maintained in 200 µL of DMEM supplemented with 10% FBS, L-glutamine, and penicillin and streptomycin overnight. The next day, 22 µL of virus stock or apical washes of hAEC were added to the first row of the plate (six replicates). Then, the virus was diluted 1:10 by mixing the media and pipetting 22 µL to the next row repeatedly, followed by 72 h incubation in 37 °C in a 5% CO2 atmosphere. Thereafter, the supernatant was aspirated, and the cells were stained with 100 µL of crystal violet solution  for 5 min. Subsequently, the crystal violet solution was aspirated, cells were washed with PBS, and the number of wells with intact or damaged cell layer was determined.VeroE6 cells were seeded at a density of 10,000 cells per well in a 96-well plate and maintained in DMEM supplemented with 10% FBS, L-glutamine, penicillin, and streptomycin overnight. Then, the medium was aspirated, and serially diluted IFNα subtypes and IFNλ3 (R&D Systems) and virus with a final concentration of 350 PFU/mL were added to the cells in a total volume of 100 µL of cell culture media, followed by 72 h incubation in 37 °C in a 5% CO50 was calculated using GraphPad Prism 6.The IC2 atmosphere. Thereafter, 100 µL of 8% ROTIHistofix (Roth)  were added for a minimum of 2 h at room temperature to fix the cells and inactivate the virus.The icELISA was performed based on the previously published protocol . Briefly2SO4 (Roth). The absorbance was measured at 450 nm with a reference wavelength of 620 nm using Spark 10M multimode microplate reader (Tecan).Afterward, the plate was washed thrice with PBS. The PBS was aspirated, 200 µL of freshly prepared permeabilization buffer  were added to the cells, and the plate was incubated for 30 min at room temperature under constant shaking. Subsequently, the permeabilization buffer was aspirated, and 200 µL of blocking buffer  were added for 1 h. Then, the blocking buffer was aspirated, and 50 µL of primary antibody solution  was added to each well. The plate was incubated overnight at 4 °C. The next day, the primary antibody solution was aspirated, and the plate was washed thrice with wash buffer . Thereafter, 50 µL of the secondary antibody solution  was added to the wells, and the plate was incubated for 2 h at room temperature. After the incubation period, the wells were washed four times with 250 µL of wash buffer. Afterward, 100 µL of tetramethylbenzidine (TMB) substrate solution (BioLegend) were added, and the plate was incubated for about 20 min at room temperature in the dark. The reaction was stopped by addition of 100 µL of 2N HTo exclude cytotoxic effects of the compounds used in our assays, a cell viability assay was performed using the Orangu Cell Counting Solution (CELL Guidance Systems) according to the manufacturer’s instructions. The cells were seeded and treated equally to the protocol that was used before without any viral infection. Afterward, 10 µL of Orangu Cell Counting Solution were added to each well, and the plate was incubated for 2 h. Then, the absorbance was measured at 450 nm with Spark 10M multimode microplate reader.VeroE6 cells were seeded and treated as described for the icELISA. After incubation with the primary antibody solution, 50 µL of secondary antibody solution  were added to each well, and the plate was incubated for 2 h at room temperature. Thereafter, the secondary antibody solution was aspirated, and the cells were counterstained for 20 min at room temperature with 50 µL of DAPI solution (0.1 µg/mL DAPI [Sigma-Aldrich] in PBS). Subsequently, the plate was washed thrice with PBS and microscopically analyzed using Leica THUNDER Imager 3D Cell Culture.Fetal tissues for reconstitution of humanized mice were obtained through anonymous donations with informed written consent via Advanced Bioscience Resources under the University of Saskatchewan Research Ethics Board Bio ID-371. All animal studies were performed under University of Saskatchewan’s Animal Research Ethics Board protocols 20180079 and 20200016 and adhered to Canadian Council on Animal Care guidelines.