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A gene cluster responsible for the degradation of nicotinic acid (NA) in Bacillus niacini has recently been identified, and the structures and functions of the resulting enzymes are currently being evaluated to establish pathway intermediates. One of the genes within this cluster encodes a flavin monooxygenase (BnFMO) that is hypothesized to catalyze a hydroxylation reaction. Kinetic analyses of the recombinantly purified BnFMO suggest that this enzyme catalyzes the hydroxylation of 2,6-dihydroxynicotinic acid (2,6-DHNA) or 2,6-dihydroxypyridine (2,6-DHP), which is formed spontaneously by the decarboxylation of 2,6-DHNA. To understand the details of this hydroxylation reaction, we determined the structure of BnFMO using a multimodel approach combining protein X-ray crystallography and cryo-electron microscopy (cryo-EM). A liganded BnFMO cryo-EM structure was obtained in the presence of 2,6-DHP, allowing us to make predictions about potential catalytic residues. The structural data demonstrate that BnFMO is trimeric, which is unusual for Class A flavin monooxygenases. In both the electron density and coulomb potential maps, a region at the trimeric interface was observed that was consistent with and modeled as lipid molecules. High-resolution mass spectral analysis suggests that there is a mixture of phosphatidylethanolamine and phosphatidylglycerol lipids present. Together, these data provide insights into the molecular details of the central hydroxylation reaction unique to the aerobic degradation of NA in Bacillus niacini .

In chronic lymphocytic leukemia (CLL), survival of neoplastic cells depends on microenvironmental signals at lymphoid sites where the crosstalk between the integrin VLA-4 (CD49d/CD29), expressed in ~40% of CLL, and the B-cell receptor (BCR) occurs. Here, BCR engagement inside-out activates VLA-4, thus enhancing VLA-4-mediated adhesion of CLL cells, which in turn obtain pro-survival signals from the surrounding microenvironment. We report that the BCR is also able to effectively inside-out activate the VLA-4 integrin in circulating CD49d-expressing CLL cells through an autonomous antigen-independent BCR signaling. As a consequence, circulating CLL cells exhibiting activated VLA-4 express markers of BCR pathway activation (phospho-BTK and phospho-PLC-γ2) along with higher levels of phospho-ERK and phospho-AKT indicating parallel activation of downstream pathways. Moreover, circulating CLL cells expressing activated VLA-4 bind soluble blood-borne VCAM-1 leading to increased VLA-4-dependent actin polymerization/re-organization and ERK phosphorylation. Finally, evidence is provided that ibrutinib treatment, by affecting autonomous BCR signaling, impairs the constitutive VLA-4 activation eventually decreasing soluble VCAM-1 binding and reducing downstream ERK phosphorylation by circulating CLL cells. This study describes a novel anchor-independent mechanism occurring in circulating CLL cells involving the BCR and the VLA-4 integrin, which help to unravel the peculiar biological and clinical features of CD49d+ CLL., (© 2024. The Author(s).)

Competing Interests: The authors declare no conflict of interest.

Chronic Lymphocytic Leukemia (CLL) is an indolent malignancy characterized by the accumulation of quiescent mature B cells. However, these cells are transcriptionally and translationally active, implicating an active metabolism. The recent literature suggests that CLL cells have an oxidative-type phenotype. Given the role of cell metabolism, which is able to influence the outcome of treatments, in other neoplasms, we aimed to assess its prognostic role in CLL patients by determining the ex vivo bioenergetic metabolic profile of CLL cells, evaluating the correlation with the patient clinical/biological characteristics and the in vivo response to BTK inhibitor treatment. Clustering analysis of primary samples identified two groups, characterized by low (CLL low) or high (CLL high) bioenergetic metabolic rates. Compared to the CLL high, CLL with lower bioenergetic metabolic rates belonged to patients characterized by a statistically significant higher white blood cell count and by unfavorable molecular genetics. More importantly, patients in the CLL low cluster displayed a better and more durable response to the BTK inhibitor ibrutinib, thus defining a bioenergetic metabolic subgroup that can benefit the most from this therapy.

