Is rituximab more effective than cyclophosphamide for treatment of granulomatosis with polyangiitis (GPA)?In this comparativeness effectiveness study based on clinical data from 194 patients with GPA, rituximab as induction therapy was associated with significantly more achievement of remission with a prednisone dose of 10 mg/d or less compared with cyclophosphamide. Rare disease registries and comparative effectiveness studies enable hitherto poorly addressed treatment comparisons for such entities.These findings may inform clinical decision-making regarding the choice of remission-inducing regimen for patients with GPA. This comparative effectiveness study compares the association of rituximab vs cyclophosphamide with inducing remission among unselected patients with granulomatosis with polyangiitis. Results of randomized clinical trials have demonstrated rituximab’s noninferiority to cyclophosphamide as induction therapy for antineutrophil cytoplasm antibody (ANCA)–associated vasculitides (AAV), with neither treatment having a specific advantage for granulomatosis with polyangiitis (GPA). However, post hoc analysis results have suggested that rituximab might be more effective than cyclophosphamide in inducing remission in patients with proteinase 3–positive AAV.To compare the effectiveness of rituximab and cyclophosphamide in inducing GPA remission in a large population of unselected patients.This comparative effectiveness study used multicenter target trial emulation observational data from 32 French hospitals in the French Vasculitis Study Group Registry. Groups were determined according to treatments received, without any intervention from the investigators. Inverse probability of treatment weighting was used to correct for baseline imbalance between groups. Participants included patients with newly diagnosed or relapsing GPA who satisfied American College of Rheumatology classification criteria and/or Chapel Hill Consensus Conference nomenclature. Data were analyzed from October 1, 2021, to May 31, 2022.At least 1 infusion of rituximab or cyclophosphamide for induction therapy between April 1, 2008, and April 1, 2018.The primary outcome was remission rate at month 6 (±2 months), with remission defined as a Birmingham Vasculitis Activity Score (BVAS) of 0 and prednisone dose of 10 mg/d or less. The BVAS is a validated tool for small-vessel vasculitis and used to assess the level of disease activity, with a numerical weight attached to each involved organ system. The BVAS has a range of 0 to 63 points; a score of 0 indicates no disease activity. Subgroup analyses included the primary outcome for patients with a new diagnosis, for most recently treated patients, and for patients with myeloperoxidase-ANCA positivity.E value for RR, 3.05). Similar results were observed in the subgroup of patients with newly diagnosed GPA and those with a more recent treatment. In the subset of 27 patients with myeloperoxidase-ANCA–positive GPA, 8 of 10 rituximab recipients and 8 of 17 cyclophosphamide recipients met the primary end point .Among 194 patients with GPA included in the analysis , 165 (85.1%) had a new diagnosis, and 147 of 182 with data available (80.8%) had proteinase 3–ANCA positivity. Sixty-one patients received rituximab and 133 received cyclophosphamide for induction therapy. In the weighted analysis, the primary outcome was reached for 73.1% of patients receiving rituximab vs 40.1% receiving cyclophosphamide (relative risk [RR], 1.82 ; risk difference, 33.0% ; In this comparativeness effectiveness study using clinical data, rituximab induction therapy for GPA was more frequently associated with remission than cyclophosphamide. These results inform clinical decision-making concerning the choice of remission induction therapy for this subset of patients with AAV. Among AAVs, granulomatosis with polyangiitis (GPA) is characterized by granulomatous inflammation, usually involving the upper and lower respiratory tract, with frequent necrotizing glomerulonephritis, and with ANCA-targeting proteinase 3 (PR3) in most patients with active disease.2—and a higher risk of relapse.4 Similarly, PR3-ANCA positivity as opposed to myeloperoxidase (MPO)-ANCA positivity is independently associated with a higher risk of relapse.4The diagnosis of GPA as opposed to microscopic polyangiitis is associated with a lower probability of achieving complete remission—defined as no evidence of clinical disease activity while not receiving glucocorticoid treatment6 showed rituximab’s noninferiority to cyclophosphamide for remission induction in severe AAVs, with neither treatment having a specific advantage for GPA in adjusted analyses. However, post hoc analysis of the trial data7 showed that rituximab-treated patients with PR3-AAV achieved complete remission at 6 months more frequently than cyclophosphamide-treated patients. Trial limitations included the enrollment of patients with GPA or microscopic polyangiitis, who might need to be studied separately; small sample size owing to the rarity of those AAVs; and highly selected patients with minor comorbidities that precluded generalization. For those reasons, registry data are useful to complement data from randomized clinical trials to investigate the external validity of drugs prescribed in routine practice. Thus, it remains to be established whether rituximab is more effective than cyclophosphamide at specifically achieving GPA remission. The