Functional diversification of innate and inflammatory immune responses mediated by antibody fragment crystallizable activities against SARS-CoV-2.

Monoclonal antibodies (mAb) targeting the SARS-CoV-2 Spike (S) glycoprotein have been exploited for the treatment of severe COVID-19. In this study, we evaluated the immune-regulatory features of two neutralizing anti-S mAbs (nAbs), named J08 and F05, with wild-type (WT) conformation or silenced Fc functions. In the presence of D614G SARS-CoV-2, WT nAbs enhance intracellular viral uptake in immune cells and amplify antiviral type I Interferon and inflammatory cytokine and chemokine production without viral replication, promoting the differentiation of CD16+ inflammatory monocytes and innate/adaptive PD-L1+ and PD-L1+CD80+ plasmacytoid Dendritic Cells. In spite of a reduced neutralizing property, WT J08 nAb still promotes the IL-6 production and differentiation of CD16+ monocytes once binding Omicron BA.1 variant. Fc-mediated regulation of antiviral and inflammatory responses, in the absence of viral replication, highlighted in this study, might positively tune immune response during SARS-CoV-2 infection and be exploited also in mAb-based therapeutic and prophylactic strategies against viral infections.

Development of an Interactive Tool to Support Shared Decision-Making in Rheumatoid Arthritis: Treatment Attribute Preference.

OBJECTIVE: An interactive tool identifying treatment attributes important to patients can enhance shared decision-making (SDM) in rheumatoid arthritis (RA). A formative survey was conducted to identify the most important treatment attributes from patients' perspective, which can be used to develop an interactive SDM tool. METHODS: The survey was performed in two phases: qualitative interviews and quantitative surveys. The qualitative interviews were conducted to inform the design of the quantitative survey. In qualitative interviews, patients with RA (n = 10) and rheumatologists (n = 10) were introduced to the SDM tool concept. Feedback on the design and scope of the SDM tool was used to develop a quantitative survey, conducted in a large sample size of patients. Patient preferences for treatment attributes (route of administration and dosing frequency, serious side effects, out-of-pocket costs, efficacy, and monitoring requirement) were assessed via adaptive conjoint exercise involving ranking of hypothetical RA treatment configurations. RESULTS: A total of 944 patients (males: 43%, females: 57%) with RA participated in the quantitative survey. Route of administration and dosing frequency (38%) followed by serious side effects (33%) were the two most important treatment attributes for individual patients. The recontact survey (n = 172/944) estimated tool stability of 72% (n = 124/172) in terms of the relative importance of treatment attributes. CONCLUSION: The findings of this survey could be used in the development of an SDM tool that can potentially provide insights into patient preferences and is generally well received by patients and rheumatologists with good agreement and reliability.

A 12-month follow-up of the immune response to SARS-CoV-2 primary vaccination: evidence from a real-world study.

A real-world population-based longitudinal study, aimed at determining the magnitude and duration of immunity induced by different types of vaccines against COVID-19, started in 2021 by enrolling a cohort of 2,497 individuals at time of their first vaccination. The study cohort included both healthy adults aged ≤65 years and elderly subjects aged >65 years with two or more co-morbidities. Here, patterns of anti-SARS-CoV-2 humoral and cell-mediated specific immune response, assessed on 1,182 remaining subjects, at 6 (T6) and 12 months (T12) after the first vaccine dose, are described. At T12 median anti-Spike IgG antibody levels were increased compared to T6. The determinants of increased anti-Spike IgG were the receipt of a third vaccine dose between T6 and T12 and being positive for anti-Nucleocapside IgG at T12, a marker of recent infection, while age had no significant effect. The capacity of T12 sera to neutralize in vitro the ancestral B strain and the Omicron BA.5 variant was assessed in a subgroup of vaccinated subjects. A correlation between anti-S IgG levels and sera neutralizing capacity was identified and higher neutralizing capacity was evident in healthy adults compared to frail elderly subjects and in those who were positive for anti-Nucleocapside IgG at T12. Remarkably, one third of T12 sera from anti-Nucleocapside IgG negative older individuals were unable to neutralize the BA.5 variant strain. Finally, the evaluation of T-cell mediated immunity showed that most analysed subjects, independently from age and comorbidity, displayed Spike-specific responses with a high degree of polyfunctionality, especially in the CD8 compartment. In conclusion, vaccinated subjects had high levels of circulating antibodies against SARS-CoV-2 Spike protein 12 months after the primary vaccination, which increased as compared to T6. The enhancing effect could be attributable to the administration of a third vaccine dose but also to the occurrence of breakthrough infection. Older individuals, especially those who were anti-Nucleocapside IgG negative, displayed an impaired capacity to neutralize the BA.5 variant strain. Spike specific T-cell responses, able to sustain immunity and maintain the ability to fight the infection, were present in most of older and younger subjects assayed at T12.