BTK and PI3K inhibitors are among the drugs approved for the treatment of patients with lymphoid neoplasms. Although active, their ability to lead to long-lasting complete remission is rather limited, especially in the lymphoma setting. This indicates that tumor cells often develop resistance to the drugs. We started from a marginal zone lymphoma cell line, Karpas-1718, kept under prolonged exposure to the PI3Kδ inhibitor idelalisib until acquisition of resistance, or with no drug. Cells underwent transcriptome, miRNA and methylation profiling, whole-exome sequencing, and pharmacologic screening, which led to the identification of the overexpression of ERBB4 and its ligands HBEGF and NRG2 in the resistant cells. Cellular and genetic experiments demonstrated the involvement of this axis in blocking the antitumor activity of various BTK/PI3K inhibitors, currently used in the clinical setting. Addition of recombinant HBEGF induced resistance to BTK/PI3K inhibitors in parental cells and in additional lymphoma models. Combination with the ERBB inhibitor lapatinib was beneficial in resistant cells and in other lymphoma models already expressing the identified resistance factors. An epigenetic reprogramming sustained the expression of the resistance-related factors, and pretreatment with demethylating agents or EZH2 inhibitors overcame the resistance. Resistance factors were also shown to be expressed in clinical specimens. In conclusion, we showed that the overexpression of ERBB4 and its ligands represents a novel mechanism of resistance for lymphoma cells to bypass the antitumor activity of BTK and PI3K inhibitors and that targeted pharmacologic interventions can restore sensitivity to the small molecules., (©2023 American Association for Cancer Research.)

Cofilin 1 is an actin depolymerizing protein playing a fundamental role in the turnover of actin filaments specifically in dendritic spines, where it has been associated with structural and functional plasticity processes. Using a differential proteomic approach, we recently identified cofilin 1 as a potential candidate for controlling plasticity levels in the mouse visual cortex. Here, we focus on analyzing the expression of cofilin 1 and of its serine-3 phosphorylated inactive form in the mouse visual cortex during postnatal development and its modulation by visual input. Western blot experiments showed that cofilin 1 decreases from the critical period to the adult stage, in correlation with the decreasing level of cortical plasticity, and that monocular deprivation increases its expression in the cortex contralateral to the deprived eye during the critical period but not in the adult stage. By immunohistochemistry, we identified that the phospho-cofilin 1 immunopositive signal is homogeneously expressed along the different layers of the mouse visual cortex and that it increases during postnatal development. Furthermore, monocular deprivation increases the phospho-cofilin 1 signal in the contralateral cortex to the deprived eye during the critical period but not in the adult stage. Altogether, these results suggest that cofilin 1 and its modification by phosphorylation are relevant players in the processes controlling experience-dependent plasticity in the mouse visual cortex., (Copyright © 2022 IBRO. Published by Elsevier Ltd. All rights reserved.)

Isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are the central five-carbon precursors to all terpenes. Despite their significance, exogenous, independent delivery of IPP and DMAPP to cells is impossible as the negatively charged pyrophosphate makes these molecules membrane impermeant. Herein, we demonstrate a facile method to circumvent this challenge through esterification of the β-phosphate with two self-immolative esters (SIEs) that neutralize the negatively charged pyrophosphate to yield membrane-permeant analogs of IPP and DMAPP. Following cellular incorporation, general esterase activity initiates cleavage of the SIEs, resulting in traceless release of IPP and DMAPP for metabolic utilization. Addition of the synthesized IPP and DMAPP precursor analogs rescued cell growth of glioblastoma (U-87MG) cancer cells concurrently treated with the HMG-CoA reductase inhibitor pitavastatin, which otherwise abrogates cell growth via blocking production of IPP and DMAPP. This work demonstrates a new application of a prodrug strategy to incorporate a metabolic intermediate and promises to enable future interrogation of the distinct biological roles of IPP and DMAPP., (© 2022 Wiley-VCH GmbH.)