objective of this study was to undertake an emulation target trial using observational data to compare rituximab vs cyclophosphamide efficacies at inducing remission in a large number of unselected patients with GPA.Results of randomized clinical trialsISPOR) reporting guideline for comparative effectiveness research. This observational analysis was designed to emulate a target trial  of induction therapy with rituximab vs cyclophosphamide to obtain GPA remission in a clinical setting.This comparative effectiveness study was conducted in compliance with the Declaration of Helsinki and approved by the Cochin University Hospital Ethics Committee. All patients in the French Vasculitis Study Group (FVSG) Registry gave their written informed consent. This study followed the International Society for Pharmacoeconomics and Outcomes Research  for remission induction between April 1, 2008, and April 1, 2018. The BVAS is a validated tool for small-vessel vasculitis and used to assess the level of disease activity, with a numerical weight attached to each involved organ system. The BVAS has a range of 0 to 63 points; a score of 0 indicates no disease activity. Only patients with clinical findings available since diagnosis and who were followed up for at least 6 months or died within 6 months of entry were included. Exclusion criteria were the coexistence of an associated autoimmune disease, active malignant disease or a history of malignant disease in the last 5 years, previous receipt of rituximab for induction therapy, receipt of oral or intravenous cyclophosphamide or rituximab within 4 months before enrollment, history of standard therapy–induced adverse events , limited GPA, alveolar hemorrhage with respiratory failure, creatinine level of greater than 4.0 mg/dL , or concomitant treatment consisting of plasma exchange(s), methotrexate, mycophenolate mofetil, or azathioprine as part of the induction regimen.We included patients with newly diagnosed or relapsing GPA, with active disease defined as a Birmingham Vasculitis Activity Score (BVAS [version 3]) of at least 311 and used in daily practice during the study period: (1) rituximab administration of 4 weekly infusions of 375 mg/m2 or 1 g, 2 weeks apart; and (2) cyclophosphamide infusion of 0.6 g/m2 on days 1, 15, and 29, then 0.7 g/m2 every 21 days. Patients were allocated to the group corresponding to the first induction agent received (rituximab or cyclophosphamide), regardless of subsequent adherence to this regimen. After achieving remission, maintenance treatments allowed for both groups were azathioprine (2 mg/kg/d), methotrexate (0.3 mg/kg/wk), or a low-dose (500 mg) preemptive rituximab infusion every 6 months from months 4 to 6 through month 24 (4-5 infusions within 18 months).We compared 2 induction therapy strategies according to French guidelinesPatients may have received, or not, 1 to 3 pulses of methylprednisolone (≤1000 mg) followed by oral prednisone equivalent to 1 mg/kg/d . The oral prednisone dose had to be tapered so that, by months 6 (±1 month) and 12 (±1 month), all patients who achieved remission without flaring received prednisone, 10 and 5 mg/d, respectively. Within these parameters, the dose of oral prednisone was allowed at the discretion of the investigator.2 Remission was defined as the absence of signs of new or worsening disease activity according to BVAS of 010 and prednisone dose of 10 mg/d or less; relapse, as the reoccurrence or new appearance of disease activity attributable to active vasculitis. Failure was defined as relapse, all-cause death, addition of or shift to rituximab (for the cyclophosphamide group) or cyclophosphamide (for the rituximab group), or treatment discontinuation. Discontinuation was defined as fewer than 6 infusions (except in documented remission) for the cyclophosphamide group and prescription of oral or intravenous cyclophosphamide within 2 to 6 weeks after induction for the rituximab group. The following manifestations were considered granulomatous manifestations: orbital masses, pulmonary masses, pachymeningitis, subglottic stenosis, or involvement of the ear, nose, and throat.12The following definitions were applied.The primary outcome measure was remission rate at month 6 (±2 months), as previously defined. Subgroup analyses included the primary outcome for patients with newly diagnosed disease, among those most recently treated, and for patients with MPO-ANCA positivity. Secondary end points were the percentage of patients with BVAS of 0 at month 6 (±2 months) at any glucocorticoid dose, retention rate without failure at 24 months, and safety at month 6 (±2 months). Follow-up for each patient started at induction onset (baseline).13 Accordingly, observations were weighted by 1 / PS for patients receiving rituximab and 1 / (1 – PS) for those receiving cyclophosphamide, where PS denotes the propensity score . The propensity score was estimated using logistic regression. Covariates were selected based on a nonparsimonious approach to account for both potential confounders and variables that could serve as proxies for unknown or unmeasured confounders. Moreover, priority was given to prognostic variables, because they allow lowering of the IPTW-estimator variance, whereas variables strongly associated with treatment but not—or weakly—with the outcome  can induce unstable weights with little or no gain in terms of bias reduction. Covariates included in the model were year