−/−γc−/−CD47−/− TKO mouse as the immunocompromised recipient mouse strain. Briefly, two pieces (∼2 mm3 to 4 mm3) of 17- to 22-wk-gestation human fetal lung (Advanced Bioscience Resource) were implanted subcutaneously onto the backs of mice. Subcutaneous wounds were closed with surgical glue. Lung organoids were allowed to grow to ∼1 cm in diameter prior to use in experiments. Mice were generated from three donors.LoM were generated as previously described , 65 exce50 assay, and sequenced. The virus stock was clarified by centrifugation at 4,700 × g for 10 min and stored at −80 °C until thawed for infections. The TKO-LoM mice were infected by direct injection of SARS-COV-2 (1 × 105 TCID50 in 50 µL) or vehicle control into each human lung organoid. Mice received daily intraperitoneal injections of 1.5 × 105 U of IFNα2 or IFNα5 or vehicle control for 4 d starting 2 h postchallenge. Mice were killed on day 5, and lung organoids were harvested for infectious virus assay.The B.1.1.7  variant was isolated on Vero76 cells (ATCC) from a clinical specimen kindly provided by Graham Tipples and Kanti Pabbaraju at Alberta Health Services, Edmonton, AB, Canada. The virus was subsequently propagated, and a p.2 stock was generated, titrated on Vero76 cells by conventional TCIDg for 5 min and then serially diluted 10-fold in DMEM supplemented with 2% heat-inactivated FBS and 2× penicillin–streptomycin. Sample volumes of 50 µL were added to 96-well plates of 95% confluent Vero76 cells in triplicate and incubated at 37 °C with 5% CO2 before scoring for the presence of cytopathic effects.Lung organoids were weighed and placed in 1 mL of DMEM supplemented with 1% heat-inactivated FBS, 1× L-glutamine before being homogenized in a Tissuelyser II Homogenizer (Qiagen) at 30 Hz for 6 min. Tissue homogenates were clarified by centrifugation at 5,000 × 2).The hAEC were obtained from lung transplant donors postmortem (ethics of University Duisburg-Essen 18-8024-BO and 19-8717-BO) or from explanted lungs . Selection criteria for donors are listed in the Eurotransplant guidelines. The hAECs from explanted lungs were cultured and differentiated as previously described . The hAE2 atmosphere. Thereafter, the inoculum was aspirated, and the cells were washed thrice with 150 µL of HBSS for 10 min. The last wash was collected and stored at −80 °C as a 0-h sample. At the indicated time points, cells were washed apically for 10 min, and the washes were subjected to an end-point dilution assay or to a plaque titration assay as described for SARS-CoV-2 and influenza, respectively.Fully differentiated hAECs were washed with Hanks’ balanced salt solution (HBSS) apically for 10 min before infection. For SARS experiments, the cells were infected apically with 30,000 PFU diluted in HBSS; for influenza, the cells were apically infected with IAV H1H1 strain A/Puerto Rico/34 (PR8) at 0.1 multiplicity of infection in 200 µL of HBSS. The cells were incubated with the inoculum for 1 h in 33 °C in a 5% COTreatment of hAECs was performed by adding the indicated amounts of IFNs or remdesivir directly to the cell culture medium on the basolateral side of the cells.For the isolation of RNA, cells were lysed using Qiagen RLT buffer (Qiagen) supplemented with 1% β-mercaptoethanol (Sigma-Aldrich).Total RNA was purified from hAECs and VeroE6 cells using the RNeasy Mini Kit (Qiagen) according to manufacturer’s instructions with preceding DNase I digestion with the RNase-Free DNase Set (Qiagen).M- or N-gene expression, 500 ng of total RNA were reverse transcribed using the PrimeScript RT Master Mix (Takara). Promega’s GoTaq Probe qPCR Master Mix was used according to the manufacturer’s instructions, with gene-specific primers and probes . RT-qPCR was performed on a LightCycler 480 II (Roche) instrument, with the following conditions: Initial denaturation was 2 min at 95 °C and a ramp rate of 4.4 °C/s, followed by 40 cycles of denaturation for 15 s at 95 °C and a ramp rate of 4.4 °C/s and amplification for 60 s at 60 °C and a ramp rate of 2.2 °C/s. To assess M- and N-gene copy numbers, the M- and N-genes were partially cloned into pCR2.1 (ThermoFisher Scientific) or pMiniT 2.0 (NEB), respectively, and a 1:10 plasmid dilution series was used as a reference.To determine relative SARS-CoV-2 2, 0.01% MgCl2, and 0.2% BSA). Cells were washed once with infection PBS, infected with 500 µL of diluted samples (virus inoculum), and incubated at 37 °C, 5% CO2 for 30 min. The inoculum was removed, and the infected monolayer was overlaid with plaque medium . Plates were kept at room temperature until the agar solidified, and were incubated upside down at 37 °C, 5% CO2 for 72 h. Plaques were quantified in terms of infectious IAV particles, and were represented as plaque-forming units per milliliter.MDCK-II cells were seeded in six-well plates, and cultured in DMEM supplemented with 5% FBS and 