PI3Kδ inhibitors are active in patients with lymphoid neoplasms and a first series of them have been approved for the treatment of multiple types of B-cell lymphoid tumors, including marginal zone lymphoma (MZL). The identification of the mechanisms underlying either primary or secondary resistance is fundamental to optimize the use of novel drugs. Here we present a model of secondary resistance to PI3Kδ inhibitors obtained by prolonged exposure of a splenic MZL cell line to idelalisib. The VL51 cell line was kept under continuous exposure to idelalisib. The study included detailed characterization of the model, pharmacological screens, silencing experiments, and validation experiments on multiple cell lines and on clinical specimens. VL51 developed resistance to idelalisib, copanlisib, duvelisib, and umbralisib. An integrative analysis of transcriptome and methylation data highlighted an enrichment of upregulated transcripts and low-methylated promoters in resistant cells, including IL-6/STAT3- and PDGFRA-related genes and surface CD19 expression, alongside the repression of the let-7 family of miRNA, and miR-125, miR-130, miR-193 and miR-20. The IL-6R blocking antibody tocilizumab, the STAT3 inhibitor stattic, the LIN28 inhibitor LIN1632, the PDGFR inhibitor masitinib and the anti-CD19 antibody drug conjugate loncastuximab tesirine were active compounds in the resistant cells as single agents and/or in combination with PI3Kδ inhibition. Findings were validated on additional in vitro lymphoma models and on clinical specimens. A novel model of resistance obtained from splenic MZL allowed the identification of therapeutic approaches able to improve the antitumor activity of PI3Kδ inhibitors in B-cell lymphoid tumors.

Approved drugs for the treatment of osteoporosis can prevent further bone loss but do not stimulate bone formation. Approaches that improve bone density in metabolic diseases are needed. Therapies that take advantage of the ability of mesenchymal stem cells (MSCs) to differentiate into various osteogenic lineages to treat bone disorders are of particular interest. Here we examine the ability of small interfering RNA (siRNA) to enhance osteoblast differentiation and bone formation by silencing the negative suppressor gene GNAS in bone MSCs. Using clinically validated lipid nanoparticle (LNP) siRNA delivery systems, we show that silencing the suppressor gene GNAS in vitro in MSCs leads to molecular and phenotypic changes similar to those seen in osteoblasts. Further, we demonstrate that these LNP-siRNAs can transfect a large proportion of mice MSCs in the compact bone following intravenous injection. Transfection of MSCs in various animal models led to silencing of GNAS and enhanced differentiation of MSCs into osteoblasts. These data demonstrate the potential for LNP delivery of siRNA to enhance the differentiation of MSCs into osteoblasts, and suggests that they are a promising approach for the treatment of osteoporosis and other bone diseases., Competing Interests: Declaration of interests P.R.C. has financial interests in Precision NanoSystems, Acuitas Therapeutics, and Mesentech., (Copyright © 2022 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

CD49d, the α4 chain of the VLA-4 integrin, is a negative prognosticator in chronic lymphocytic leukemia (CLL) with a key role in CLL cell-microenvironment interactions mainly occurring via its ligands VCAM-1 and fibronectin. In the present study, we focused on EMILIN-1 (Elastin-MIcrofibriL-INterfacer-1), an alternative VLA-4 ligand whose role has been so far reported only in non-hematological settings, by investigating: i) the distribution of EMILIN-1 in CLL-involved tissues; ii) the capability of EMILIN-1 to operate, via its globular C1q (gC1q) domain, as additional adhesion ligand in CLL; iii) the functional meaning of EMILIN-1 gC1q/VLA-4 interactions in CLL. EMILIN-1 is widely present in the CLL-involved areas of bone marrow biopsies (BMBs) without difference between CD49d negative and positive cases, displaying at least three different expression patterns: "fibrillar", "dot-like" and "mixed". The lack in CLL-BMB of neutrophil elastase, whose proteolytic activity degrades EMILIN-1 and impairs EMILIN-1 function, suggests full functional EMILIN-1 in CLL independently of its expression pattern. Functionally, EMILIN-1 gC1q domain promotes adhesion of CLL cells through specific interaction with VLA-4, and releases pro-survival signals for CLL cells, as demonstrated by enhanced ERK and AKT phosphorylation and impairment of in-vitro-induced apoptosis. EMILIN-1/VLA-4 interaction can efficiently contribute to the maintenance of the neoplastic clone in CLL., (© 2021 John Wiley & Sons Ltd.)