of induction treatment, pure granulomatous disease, BVAS, age, self-reported sex, hematuria, PR3-ANCA positivity, ischemic abdominal pain, stroke, mononeuritis multiplex, severe alveolar hemorrhage or acute respiratory distress syndrome, sensorial deafness, motor peripheral neuropathy, scleritis, creatinine clearance of greater than 30 mL/min, and cranial nerve involvement.Data were analyzed from October 1, 2021, to May 31, 2022. An inverse probability of treatment weighting (IPTW) approach was used to account for differences in baseline variables between treatment groups.15 and served as a target in the propensity score construction. Another key requirement for IPTW was the positivity assumption, that is, the propensity score must be bounded away from 0 and 1. Positivity was assessed by determining the mean stabilized weights and their SD, minimum, and maximum. A mean far from 1 or the presence of very extreme values can indicate nonpositivity or misspecification of the propensity score model.16 The overlap of propensity score distributions by group was also assessed graphically, including when the analysis was restricted to patients with newly diagnosed GPA.To check the ability of IPTW to obtain well-balanced arms, standardized differences between arms before and after weighting were computed. A standardized difference less than 10% indicated successful balance17 A truncation at the 99th percentile of the propensity score was also applied to avoid extreme weights. The primary outcome analysis relied on the IPTW relative risk (RR), with 95% CI derived from a Poisson model with robust variance. The absolute risk difference and 95% CI were also estimated using a robust Poisson model with identity link. Two sensitivity analyses were computed for the primary outcome. First, estimates were computed in the unweighted sample. Second, a double-robust estimator for the RR was used. That latter approach combines 2 models: the propensity score model for the IPTW and a Poisson model relating the outcome to the treatment and covariates. This allows an unbiased RR estimate if either the Poisson models for the outcome or the propensity score model is correctly specified, but not necessarily both (double-robustness property). We also evaluated how sensitive the results were to unmeasured confounding using the E value, which measures the minimum strength of association that an unmeasured confounder should have with the treatment and outcome to fully exclude the treatment effect.18To preserve the sample size of the original data, stabilized weights were used.A preplanned subgroup analysis examined the primary outcome for the subset of patients with newly diagnosed GPA, for those most recently treated, or for those positive for MPO-ANCA. Because the sample size was too small to calculate relevant weights for the latter, we calculated only the unweighted RR (on imputed data sets) based on a Poisson model. The rate of patients with BVAS of 0 at month 6 (±2 months) at any glucocorticoid dose was analyzed using the same approach as the primary outcome. As an exploratory analysis, the hazard ratio with 95% CI for survival without failure at 24 months was estimated in the IPTW sample using a Cox proportional hazards model with robust variance.19 All tests applied a 2-sided P ≤ .05 significance level. The analyses were computed using R software, version 4.0.3 .Missing baseline variables and outcomes were handled by multiple imputation by chained equation, using all baseline propensity score model variables and the treatment received in the imputation model. The number of independent imputed data sets was determined by rounding the percentage of observations with missing data up to the nearest integer (n = 42). For each data set, a propensity score was estimated, and the linear predictive factors were then pooled according to Rubin rules.Among 281 patients with GPA assessed for eligibility, 194  were included ; 61 receE value for RR, 3.05)  than cyclophosphamide-treated patients (42 of 107 [39.3%]) achieved BVAS of  0 at 6 months (±2 months) with a prednisone dose of 10 mg/d or less (primary outcome). The weighted analysis on the multiply imputed data sets found that the primary outcome was reached for 73.1% of rituximab recipients vs 40.1% of cyclophosphamide recipients  . The resR, 3.05) .The overlap of propensity score distributions by group is shown graphically , regardless of prednisone dose, was 47 of 55 (85.5%); in the cyclophosphamide group, 95 of 115 (82.6%). The comparative imputed analyses are shown in Among rituximab induction recipients, rituximab was prescribed as maintenance therapy for 48 of 55 (87.3%); azathioprine, for 2 of 47 (4.3%); methotrexate, for 1 of 48 (2.1%); and mycophenolate mofetil, for 1 of 48 (2.1%). Among cyclophosphamide induction recipients, rituximab was prescribed as maintenance therapy for 57 of 121 (47.1%); azathioprine, for 57 of 117 (48.7%); methotrexate, for 8 of 110 (7.3%); and mycophenolate mofetil, for 7 of 116 (6.0%). Postinclusion treatment failures at 24 months occurred in 7 rituximab recipients and 51 cyclophosphamide recipients, with most due to relapses . Descriptive analysis of treatment failures at 24 months and comparative analyses of survival without failure at 24 months are reported in eTables 4 and 5, respectively, in The results of this target trial emulation based on clinical data of patients with newly diagnosed or relapsing GPA showed that