Clinical Outcomes in Patients with Rheumatoid Arthritis After Switching Between Interleukin-6-Receptor Inhibitors and Janus Kinase Inhibitors: Findings from an Observational Study.

INTRODUCTION: This observational study evaluated response in patients with rheumatoid arthritis (RA) who switched from an interleukin-6 receptor inhibitor (IL-6Ri) to a Janus kinase inhibitor (JAKi) and vice versa. METHODS: Adult patients with RA, who initiated IL-6Ri or JAKi (following discontinuation of JAKi or IL-6Ri, respectively) during/after December 2012 and had a 6-month follow-up visit were enrolled. Clinical outcomes were evaluated at baseline and the follow-up visit. Continuous outcomes included Clinical Disease Activity Index (CDAI), Health Assessment Questionnaire (HAQ), pain, fatigue, tender joint count, swollen joint count, Physician Global Assessment (MDGA), Patient Global Assessment (PtGA), and morning stiffness duration. Categorical outcomes included the proportion of patients achieving CDAI low disease activity (LDA), remission, and minimal clinically important differences (MCIDs) for HAQ, pain, fatigue, MDGA, and PtGA. Continuous outcomes were summarized as mean changes from baseline, and categorical outcomes as response rates. Differences in the outcome measures between groups were evaluated using linear and logistic regression models. RESULTS: Between IL-6Ri (n = 100) and JAKi initiators (n = 129), no significant differences were noted for continuous outcomes. Within both groups, a significant proportion of patients achieved LDA, remission, and MCIDs for other measures, although the odds of achieving LDA were higher among IL-6Ri (vs. JAKi) initiators with moderate-to-severe disease (adjusted odds ratio: 3.30 [1.01, 10.78]). CONCLUSIONS: Patients with RA can achieve improvement in response when switching between IL-6Ri and JAKi. Although both therapies affect the IL-6 pathway, there are distinct mechanisms of action, which likely contribute to their clinical improvement, when reciprocally switched as follow-on treatments.

Real-World Sarilumab Use and Rule Testing to Predict Treatment Response in Patients with Rheumatoid Arthritis: Findings from the RISE Registry.

INTRODUCTION: Clinical trial findings may not be generalizable to routine practice. This study evaluated sarilumab effectiveness in patients with rheumatoid arthritis (RA) and tested the real-world applicability of a response prediction rule, derived from trial data using machine learning (based on C-reactive protein [CRP] > 12.3 mg/l and seropositivity [anticyclic citrullinated peptide antibodies, ACPA +]). METHODS: Sarilumab initiators from the ACR-RISE Registry, with ≥ 1 prescription on/after its FDA approval (2017-2020), were divided into three cohorts based on progressively restrictive criteria: Cohort A (had active disease), Cohort B (met eligibility criteria of a phase 3 trial in RA patients with inadequate response/intolerance to tumor necrosis factor inhibitors [TNFi]), and Cohort C (characteristics matched to the phase 3 trial baseline). Mean changes in Clinical Disease Activity Index (CDAI) and Routine Assessment of Patient Index Data 3 (RAPID3) were evaluated at 6 and 12 months. In a separate cohort, predictive rule was tested based on CRP levels and seropositive status (ACPA and/or rheumatoid factor); patients were categorized into rule-positive (seropositive with CRP > 12.3 mg/l) and rule-negative groups to compare the odds of achieving CDAI low disease activity (LDA)/remission and minimal clinically important difference (MCID) over 24 weeks. RESULTS: Among sarilumab initiators (N = 2949), treatment effectiveness was noted across cohorts, with greater improvement noted for Cohort C at 6 and 12 months. Among the predictive rule cohort (N = 205), rule-positive (vs. rule-negative) patients were more likely to reach LDA (odds ratio: 1.5 [0.7, 3.2]) and MCID (1.1 [0.5, 2.4]). Sensitivity analyses (CRP > 5 mg/l) showed better response to sarilumab in rule-positive patients. CONCLUSIONS: In real-world setting, sarilumab demonstrated treatment effectiveness, with greater improvements in the most selective population, mirroring phase 3 TNFi-refractory and rule-positive RA patients. Seropositivity appeared a stronger driver for treatment response than CRP, although optimization of the rule in routine practice requires further data.