1% penicillin–streptomycin until 100% confluent. On the day of infection, 10-fold dilutions of apical washes were prepared in infection PBS  according to the manufacturer’s instruction.SI Appendix, Table S5). Expression levels were normalized by 2-ΔΔCT method (Complementary DNA was synthesized from isolated RNA using HiScript II RT SuperMix for qPCR (Vazyme) according to the manufacturer’s instructions. ISG expression levels were quantified by qPCR with Luna Universal qPCR Master Mix and the respective primer pairs . Cells were centrifuged for 15 min at 16,100 × Tryptic digestion was performed on 20 µL of cell lysate. Disulfide bonds were reduced by adding a final 5 mM dithiothreitol for 15 min at 50 °C before thiols were alkylated by a final 15 mM IAA (iodoacetamide) for 15 min in the dark. Hydrophilic and hydrophobic Cytiva Sera-Mag Carboxyl-Magnet-Beads  were mixed 1:1, and 2 µL of beads (25 µg/µL) were added per sample. The samples were filled up to 70% ACN (acetonitrile) and incubated for 15 min to ensure protein binding to the beads. Subsequently, beads were washed two times with 70% EtOH followed by washing with 100% ACN. Beads were resuspended in 100 mM ammonium bicarbonate carbonate containing 0.2 µg of trypsin (SERVA) per sample and incubated overnight at 37 °C. The peptides were transferred into a new reaction tube, vacuum dried, and dissolved in 0.1% trifluoroacetic acid.m/z within a mass range of 350 m/z to 1,400 m/z. The 10 highest abundant peptide ions were fragmented by higher-energy collisional dissociation , and MS/MS spectra were acquired at a resolution of 35,000.Then 400 ng of tryptic peptides per sample were analyzed using an Ultimate 3000 RSLCnano HPLC (Dionex) coupled to a Q Exactive HF Orbitrap (Thermo Fisher Scientific). Samples were preconcentrated on a C18 trap column  within 7 min at a flow rate of 30 μL/min with 0.1% trifluoric acid and subsequently transferred to a Nano Viper C18 analytical column . Peptide separation was performed by a gradient from 5 to 30% solvent B over 120 min at 400 nL/min . Full-scan mass spectra were acquired in profile mode at a resolution of 70,000 at 400 Homo sapiens or Homo sapiens and SARS-CoV-2. Search parameters were default, label-free quantification was used for peak quantification, and normalization was enabled. Peptides were considered for quantification irrespective of modifications. Match between runs was enabled when the analysis was performed considering human proteins only. Statistical data analysis was conducted using R (v.3.6.2). Differences between the experimental groups were assessed using t tests , and proteins quantified in a minimum of three out of four donors per group with a minimum of two unique peptides, a P value ≤ 0.05, and a ratio of mean abundances ≥ 1.5 or ≤ 0.67 were considered statistically significant. Proteins that were quantified in one experimental group but not detected at all in an opposed group were defined as On-Offs between these groups. GO annotation and enrichment analyses were performed using STRING (v.11). Data visualization was done using R and Cytoscape (v.3.8.2).Peptide identification and quantification were performed using MaxQuant (v.1.6.17) searching UniProtKB/SwissProt  restricted to either 7 reads per RNA sample.Quality and integrity of total RNA was controlled on 5200 Fragment Analyzer System (Agilent Technologies)). The RNA sequencing library was generated from 50 ng of total RNA using NEBNext Single Cell/Low Input RNA Library to manufactureŕs protocols. The libraries were treated with Illumina Free Adapter Blocking and were sequenced on Illumina NovaSeq 6000 using NovaSeq 6000 S1 Reagent Kit  with an average of 3 × 10https://digitalinsights.qiagen.com/products-overview/discovery-insights-portfolio/analysis-and-visualization/qiagen-ipa/) for biological analysis using the cutoffs P value < 0.05, fold change (fc) > |1.5|, and mean counts min > 5. IPA statistics is based on two outputs. A P value derived from a right-tailed Fisher’s exact test estimates the probability that the association between a function or pathway and a set of molecules might be due to random chance, but does not consider directional changes. This is, however, predicted for a disease and/or function, canonical pathway, or upstream regulator (activation or inhibition) by the activation z-score algorithm. The z score describes the number of SDs data lie above or below the mean. A z score > 2 was considered significantly increased, whereas a z