those who received rituximab for induction treatment more frequently achieved remission with a prednisone dose of 10 mg/d or less compared with cyclophosphamide-treated patients. This comparative effectiveness analysis using observational data from a prospective rare disease registry allowed treatment comparisons—so far poorly addressed—and confirmed the hypothesis that rituximab could be superior to cyclophosphamide for induction of GPA remission.6 In that study, patients with AAV allocated to receive rituximab experienced proportionally (but not significantly) more frequent remissions with successful completion of prednisone tapering at 6 months (64%), compared with patients allocated to receive cyclophosphamide (53%). Their sensitivity analysis restricted to patients with GPA, in line with our observations, highlighted that more rituximab recipients (63% vs 50% of cyclophosphamide recipients) achieved complete remission with successful completion of prednisone tapering at 6 months, even in the absence of a statistically significant difference. Furthermore, a post hoc analysis of trial data from 131 patients with PR3-AAV (127 of whom were classified as having GPA)7 showed that patients with PR3-AAV were more than twice as likely to achieve complete remission at 6 months when treated with rituximab rather than with cyclophosphamide . Differences in key AAV outcomes may not be apparent in clinical trials owing to their own limitations, including the fact that more than 1 disease had often been included to overcome their rarity, and, hence, they were not powered to detect significant differences about a given intervention’s efficacy within a single disease entity.20 Our clinical data from a larger subset of patient with GPA indicated a specific advantage of rituximab over cyclophosphamide induction therapy for patients classified as having GPA, most of whom had PR3-ANCA–positive disease.Our findings are consistent with those of the pivotal trial on rituximab induction treatment for AAVs.6 The higher remission rate among rituximab recipients was not owing to more of them having relapsing disease. Our analysis confirmed that remission rate remained significantly higher for rituximab recipients than cyclophosphamide recipients when the analysis was restricted to those with newly diagnosed GPA, a finding not yet reported.Our small number of patients with relapsing GPA prescribed cyclophosphamide induction treatment post relapse may be attributed to the known lower cyclophosphamide efficacy to induce AAV remission in such patients found in the pivotal trial.21 rather than using the BVAS alone. The absence of differences for patients with MPO-GPA may only reflect the small sample size. Although secondary outcome analysis results also gave the advantage to rituximab over cyclophosphamide, the data are unclear because more recipients of rituximab induction treatment also received rituximab maintenance therapy, known to be more effective. No toxicity signals were observed in either group.Between-group differences in the numbers of patients achieving 6-month BVAS of 0 at any glucocorticoid dose was not observed, which might be attributed to residual vasculitis activity that was undetectable because of glucocorticoid masking. It may be easier to demonstrate treatment efficacy by including the glucocorticoid dose in the definition of the primary end point, as recommended,Our study has major strengths. The FVSG Registry collected the clinical characteristics and outcomes of 194 patients originating from several medical specialties, thereby enabling analysis of a truly representative distribution of different GPA presentations and evolutions. The thoroughness of these data allowed detailed characterizations of both treatment groups, with well-phenotyped baseline features and outcome ascertainment and close matching according to key confounders. Analyses benefited from multiple methods to address potential confounding by indication, and subgroup and sensitivity analyses were concordant for the primary outcome, bolstering confidence in the main results. The demographic composition of the FVSG Registry allowed inclusion of patients with comorbidities and/or with MPO-ANCA–positive and ANCA-negative disease, who are often underrepresented.This study has some limitations. First, as in any observational analysis, assignment to a particular treatment was not randomized. The higher number of rituximab recipients with relapsing disease could not be balanced because of the small number of such cyclophosphamide recipients. This enrichment cannot explain the overall higher remission rate of recipients of rituximab induction treatment, as supported by the analysis restricted to newly diagnosed patients. In addition, we rigorously balanced the rituximab and cyclophosphamide groups with respect to all other demographic characteristics, medical history, and all covariates a priori thought to be associated with the likelihood of receiving rituximab or cyclophosphamide and achieving remission. However, we agree that other unmeasured confounding that could affect the primary outcome might still persist. Second, although the enrolment period was limited to 10 years, it cannot be excluded that the physicians gradually attempted a transition toward lower levels of glucocorticoid use for patients with AAV during the induction phase, which might have benefited rituximab recipients, who had received induction