Rheumatoid arthritis (RA) is a condition that may cause joint damage, if untreated. Sarilumab is an advanced medication, approved for treating moderate-to-severe RA in patients not responding to initial standard medicines. Clinical trials have shown that sarilumab improves RA symptoms; however, some people do not respond. This is a common problem in RA treatment. Physicians measure proteins in people's blood (called biomarkers; e.g., anticyclic citrullinated peptide antibodies [ACPA], C-reactive protein [CRP], and rheumatoid factor [RF]) to predict a medicine's response. A previous study showed that people with positive blood tests for ACPA and CRP (> 12.3 mg/l) responded well to sarilumab; this study was based on machine learning (a branch of science using computers) and identified factors that could be linked to treatment benefits. The present study analyzed routine data of 2949 people from the ACR-RISE Registry and showed an improvement in RA symptoms after 6 and 12 months of sarilumab, with a greater improvement noted in patients previously treated with other medicines. Biomarkers were tested in 205 people to check whether they could predict treatment response in day-to-day life. People were called rule-positive if they tested positive for RF and/or ACPA with CRP > 12.3 mg/l, and otherwise rule-negative. After 24 weeks of treatment, rule-positive people had a greater chance of disease improvement than rule-negative people. These results showed the benefits of sarilumab in RA in routine care and suggested the usefulness of machine learning in identifying biomarkers that physicians can use to make treatment decisions.

Association of hemoglobin levels with radiographic progression in patients with rheumatoid arthritis: an analysis from the BRASS registry.

BACKGROUND: To evaluate baseline hemoglobin (Hb) and radiographic progression over time in patients enrolled in the Brigham and Women's Rheumatoid Arthritis Sequential Study (BRASS) registry. METHODS: The BRASS is a prospective observational registry of patients with rheumatoid arthritis. BRASS Hb data and total sharp score data were matched with the main BRASS patients. Hb at baseline was categorized per the World Health Organization guidelines. Mean Hb, mean total sharp score, and mean changes over time from baseline to month 120 were summarized (overall, by low/normal Hb, and by current medication at baseline). All analyses were descriptive. RESULTS: Out of the total (N = 1114) rheumatoid arthritis patients included in the analysis, patients with low Hb at baseline (n = 224 [20%]) had longer disease duration and higher disease activity and reported more pain compared with patients with normal Hb at baseline (n = 890 [80%]). Patients with low Hb at baseline continued to have lower Hb than patients with normal Hb throughout 10 years; although, on average, patients in the low Hb subgroup exhibited a steady increase in Hb levels. A larger increase in total sharp score over time was observed for patients with low Hb than for patients with normal Hb. No meaningful differences potentially attributable to medication at baseline were detected. CONCLUSIONS: Patients with low Hb levels at baseline tended to have increased radiographic progression as measured by total sharp score compared with patients with rheumatoid arthritis having normal Hb levels. Patients with low Hb experienced sustained improvements in Hb levels over time, regardless of the class of medication used. TRIAL REGISTRATION: ClinicalTrials.gov NCT01793103.

Comparative effectiveness of TNF inhibitor vs IL-6 receptor inhibitor as monotherapy or combination therapy with methotrexate in biologic-experienced patients with rheumatoid arthritis: An analysis from the CorEvitas RA Registry.