score < −2 was considered significantly decreased (FASTQ files of RNA sequencing files were imported into the Array Studio software v10.2.5.9 (QIAGEN) package for further data analysis. All FASTQ files were aligned to the gene model Ensembl v96 and to the reference library Human B38 using the proprietary OmicSoft Aligner OSA . Differeecreased . We perfecreased .P values < 0.05 were considered significant.Differences in transformed data were tested for significance using GraphPad Prism v8.4.2 for Windows (GraphPad). Statistically significant differences between the IFNα-treated groups and the untreated group were analyzed using nonparametric Kruskal–Wallis test with Dunn’s multiple comparison test. Supplementary File


To evaluate the effect of the combination of calcium hydroxide (Ca(OH)2, OxOral®, and OxOral® plus Ca(OH)2.  Sixty human teeth were used, from which root canals were infected and randomly divided into the following treatment groups: saline solution, saline solution plus Ca(OH)2 treatment from 4.4 ± 0.074 log10 CFU/mL to 0.0 ± 0.001 log10 CFU/mL. In addition, alkaline conditions maintenance was observed from application time (pH = 12.2 ± 0.033) to 18 d posttreatment (pH = 12.6 ± 0.083).  A permanent reduction in bacterial growth was observed at days 1, 6, 12, and 18 after OxOral® plus Ca(OH)2 provides an alkaline pH and inhibits E. faecalis growth into the root canals. Our study opens the possibility for further research on the use of OxOral® in endodontic therapy. The combination of OxOral® and Ca(OH) Untreated dental caries, crown, or root fractures of the tooth, conservative dental treatments that have not been properly applied, and exposure of pulp tissue to chemical agents or dental materials may lead to inflammation of the dental pulp tissue, known as pulpitis. Its progression leads to an irreversible condition, where the inflammation spreads to the tooth apices, periodontium, dentinal tubules, and blood vessels .2), which may be dissolved in saline solution or anesthesia, to keep clean and aseptic [2 is an odorless white powder obtained by calcination of calcium carbonate and its transformation into calcium oxide, which possesses a molecular weight of 74.08 g/mol, low solubility in water with a pH = 12.5 − 12.8, and is insoluble in alcohol. Many studies have demonstrated the properties of Ca(OH)2-based sealants, such as physical properties, biocompatibility, leaks, adhesion, antibacterial, and periapical healing effects [2 in a dressing or intracanal medication are stimulation of the periapical tissues to maintain health and promotion of healing and its antimicrobial effects. Although the exact mechanisms are unknown, the following ones have been proposed: (a) Ca(OH)2 is antibacterial depending on the availability of free hydroxyl ions that encourages repair and active calcification [The endodontic treatment is commonly recommended, when there is irreversible pulpitis or pulp necrosis. Therapy consists of the total removal of the dental pulp and the three-dimensional sealing of the root canal to restore the function of the teeth within the masticatory apparatus , 3. Duri aseptic . Ca(OH)2 effects , 7. The fication . Furtherfication , (b) ?adfication , (c) it fication .2 endodontic treatments failure in teeth has been reported. Enterococcus faecalis has been reported as the bacterial microorganism responsible for the failure of endodontic therapies [. Enterococcus faecaliscolonizes inside dentinal tubules up to 300 microns, a site that is difficult to access for the action of Ca(OH)2, affecting its effectiveness in endodontic therapy [2 with an appropriate solvent may increase its solubility, antimicrobial activity, and access to complex root canal sites. The solvent used for Ca(OH)2 influences both the physical and chemical properties of the material, including its viscosity and ion release pattern. A range of water-based materials has traditionally been used but there are alternative vehicles [2 solvent for the endodontic treatment. ESS has a neutral pH (6.4–7.5) and is produced from the electrolysis of sodium chloride (NaCl) solution in an electrolytic cell where a diaphragm (partition or membrane) separates the anode and the cathode. Cations are drawn towards the electrode negative, where they receive electrons, forming a negatively charged antioxidant solution . At the positive electrode, anions are attracted, which give up their additional electrons to create a positively charged oxidant solution (acid solution) [−).The reintroduction of the alkaline solution back into the acidic solution allows adjusting the pH up to neutral. Therefore, ESS is mainly composed of hypochlorous acid (HOCl), OCl−, chlorine (Cl−), and sodium hypochlorite (NaOCl). Electrolysis may produce H+ and OH ions, H and OH radicals, H2, O2, HO2, and O3 due to redox reactions. As a result, hydrogen and ozone gas are released, and a percentage of hydroxides remains in the solution in various forms, including but not limited to hydrogen peroxide (H2O2) [Despite its advantages, Ca(OH)herapies . Enteroc therapy , 16. Comvehicles . This stolution) . For ESSe (H2O2) , 20.