The main purpose of this paper is to investigate the role of social and emotional learning (SEL) skills and resilience in explaining mental health in male and female adolescents, during the COVID-19 pandemic. Three self-report questionnaires were administered to 778 participants aged between 11 and 16 years (mean age = 12.73 years; SD = 1.73) and recruited from 18 schools in Northern Italy. The SSIS-SEL b -S and the CD-RISC 10 assessed SEL and resilience skills respectively, while the Strengths and Difficulties Questionnaire (SDQ) was used to measure mental health in terms of internalizing problems, externalizing problems, and prosocial behavior. We found that SEL and resilience skills were positively and significantly associated with each other, negatively associated with internalizing and externalizing problems, and positively related to prosocial behavior. Three linear regression analyses showed the significant role of resilience, age, and gender in explaining the variance of internalizing problems; the significant role of SEL skills, resilience, age, and gender in explaining the variance of externalizing problems; and the role of SEL skills, age, and gender in explaining prosocial behavior. Importantly, we found that resilience fully mediated the relationship between SEL skills and internalizing problems, partially mediated the relationship between SEL skills and externalizing problems and didn't mediate the relationship between SEL skills and prosocial behavior. The paper concludes with a discussion of the limitations of the study as well as its practical implications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Grazzani, Agliati, Cavioni, Conte, Gandellini, Lupica Spagnolo, Ornaghi, Rossi, Cefai, Bartolo, Camilleri and Oriordan.)

Chronic lymphocytic leukemia (CLL) is characterized by a low CD20 expression, in part explained by an epigenetic-driven downregulation triggered by mutations of the NOTCH1 gene. In the present study, by taking advantage of a wide and well-characterized CLL cohort (n=537), we demonstrate that CD20 expression is downregulated in SF3B1-mutated CLL in an extent similar to NOTCH1-mutated CLL. In fact, SF3B1-mutated CLL cells show common features with NOTCH1-mutated CLL cells, including a gene expression profile enriched of NOTCH1-related gene sets and elevated expression of the active intracytoplasmic NOTCH1. Activation of the NOTCH1 signaling and down-regulation of surface CD20 in SF3B1-mutated CLL cells correlate with over-expression of an alternatively spliced form of DVL2, a component of the Wnt pathway and negative regulator of the NOTCH1 pathway. These findings are confirmed by separately analyzing the CD20-dim and CD20-bright cell fractions from SF3B1-mutated cases as well as by DVL2 knock-out experiments in CLL-like cell models. Altogether, the clinical and biological features that characterize NOTCH1-mutated CLL may also be recapitulated in SF3B1-mutated CLL, contributing to explain the poor prognosis of this CLL subset and providing the rationale for expanding novel agents-based therapies to SF3B1-mutated CLL.

The major capsid protein VP1 of JC Polyomavirus assembles into pentamers that serve as a model for studying viral entry of this potentially severe human pathogen. Previously, labeling of viral proteins utilized large fusion proteins or non-specific amine- or cysteine-functionalization with fluorescent dyes. Imaging of these sterically hindered fusion proteins or heterogeneously labeled virions limits reproducibility and could prevent the detection of subtle trafficking phenomena. Here we advance the π-clamp-mediated cysteine conjugation for site-selective fluorescent labeling of VP1-pentamers. We demonstrate a one-step synthesis of a probe consisting of a bio-orthogonal click chemistry handle bridged to a perfluoro-biphenyl π-clamp reactive electrophile by a polyethylene glycol linker. We expand the scope of the π-clamp conjugation by demonstrating selective labeling of an internal, surface exposed loop in VP1. Thus, the π-clamp conjugation offers a general method to selectively bioconjugate tags-of-interest to viral proteins without impeding their ability to bind and enter cells., (© 2021 Wiley-VCH GmbH.)