OBJECTIVE: Randomized controlled trials (RCTs) in biologic-naïve rheumatoid arthritis (RA) patients with high disease activity and inadequate response/intolerance to methotrexate have shown interleukin-6 (IL-6) receptor inhibitors (IL-6Ri) to be superior to tumor necrosis factor inhibitors (TNFi) as monotherapy. This observational study aimed to compare the effectiveness of TNFi vs IL-6Ri as mono- or combination therapy in biologic/targeted synthetic (b/ts) -experienced RA patients with moderate/high disease activity. METHODS: Eligible b/ts-experienced patients from the CorEvitas RA registry were categorized as TNFi and IL-6Ri initiators, with subgroups initiating as mono- or combination therapy. Mixed-effects regression models evaluated the impact of treatment on Clinical Disease Activity Index (CDAI), patient-reported outcomes, and disproportionate pain (DP). Unadjusted and covariate-adjusted effects were reported. RESULTS: Patients initiating IL-6Ri (n = 286) vs TNFi monotherapy (n = 737) were older, had a longer RA history and higher baseline CDAI, and were more likely to initiate as third-line therapy; IL-6Ri (n = 401) vs TNFi (n = 1315) combination therapy initiators had higher baseline CDAI and were more likely to initiate as third-line therapy. No significant differences were noted in the outcomes between TNFi and IL-6Ri initiators (as mono- or combination therapy). CONCLUSION: This observational study showed no significant differences in outcomes among b/ts-experienced TNFi vs IL-6Ri initiators, as either mono- or combination therapy. These findings were in contrast with the previous RCTs in biologic-naïve patients and could be explained by the differences in the patient characteristics included in this study. Further studies are needed to help understand the reasons for this discrepancy in the real-world b/ts-experienced population.

Meaningful Improvement and Worsening in Patients Who Do Not Achieve Low Disease Activity and Switch Therapy to a New Biologic or Targeted Disease-Modifying Antirheumatic Drug: Results From the CorEvitas RA Registry.

OBJECTIVE: The aim of this study was to assess the change in disease activity associated with switching from 1 biologic/targeted synthetic disease-modifying antirheumatic drug (b/tsDMARD) to another in patients with rheumatoid arthritis who did not achieve low disease activity (LDA) after 6 to 12 months of their initial treatment. METHODS: This observational study included patients from the CorEvitas Rheumatoid Arthritis Registry, who initiated a b/tsDMARD at the index visit (prebaseline), had any clinical disease activity index (CDAI) improvement but did not achieve LDA/remission at the subsequent visit (baseline), and switched therapy at baseline or between baseline and follow-up visits. Regardless of the preswitch CDAI value, 2 thresholds of CDAI change were used to define meaningful improvement and worsening for all patients: ≥6 units and ≥12 units; no meaningful change was defined as any change between -6 to +6 units and -12 to +12 units, based on respective thresholds. RESULTS: Of 1226 patients fulfilling the inclusion criteria, 93 (7.6%) switched therapy at baseline or between baseline and follow-up, after an inadequate response at the baseline visit. At follow-up, meaningful worsening occurred in 30.1% and 12.9% of switchers, whereas the remaining switchers achieved meaningful improvement (34.4% and 20.4%) or had no meaningful change (35.5% and 66.7%), based on the thresholds of ≥6 and ≥12 units, respectively. CONCLUSIONS: Rheumatoid arthritis patients, who had not achieved LDA and switched b/tsDMARD, were more likely to have meaningful improvement or no change, rather than meaningful worsening. These data may help some patients overcome their hesitancy to switch therapy, potentially improving clinical outcomes.

Novel evidence of Thymosin α1 immunomodulatory properties in SARS-CoV-2 infection: Effect on innate inflammatory response in a peripheral blood mononuclear cell-based in vitro model.