− and a high concentration of oxidation-reduction potential (ORP) are responsible for the antimicrobial activity of ESS. However, other studies suggest that HOCl is the most active of compounds [ESS has become a new alternative in tissue asepsis in different areas of medicine and the food industry for its high antimicrobial activity, stability, and safety , 22. Somompounds , 24.E. faecalis in root canals has been previously reported [2 with an ESS with neutral pH called OxOral®, in E. faecalis-infected root canals.A significant antimicrobial activity of ESS in removing biofilms from reported –29. The E. faecalis (ATCC® 29212™) cells was rapidly thawed in a prewarmed 37 °C water, and cells were placed into 100 mL brain heart infusion broth  supplemented with 5 μg/mL hemin and 0.5 μg/mL menadione at 0.5 McFarland. The culture was incubated at 37 °C for 24 h under anaerobic conditions. Afterwards, 1 mL of the preculture of E. faecalis was collected, placed in 100 mL of BHI-supplemented broth, and incubated until reaching the exponential, logarithmic phase.One cryovial containing nd premolar teeth were obtained from human volunteers of the Endodontic Postgraduate Program of the School of Dentistry at the Autonomous University of Nuevo Leon. The study was conducted following the Declaration of Helsinki, the Institutional Ethics Committee approved the protocol (SPSI-01613/00249), and informed consent was obtained from each volunteer. The dental crowns were sectioned with a diamond disc and press. The working length was determined with a #15 dental file (K-Flex) of 25 mm; 1 mm was subtracted from its exit from the apical foramen. Instrumentation began with Triple-Flex Files Stainless Steel files  up to file #40 using 5.2% NaOCl as an irrigating agent between each file change to maintain conduit permeability.Sixty human maxillary 2The apical region of each tooth was sealed with composite resin and externally with a layer of nail varnish, excluding the cervical opening, which was enlarged with Gates GLIDDEN files to be later autoclaved at 121°C for 15 min in 1.5 mL tubes. All root portions and materials used  were autoclaved.μL of E. faecalis with a bacterial concentration of 5 log10 CFU/mL and incubated in an anaerobic atmosphere at 37 °C for 21 d to achieve the formation of E. faecalis biofilms within the root canal, after which 10 μl of supplemented BHI broth were added every two days to avoid teeth dehydration and promote bacterial growth.Each root canal was inoculated with 15 After incubation, 12 samples were randomly selected and plated on BHI agar to verify contamination of the root canals by CFU counting.μL of the treatment was applied within the root canal based on the following treatment groups: group 1, 0.9% NaCl; group 2, 0.9% NaCl plus Ca(OH)2 paste; group 3, OxOral®; and group 4, OxOral® plus Ca(OH)2 paste. Ca(OH)2 medication pastes were prepared by mixing 0.5 g of Ca(OH)2 powder plus three drops of saline solution (0.9% NaCl) or OxOral®. All treated teeth were incubated for up to 18 d.Sixty root canals were randomly divided into four groups, and 10 E. faecalis growth evaluation, aliquots of 100 μL were taken at days 1, 6, 12, and 18 post-treatment, serially diluted, and plated onto BHI-supplemented agar using a glass spreader. Next, plates were incubated at 37 °C for 24 h, and CFU/mL were calculated; all measurements were performed in triplicate.For μL of E. faecalis cells at ~5 log10 CFU/mL at 37 °C for 24 h. Tubes were then randomly divided into four groups and 9 mL of the different treatments were added. pH values were measured at days 1, 6, 12, and 18 post-treatment, by a digital pH meter  calibrated with buffer solutions  before each experiment. After removing the specimens, the container was placed in an orbital shaker  for 5 sec before measuring. The room