Purpose: In chronic lymphocytic leukemia (CLL), TP53 mutations are associated with reduced survival and resistance to standard chemoimmunotherapy (CIT). Nevertheless, the clinical impact of subclonal TP53 mutations below 10% to 15% variant allele frequency (VAF) remains unclear., Experimental Design: Using a training/validation approach, we retrospectively analyzed the clinical and biological features of TP53 mutations above (high-VAF) or below (low-VAF) the previously reported 10.0% VAF threshold, as determined by deep next-generation sequencing. Clinical impact of low-VAF TP53 mutations was also confirmed in a cohort ( n = 251) of CLL treated with fludarabine-cyclophosphamide-rituximab (FCR) or FCR-like regimens from two UK trials., Results: In the training cohort, 97 of 684 patients bore 152 TP53 mutations, while in the validation cohort, 71 of 536 patients had 109 TP53 mutations. In both cohorts, patients with the TP53 mutation experienced significantly shorter overall survival (OS) than TP53 wild-type patients, regardless of the TP53 mutation VAF. By combining TP53 mutation and 17p13.1 deletion (del17p) data in the total cohort ( n = 1,220), 113 cases were TP53 mutated only (73/113 with low-VAF mutations), 55 del17p/ TP53 mutated (3/55 with low-VAF mutations), 20 del17p only, and 1,032 (84.6%) TP53 wild-type. A model including low-VAF cases outperformed the canonical model, which considered only high-VAF cases (c-indices 0.643 vs. 0.603, P < 0.0001), and improved the prognostic risk stratification of CLL International Prognostic Index. Clinical results were confirmed in CIT-treated cases ( n = 552) from the retrospective cohort, and the UK trials cohort., Conclusions: TP53 mutations affected OS regardless of VAF. This finding can be used to update the definition of TP53 mutated CLL for clinical purposes., (©2021 American Association for Cancer Research.)

The introduction of agents inhibiting the BCR-associated kinases such as ibrutinib has dramatically changed treatments algorithms of chronic lymphocytic leukaemia (CLL) as well as the role of different adverse prognosticators. We evaluated the efficacy of ibrutinib as single agent, in a real-life context, on 180 patients with CLL mostly pre-treated, recruited from three independent cohorts from Italy. Patients received 420 mg oral ibrutinib once daily until progression or occurrence of unacceptable side effects. Seventy-three patients discontinued ibrutinib for progression or for adverse events. NOTCH1 mutations (M) were correlated with a reduced redistribution lymphocytosis, calculated at 3 months on ibrutinib (p=0.022). Moreover, NOTCH1 mutated patients showed inferior nodal response at 6 months on ibrutinib compared to NOTCH1 wild type patients (p<0.0001). Significant shorter progression free survival (PFS) and overall survival (OS) were observed in NOTCH1 mutated patients (p=0.00002 and p=0.001). Interestingly, NOTCH1 M plus lower bax/bcl-2 ratio identified a CLL subset showing the worst PFS and OS (p=0.0002 and p=0.005). In multivariate analysis of PFS and OS, NOTCH1 M were confirmed an independent prognosticator (p=0.00006 and p=0.0039). In conclusion, NOTCH1 M are strongly associated with lower bax/bcl-2 ratio, consistent with a defective apoptosis, lower redistribution lymphocytosis and lower nodal shrinkage under ibrutinib treatment, this last responsible for partial responses, subsequent relapses, shorter PFS and OS. The therapeutic options for NOTCH1 mutated patients could be represented by either new small molecules combination approaches or from antibodies targeting NOTCH1.