The peculiar property of Thymosin alpha 1 (Tα1) to act as master regulator of immune homeostasis has been successfully defined in different physiological and pathological contexts ranging from cancer to infection. Interestingly, recent papers also demonstrated its mitigating effect on the "cytokine storm" as well as on the T-cell exhaustion/activation in SARS-CoV-2 infected individuals. Nevertheless, in spite of the increasing knowledge on Tα1-induced effects on T cell response confirming the distinctive features of this multifaceted peptide, little is known on its effects on innate immunity during SARS-CoV-2 infection. Here, we interrogated peripheral blood mononuclear cell (PBMC) cultures stimulated with SARS-CoV-2 to disclose Tα1 properties on the main cell players of early response to infection, namely monocytes and myeloid dendritic cells (mDC). Moving from ex vivo data showing an enhancement in the frequency of inflammatory monocytes and activated mDC in COVID-19 patients, a PBMC-based experimental setting reproduced in vitro a similar profile with an increased percentage of CD16+ inflammatory monocytes and mDC expressing CD86 and HLA-DR activation markers in response to SARS-CoV-2 stimulation. Interestingly, the treatment of SARS-CoV-2-stimulated PBMC with Tα1 dampened the inflammatory/activation status of both monocytes and mDC by reducing the release of pro-inflammatory mediators, including TNF-α, IL-6 and IL-8, while promoting the production of the anti-inflammatory cytokine IL-10. This study further clarifies the working hypothesis on Tα1 mitigating action on COVID-19 inflammatory condition. Moreover, these evidence shed light on inflammatory pathways and cell types involved in acute SARS-CoV-2 infection and likely targetable by newly immune-regulating therapeutic approaches.

The effect of targeted rheumatoid arthritis therapeutics on systemic inflammation and anemia: analysis of data from the CorEvitas RA registry.

BACKGROUND: To evaluate the effects of tumor necrosis factor inhibitors (TNFi), interleukin-6 receptor inhibitors (IL-6Ri), and Janus kinase inhibitors (JAKi) on hemoglobin (Hb) and C-reactive protein (CRP) levels in adults enrolled in CorEvitas (formerly Corrona), a large US rheumatoid arthritis (RA) registry. METHODS: Patients who initiated TNFi, IL-6Ri, or JAKi treatment during or after January 2010, had Hb and CRP measurements at baseline and 6-month follow-up (± 3 months) and had continued therapy at least until that follow-up, through March 2020, were included in the analysis. Changes in Hb and CRP were assessed at month 6. Abnormal Hb was defined as < 12 g/dL (women) or < 13 g/dL (men); abnormal CRP was ≥ 0.8 mg/dL. Differences in Hb and CRP levels were evaluated using multivariable regression. RESULTS: Of 2772 patients (TNFi, 65%; IL-6Ri, 17%; JAKi, 17%) evaluated, 1044 (38%) had abnormal Hb or CRP at initiation; an additional 252 (9%) had both abnormal Hb and CRP. At month 6, the IL-6Ri group had a greater Hb increase than the TNFi (mean difference in effect on Hb: 0.28 g/dL; 95% CI 0.19-0.38) and JAKi (mean difference in effect on Hb: 0.47 g/dL; 95% CI 0.35-0.58) groups, regardless of baseline Hb status (both p < 0.001). The CRP decrease at month 6 was greater with IL-6Ri compared with TNFi and JAKi, regardless of baseline CRP status (both p < 0.05). CONCLUSION: These real-world results align with the mechanism of IL-6R inhibition and may inform treatment decisions for patients with RA.

Evaluation of humoral and cellular response to four vaccines against COVID-19 in different age groups: A longitudinal study.

To date there has been limited head-to-head evaluation of immune responses to different types of COVID-19 vaccines. A real-world population-based longitudinal study was designed with the aim to define the magnitude and duration of immunity induced by each of four different COVID-19 vaccines available in Italy at the time of this study. Overall, 2497 individuals were enrolled at time of their first vaccination (T0). Vaccine-specific antibody responses induced over time by Comirnaty, Spikevax, Vaxzevria, Janssen Ad26.COV2.S and heterologous vaccination were compared up to six months after immunization. On a subset of Comirnaty vaccinees, serology data were correlated with the ability to neutralize a reference SARS-CoV-2 B strain, as well as Delta AY.4 and Omicron BA.1. The frequency of SARS-CoV-2-specific CD4+ T cells, CD8+ T cells, and memory B cells induced by the four different vaccines was assessed six months after the immunization. We found that mRNA vaccines are stronger inducer of anti-Spike IgG and B-memory cell responses. Humoral immune responses are lower in frail elderly subjects. Neutralization of the Delta AY.4 and Omicron BA.1 variants is severely impaired, especially in older individuals. Most vaccinees display a vaccine-specific T-cell memory six months after the vaccination. By describing the immunological response during the first phase of COVID-19 vaccination campaign in different cohorts and considering several aspects of the immunological response, this study allowed to collect key information that could facilitate the implementation of effective prevention and control measures against SARS-CoV-2.