temperature during the test was 25 °C.For pH measurements, 60 tubes (15 mL) containing 2 mL of BHI-supplemented broth were inoculated with 10 p < 0.05), using the SPSS statistics software version 22.Results were expressed as means ± standard deviation (SD) of the response of three replicate determinations per treatment from three independent experiments. The significance level was assessed by the ANOVA and Tukey's tests  as the dependent variable and the treatment groups and the time as independent variables. The results show significant differences in the treatment groups and the time, with a p = 0.001 and p = 0.012, respectively.2, OxOral®, and OxOral® plus Ca(OH)2 at 24 h with 0.051 ± 0.072, 0.044 ± 0.07, and 0.000032 ± 0.072 log10 CFU/mL, respectively. In addition, a sustained inhibitory effect was observed in saline solution plus Ca(OH)2 and OxOral® until day 6 posttreatment. However, only the combination of OxOral® plus Ca(OH)2 maintained its inhibitory potential against E. faecalis after 18 d post-treatment, as compared with the other treatments (p < 0.05), whereas saline solution showed a progressive increase in bacterial growth from 4.55 ± 0.071 log10 CFU/mL (day 0 post-treatment) to 10.05 ± 0.075 (day 18 post-treatment), as expected(Our results showed significant (p < 0.05) antimicrobial activity in the root canals treated with saline solution plus Ca(OH) expected.p > 0.05).The pH variation of the different treatments at days 0, 1, 6, 12, and 18 was also evaluated. One-way ANOVA test was performed, where the test variable was pH in function of the treatment groups and time. We observed a statistically significant (p < 0.05) difference only in the treatment group but no difference was observed with regard to time 2 showed an alkaline pH from day 0 (baseline) with 12.149 ± 0.021 and 12.22 ± 0.033 until day 18 with 12.522 ± 0.02 and 12.586 ± 0.083, respectively. We did not observe statistical differences in pH values between these treatment groups  with 6.25 ± 0.075 and 7.003 ± 0.321 until day 18 post-treatment with 7.41 ± 0.291 and 7.456 ± 0.196, respectively, whereas root canals treated with saline solution plus Ca(OH)t groups .2 and OxOral® plus Ca(OH)2, where we observe a higher solubility and pasty consistencies compared with the traditional combination.2 has been widely used since the 1920s for its biocompatibility, antimicrobial potential, and ftissue restoration support in endodontic therapies [One of the requirements of an endodontic root canal sealer is that it should not be cytotoxic and immunologically compatible with peripheral tissue. Therefore, a specific root canal sealer biocompatibility and antimicrobial activity remain one of the major considerations for selecting an appropriate sealer for a dental restoration. Ca(OH)herapies .2 antimicrobial activity.Therefore, the efficacy of any product varies according to the availability of these ions in solution, which in turn reflects the nature of the solvent used. Most existing commercial Ca(OH)2 use vehicles of water. However, other agents have recently been mixed with water, such as glycerin or polyethylene glycol (PEG) [Hydroxyl ions are responsible for Ca(OH)ol (PEG) .2 products that use vehicles of water such as Calasept Plus™, Calcipulpe™, DT Temp™, Pulpdent™, and Ultracal XS™ with pH values between 11.8 and 12.7 [2 products, where a mixture of water with PEG is used, such as Calmix™ [The pH values from using OxOral are similar to some commercial Ca(OH)and 12.7 , 31. How Calmix™ .2 pastes. For example, Tronstad et al. used a Ringer's solution  combined with Ca(OH)2 and filled the root canal of green monkey teeth (Cercopithecus aethiops sabaeus), and they observed maintenance of alkalinity conditions with a pH = 12.2 after four weeks [2.There are previous studies, where the use of neutral pH solutions is reported as a vehicle for the root canal Ca(OH)ur weeks . However2 is a slow-acting antimicrobial, which requires at least 24 hours to produce a bactericidal effect against Enterococci spp. Furthermore, Ca(OH)2 hydroxide hydrolyzes the lipid moiety of bacterial LPS, causing its biological inactivation, the desired effect to prevent an inflammatory reaction in the periapical tissue [Some studies suggest that Ca(OH)l tissue , 35.2-based pastes being the most widely used.However, the permanence in time of these pastes is one of the main discrepancies between the authors, mainly in the cases of the teeth with pulp necrosis and periapical lesion [2 