Objective: While the contribution of B-cells to SLE is well established, its role in chronic cutaneous lupus erythematosus (CCLE) remains unclear. Here, we compare B-cell and serum auto-antibody profiles between patients with systemic lupus erythematosus (SLE), CCLE, and overlap conditions., Methods: B-cells were compared by flow cytometry amongst healthy controls, CCLE without systemic lupus (CCLE+/SLE-) and SLE patients with (SLE+/CCLE+) or without CCLE (SLE+/CCLE-). Serum was analyed for autoreactive 9G4+, anti-double-stranded DNA, anti-chromatin and anti-RNA antibodies by ELISA and for anti-RNA binding proteins (RBP) by luciferase immunoprecipitation., Results: Patients with CCLE+/SLE- share B-cell abnormalities with SLE including decreased unswitched memory and increased effector B-cells albeit at a lower level than SLE patients. Similarly, both SLE and CCLE+/SLE- patients have elevated 9G4+ IgG autoantibodies despite lower levels of anti-nucleic acid and anti-RBP antibodies in CCLE+/SLE-. CCLE+/SLE- patients could be stratified into those with SLE-like B-cell profiles and a separate group with normal B-cell profiles. The former group was more serologically active and more likely to have disseminated skin lesions., Conclusion: CCLE displays perturbations in B-cell homeostasis and partial B-cell tolerance breakdown. Our study demonstrates that this entity is immunologically heterogeneous and includes a disease segment whose B-cell compartment resembles SLE and is clinically associated with enhanced serological activity and more extensive skin disease. This picture suggests that SLE-like B-cell changes in primary CCLE may help identify patients at risk for subsequent development of SLE. B-cell profiling in CCLE might also indentify candidates who would benefit from B-cell targeted therapies., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Group 2 innate lymphoid cells (ILC2s) are tissue resident in the lung and activated by inhaled allergens via epithelial-derived alarmins including IL-33. Activated ILC2s proliferate, produce IL-5 and IL-13, and induce eosinophilic inflammation. Here, we report that intranasal IL-33 or the protease allergen papain administration resulted in increased numbers of ILC2s not only in the lung but also in peripheral blood and liver. Analyses of IL-33 treated parabiosis mice showed that the increase in lung ILC2s was due to proliferation of lung resident ILC2s, whereas the increase in liver ILC2s was due to the migration of activated lung ILC2s. Lung-derived ILC2s induced eosinophilic hepatitis and expression of fibrosis-related genes. Intranasal IL-33 pre-treatment also attenuated concanavalin A-induced acute hepatitis and cirrhosis. These results suggest that activated lung resident ILC2s emigrate from the lung, circulate, settle in the liver and promote type 2 inflammation and attenuate type 1 inflammation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mathä, Romera-Hernández, Steer, Yin, Orangi, Shim, Chang, Rossi and Takei.)

Alzheimer's disease (AD) is the most common cause of dementia in humans and, currently, a valid treatment is lacking. Our goal is to demonstrate the importance and benefits of the relationship with companion animals (considered as co-therapists), intended as a means of facilitating social relations and promoting evident wellbeing in AD patients. The study involved 30 randomly chosen patients with Alzheimer's disease (group T) and three dogs. The group participated in a total of 24 animal-assisted interventions (AAIs) sessions over a span of 12 weeks, using the Mini-Mental State Examination (MMSE), Wellness and Cognitive Ability Questionnaire (Brief Assessment Cognition or BAC), and Alzheimer's Disease Assessment Scale (ADAS) as assessment tests. A second group (group C), consisting of 10 people with AD, was enrolled as control group and underwent the same assessment tests but did not benefit from the presence of the dogs. Tests were carried out at time T0 (before starting sessions), T1 (end of sessions), and T2 (two months after last session). People belonging to group T achieved an overall improvement in their perceived state of wellbeing, even on a cognitive and mnemonic plane. However, two months after the end of the sessions, the test results in people suffering from AD decreased towards the baseline (T0). The study shows how such progress can be achieved through activities based on the relationship with an animal, as long as the animal is a steady presence in the life of the patient receiving the intervention. Dogs involved in other dog-assisted therapies have been found suitable also for assisting patients with AD.

Objectives: To compare the capacity of ibrutinib (IB) and idelalisib-rituximab (IDELA-R) of prolonging overall survival (OS) as in CLL patients, previously treated with chemotherapy only., Methods: A real-life cohort of 675 cases has been identified and investigated in the database of the groups participating in the study., Results: At an unadjusted univariate analysis, a significant death risk reduction was observed favoring IB (IDELA-R vs IB HR = 0.5, 95% CI = 0.36-0.71) although with some limitations due to the non-randomized and retrospective nature of the study and to the lower number of patients in the IDELA-R group (112 cases) related to the current prescribing practice. To overcome the potential problem of confounding by indication, we adjusted the association between the type of therapy and mortality for all variables significantly associated with OS at Cox univariate analysis. Furthermore, those variables, differently distributed between the two study groups, were introduced into the multivariate Cox model to improve the effectiveness of the analysis. By introducing all these variables into the multiple Cox regression model, we confirmed the protective effect of IB vs IDELA-R (HR = 0.67, 95% CI = 0.45-0.98, P = .04) independent of potential confounders., Conclusions: Although our analysis presents some constraints, that is, the unavailability of additional potential confounders, and the retrospective nature of the study, this observation may be of help for the daily clinical practice, particularly in the absence of randomized trials comparing the two schedules., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)