Association between low hemoglobin, clinical measures, and patient-reported outcomes in patients with rheumatoid arthritis: results from post hoc analyses of three phase III trials of sarilumab.

BACKGROUND: Anemia is common in patients with rheumatoid arthritis (RA). Higher hemoglobin (Hb) levels may be associated with better clinical outcomes and patient-reported outcomes (PROs). To assess this hypothesis, we conducted two post hoc analyses in three sarilumab phase III studies: TARGET, MOBILITY, and MONARCH. METHODS: Pooled data from combination therapy from placebo-controlled MOBILITY (sarilumab + methotrexate) and TARGET (sarilumab + conventional synthetic disease-modifying antirheumatic drugs [csDMARDs]) and monotherapy data from active-controlled MONARCH (sarilumab vs. adalimumab) studies were included. Associations between Hb levels and clinical measures and PROs were assessed over 24 weeks. The mean changes from baseline in clinical outcomes and PROs (to week 24) and radiographic outcomes (to week 52) were evaluated between low and normal Hb levels (based on the World Health Organization [WHO] criteria). RESULTS: From TARGET, MOBILITY, and MONARCH, 546, 1197, and 369 patients, respectively, were stratified according to Hb levels (low vs. normal). Over 24 weeks, higher Hb levels were found to be consistently associated with better clinical outcomes and PROs in combination therapy and monotherapy groups and were more pronounced among the patients treated with sarilumab than those treated with placebo and adalimumab. The mean change from baseline to week 24 in clinical efficacy measures and PROs was similar in patients with low vs. normal Hb at baseline. Differences between sarilumab and/or adalimumab, for all outcomes, were larger for low Hb subgroups. In MOBILITY, by week 52, the inhibition of progression of structural damage (assessed via Modified Total Sharp Score [mTSS]) was 84% (sarilumab 200 mg) and 68% (sarilumab 150 mg) vs. placebo in patients with low Hb and 97% (sarilumab 200 mg) and 68% (sarilumab 150 mg) vs. placebo in patients with normal Hb. Similar results were observed for other radiographic outcomes. CONCLUSIONS: In these post hoc analyses, a consistent relationship was observed between higher Hb levels and better clinical outcomes and PROs in patients with RA. Irrespective of the baseline Hb levels, sarilumab treatment was associated with improvements in clinical measures and PROs over 24 weeks (improvements were more pronounced than those with adalimumab treatment) and mitigation of joint damage progression over 52 weeks. TRIAL REGISTRATION: ClinTrials.gov NCT01061736, NCT01709578, and NCT02332590.

Application of the Adverse Outcome Pathway Concept to In Vitro Nephrotoxicity Assessment: Kidney Injury due to Receptor-Mediated Endocytosis and Lysosomal Overload as a Case Study.

Application of adverse outcome pathways (AOP) and integration of quantitative in vitro to in vivo extrapolation (QIVIVE) may support the paradigm shift in toxicity testing to move from apical endpoints in test animals to more mechanism-based in vitro assays. Here, we developed an AOP of proximal tubule injury linking a molecular initiating event (MIE) to a cascade of key events (KEs) leading to lysosomal overload and ultimately to cell death. This AOP was used as a case study to adopt the AOP concept for systemic toxicity testing and risk assessment based on in vitro data. In this AOP, nephrotoxicity is thought to result from receptor-mediated endocytosis (MIE) of the chemical stressor, disturbance of lysosomal function (KE1), and lysosomal disruption (KE2) associated with release of reactive oxygen species and cytotoxic lysosomal enzymes that induce cell death (KE3). Based on this mechanistic framework, in vitro readouts reflecting each KE were identified. Utilizing polymyxin antibiotics as chemical stressors for this AOP, the dose-response for each in vitro endpoint was recorded in proximal tubule cells from rat (NRK-52E) and human (RPTEC/TERT1) in order to (1) experimentally support the sequence of key events (KEs), to (2) establish quantitative relationships between KEs as a basis for prediction of downstream KEs based on in vitro data reflecting early KEs and to (3) derive suitable in vitro points of departure for human risk assessment. Time-resolved analysis was used to support the temporal sequence of events within this AOP. Quantitative response-response relationships between KEs established from in vitro data on polymyxin B were successfully used to predict in vitro toxicity of other polymyxin derivatives. Finally, a physiologically based kinetic (PBK) model was utilized to transform in vitro effect concentrations to a human equivalent dose for polymyxin B. The predicted in vivo effective doses were in the range of therapeutic doses known to be associated with a risk for nephrotoxicity. Taken together, these data provide proof-of-concept for the feasibility of in vitro based risk assessment through integration of mechanistic endpoints and reverse toxicokinetic modelling.