plus saline solution or OxOral® pastes for up to 18 d.For many years, different compounds have been evaluated for root canal disinfection, Ca(OH)l lesion , 37. OurRoot treatment consists of debriding and disinfecting the entire root canal system, which requires eliminating the pulp tissue and the microorganisms causing the infection, adopting instrumentation and chemical irrigation mechanisms, and subsequently a medication in the root canal between treatment sessions. The success of endodontic treatment depends on the removal of microbes from the root canals and avoidance of reinfection.2O2, 5.2% NaOCl, and 0.9% NaCl, although saline has no antibacterial activity [E. faecalis [Paudel et al. found that 0.9% NaCl was as effective as sequential use of 3% Hactivity . In addifaecalis . Therefo2 paste has a granular consistency due to its low solubility (1.73 g/L at 20 °C). One of the most interesting observations in the present study was the ease of removing the paste composed of Ca(OH)2 plus OxOral®, unlike that composed of Ca(OH)2 plus saline solution. However, other studies are necessary to evaluate any physical-chemical changes.With a significant amount of calcium carbonate, the Ca(OH)Enterococcus faecalis [2O2 compared with the action of 5.25% and 2.5% NaOCl. All combinations of CHX and H2O2 except 0.1% CHX plus 3% H2O2 were efficient irrigants against planktonic E. faecalis and had a better cytocompatibility with PDL cells than 5.25% and 2.5% NaOCl [On the other hand, reactive oxygen and chlorine species are derived during the electrolysis process to produce electrolyzed water and have been previously evaluated in endodontic treatment. In this regard, Zhou et al. reported that a plasma jet with or without helium flowing through 3% hydrogen peroxide effectively sterilized root canals infected with faecalis . Another5% NaOCl .Escherichia coli and Pseudomonas aeruginosa beta-lactam resistant) and Gram-positive . The results showed that Estericide Qx® provides a broad-spectrum antibacterial activity mainly in Gram-negative [H. pylori activity of OxOral® by microdilution assays, and Rivera-Garcia et al. observed a significant antimicrobial activity against Listeria monocytogenes on eggshells [Some studies have reported a significant antimicrobial activity of electrolyzed superoxidized water , 43. Difnegative . Other sggshells , 22.S. aureus, E. coli, P. aeruginosa, Salmonella typhi, and Candida albicans with Microcyn® and found it was active on all microorganisms tested [Landa-Solis et al. treated pure cultures of s tested . In addis tested .According to many studies, chlorine and a high concentration of ORP in ESS seem to be responsible for its antimicrobial activity. Active chlorine compounds destroys the membranes of microorganisms. Other modes of chlorine action  also have been proposed , 47. Stu2 are its limited solubility, high pH, and use as a broad-spectrum antimicrobial agent. Despite the benefits and advantages of Ca(OH)2, its use is cumbersome. Proper handling and placement are challenging for the dentist. In addition, its removal from the canal is usually incomplete, with 20-45% residue on the canal wall, even after copious irrigation using saline, sodium hypochlorite, or EDTA. Residual Ca(OH)2may shorten the setting time of zinc oxide-eugenol endodontic sealants. In particular, it may interfere with the sealing of a root filling and affect the quality of the treatment.The main characteristics of Ca(OH)2 to eliminate bacteria from the root canal has recently been challenged. Some in vitro studies have reported that dentin inactivates the antimicrobial activity of Ca(OH)2, and a clinical study reported an increase in bacterial growth after its application. Our findings agree with these studies since the combination of Ca(OH)2 plus saline solution showed an increase in bacterial growth of E. faecalis after day six of treatment. However, in the root canals treated with the combination of Ca(OH)2 plus OxOral®, we did not observe E. faecalis growth. Therefore, it is possible that OxOral® may have a synergistic effect with Ca(OH)2.On the other hand, the potential of Ca(OH)2 showed a significant antimicrobial activity and solubility of Ca(OH)2, prolonging the alkaline pH conditions. Our results open the possibility for further research on the use of OxOral® for sealing root canals, during endodontic treatment.Taken together, the results of the present study demonstrated that the combination of OxOral® plus Ca(OH)