Analysis of Genomic Characteristics of SARS-CoV-2 in Italy, 29 January to 27 March 2020.

We performed next-generation sequencing (NGS), phylogenetic analysis, gene flows, and N- and O-glycosylation prediction on SARS-CoV-2 genomes collected from lab-confirmed cases from different Italian regions. To this end, a total of 111 SARS-CoV-2 genomes collected in Italy between 29 January and 27 March 2020 were investigated. The majority of the genomes belonged to lineage B.1, with some descendant lineages. The gene flow analysis showed that the spread occurred mainly from the north to the center and to the south of Italy, as confirmed by epidemiological data. The mean evolutionary rate estimated here was 8.731 × 10-4 (95% highest posterior density, HPD intervals 5.809 × 10-4 to 1.19 × 10-3), in line with values reported by other authors. The dated phylogeny suggested that SARS-CoV-2 lineage B.1 probably entered Italy between the end of January and early February 2020. Continuous molecular surveillance is needed to trace virus circulation and evolution.

The BNT162b2 vaccine induces humoral and cellular immune memory to SARS-CoV-2 Wuhan strain and the Omicron variant in children 5 to 11 years of age.

SARS-CoV-2 mRNA vaccines prevent severe COVID-19 by generating immune memory, comprising specific antibodies and memory B and T cells. Although children are at low risk of severe COVID-19, the spreading of highly transmissible variants has led to increasing in COVID-19 cases and hospitalizations also in the youngest, but vaccine coverage remains low. Immunogenicity to mRNA vaccines has not been extensively studied in children 5 to 11 years old. In particular, cellular immunity to the wild-type strain (Wuhan) and the cross-reactive response to the Omicron variant of concern has not been investigated. We assessed the humoral and cellular immune response to the SARS-CoV-2 BNT162b2 vaccine in 27 healthy children. We demonstrated that vaccination induced a potent humoral and cellular immune response in all vaccinees. By using spike-specific memory B cells as a measurable imprint of a previous infection, we found that 50% of the children had signs of a past, undiagnosed infection before vaccination. Children with pre-existent immune memory generated significantly increased levels of specific antibodies, and memory T and B cells, directed against not only the wild type virus but also the omicron variant.

Differential plasmacytoid dendritic cell phenotype and type I Interferon response in asymptomatic and severe COVID-19 infection.

SARS-CoV-2 fine-tunes the interferon (IFN)-induced antiviral responses, which play a key role in preventing coronavirus disease 2019 (COVID-19) progression. Indeed, critically ill patients show an impaired type I IFN response accompanied by elevated inflammatory cytokine and chemokine levels, responsible for cell and tissue damage and associated multi-organ failure. Here, the early interaction between SARS-CoV-2 and immune cells was investigated by interrogating an in vitro human peripheral blood mononuclear cell (PBMC)-based experimental model. We found that, even in absence of a productive viral replication, the virus mediates a vigorous TLR7/8-dependent production of both type I and III IFNs and inflammatory cytokines and chemokines, known to contribute to the cytokine storm observed in COVID-19. Interestingly, we observed how virus-induced type I IFN secreted by PBMC enhances anti-viral response in infected lung epithelial cells, thus, inhibiting viral replication. This type I IFN was released by plasmacytoid dendritic cells (pDC) via an ACE-2-indipendent but Neuropilin-1-dependent mechanism. Viral sensing regulates pDC phenotype by inducing cell surface expression of PD-L1 marker, a feature of type I IFN producing cells. Coherently to what observed in vitro, asymptomatic SARS-CoV-2 infected subjects displayed a similar pDC phenotype associated to a very high serum type I IFN level and induction of anti-viral IFN-stimulated genes in PBMC. Conversely, hospitalized patients with severe COVID-19 display very low frequency of circulating pDC with an inflammatory phenotype and high levels of chemokines and pro-inflammatory cytokines in serum. This study further shed light on the early events resulting from the interaction between SARS-CoV-2 and immune cells occurring in vitro and confirmed ex vivo. These observations can improve our understanding on the contribution of pDC/type I IFN axis in the regulation of the anti-viral state in asymptomatic and severe COVID-19 patients.

Targeted mutagenesis in Nicotiana tabacum ADF gene using shockwave-mediated ribonucleoprotein delivery increases osmotic stress tolerance.

DNA-free genome editing involves the direct introduction of ribonucleoprotein (RNP) complexes into cells, but this strategy has rarely been successful in plants. In the present study, we describe a new technique for the introduction of RNPs into plant cells involving the generation of cavitation bubbles using a pulsed laser. The resulting shockwave achieves the efficient transfection of walled cells in tissue explants by creating transient membrane pores. RNP-containing cells were rapidly identified by fluorescence microscopy, followed by regeneration and the screening of mutant plants by high-resolution melt analysis. We used this technique in Nicotiana tabacum to target the endogenous phytoene desaturase (PDS) and actin depolymerizing factor (ADF) genes. Genome-edited plants were produced with an efficiency of 35.2% for PDS and 16.5% for ADF. Further we evaluated the physiological, cellular and molecular effects of ADF mutations in T2 mutant plants under drought and salinity stress. The results suggest that ADF acts as a key regulator of osmotic stress tolerance in plants.

Feature tracking myocardial strain analysis in patients with bileaflet mitral valve prolapse: relationship with LGE and arrhythmias.

OBJECTIVES: Anatomical substrate and mechanical trigger co-act in arrhythmia's onset in patients with bileaflet mitral valve prolapse (bMVP). Feature tracking (FT) may improve risk stratification provided by cardiac magnetic resonance (CMR). The aim was to investigate differences in CMR and FT parameters in bMVP patients with and without complex arrhythmias (cVA and no-cVA). METHODS: In this retrospective study, 52 patients with bMVP underwent 1.5 T CMR and were classified either as no-cVA (n = 32; 12 males; 49.6 ± 17.4 years) or cVA (n = 20; 3 males; 44.7 ± 11.2 years), the latter group including 6 patients (1 male; 45.7 ± 12.7 years) with sustained ventricular tachycardia or ventricular fibrillation (SVT-FV). Twenty-four healthy volunteers (11 males, 36.2 ± 12.5 years) served as control. Curling, prolapse distance, mitral annulus disjunction (MAD), and late gadolinium enhancement (LGE) were recorded and CMR-FT analysis performed. Statistical analysis included non-parametric tests and binary logistic regression. RESULTS: LGE and MAD distance were associated with cVA with an odds ratio (OR) of 8.51 for LGE (95% CI 1.76, 41.28; p = 0.008) and of 1.25 for MAD (95% CI 1.02, 1.54; p = 0.03). GLS 2D (- 11.65 ± 6.58 vs - 16.55 ± 5.09 1/s; p = 0.04), PSSR longitudinal 2D (0.04 ± 1.62 1/s vs - 1.06 ± 0.35 1/s; p = 0.0001), and PSSR radial 3D (3.95 ± 1.97 1/s vs 2.64 ± 1.03 1/s; p = 0.0001) were different for SVT-VF versus the others. PDSR circumferential 2D (1.10 ± 0.54 vs. 0.84 ± 0.34 1/s; p = 0.04) and 3D (0.94 ± 0.42 vs. 0.69 ± 0.17 1/s; p = 0.04) differed between patients with and without papillary muscle LGE. CONCLUSIONS: CMR-FT allowed identifying subtle myocardial deformation abnormalities in bMVP patients at risk of SVT-VF. LGE and MAD distance were associated with cVA. KEY POINTS: • CMR-FT allows identifying several subtle myocardial deformation abnormalities in bMVP patients, especially those involving the papillary muscle. • CMR-FT allows identifying subtle myocardial deformation abnormalities in bMVP patients at risk of SVT and VF. • In patients with bMVP, the stronger predictor of cVA is LGE (OR = 8.51; 95% CI 1.76, 41.28; p = 0.008), followed by MAD distance (OR = 1.25; 95% CI 1.02, 1.54; p = 0.03).