treatment at a later median date than the cyclophosphamide group. However, the year in which patients received their induction treatment was taken into account in the statistical analysis. Third, patients with limited GPA, alveolar hemorrhage with respiratory failure, or creatinine level greater than 4.0 mg/dL  or who received concomitant treatment with plasma exchange(s), methotrexate, or azathioprine as part of their induction regimen were excluded. The comparative efficacy of rituximab vs cyclophosphamide regimens is less clear in these patients. However, they represent only a minority of our patients who were recruited from 32 vasculitis centers, including several medical specialties. Thus, we think that our data are likely to be representative of the entire GPA spectrum and that our sample is a good representation of patients in whom the question of induction with rituximab or cyclophosphamide arises. Additionally, the secondary outcome analysis results also supported rituximab over cyclophosphamide, but the data remain uncertain, because more rituximab induction recipients also received rituximab for maintenance, and the analyses did not account for treatments given after the exposure of interest.The findings of this target trial emulation suggest that patients with GPA obtained remission with a prednisone dose of 10 mg/d or less more frequently when they had received rituximab rather than cyclophosphamide for induction therapy. Our results may inform clinical decision-making concerning the choice of remission induction therapy for this subset of patients with AAV.


APOB) gene is causal to the inherited genetic defect cholesterol deficiency (CD) observed in neonatal and young calves. Affected calves suffer from developmental abnormalities, symptoms of incurable diarrhoea and often die within weeks to a few months after birth. Neither the detailed effects of the LTR insertion on APOB expression profile nor the specific mode of inheritance nor detailed phenotypic consequences of the mutation are undisputed. In our study, we analysed German Holstein dairy heifers at the peak of hepatic metabolic load and exposed to an additional pathogen challenge for clinical, metabolic and hepatic transcriptome differences between wild type (CDF) and heterozygote carriers of the mutation (CDC). Our data revealed that a divergent allele-biased expression pattern of the APOB gene in heterozygous CDC animals leads to a tenfold higher expression of exons upstream and a decreased expression of exons downstream of the LTR insertion compared to expression levels of CDF animals. This expression pattern could be a result of enhancer activity induced by the LTR insertion, in addition to a previously reported artificial polyadenylation signal. Thus, our data support a regulatory potential of mobile element insertions. With regard to the phenotype generated by the LTR insertion, heterozygote CDC carriers display significantly differential hepatic expression of genes involved in cholesterol biosynthesis and lipid metabolism. Phenotypically, CDC carriers show a significantly affected lipomobilization compared to wild type animals. These results reject a completely recessive mode of inheritance for the CD defect, which should be considered for selection decisions in the affected population. Exemplarily, our results illustrate the regulatory impact of mobile element insertions not only on specific host target gene expression but also on global transcriptome profiles with subsequent biological, functional and phenotypic consequences in a natural in-vivo model of a non-model mammalian organism.The insertion of an endogenous retroviral long terminal repeat (LTR) sequence into the bovine apolipoprotein B ( CD-affected neonatal calves suffered from insufficient development in combination with symptoms of incurable diarrhoea and generally died within the first 6 months after birth1. Concentrations of cholesterol and triglycerides in the blood were markedly reduced in affected individuals, indicating a disorder of lipid metabolism5. Pedigree analysis of affected calves initially suggested an autosomal recessive mode of inheritance5, and the insertion of a transposable long terminal repeat (LTR) element in exon 5 of the apolipoprotein B (APOB) gene was found as causal mutation7. The encoded APOB protein is a crucial key component for the transport of lipid molecules, including cholesterol, through the body and into cells. Initial results indicated that the insertion comprised a 1299 bp solo-long terminal repeat (LTR) insertion, which was predicted to result in a premature stop codon causing a protein that is 97% truncated compared to its wild-type sequence. Causal mutation data were later updated by a report describing that indeed a full-length bovine endogenous retroviral K element (ERV-K) had integrated into the coding part of the gene at position 11:77,891,7398. The ERV-K was flanked by two identical full-length LTRs, and the open reading frames within the retroposon genes were altered (loss-of-function)8. Menzi et al.7 provided data that indicates a complete lack of transcribed sequence for the APOB gene downstream of the LTR insertion, and Harland8 reported a polyadenylation signal in the 5′ERV-LTR, which presumably generates a premature poly(A) tail and subsequent termination of transcription. ERV-LTRs are known to exert regulatory activities as reviewed by Chuong et al.9, and due to their autonomous promoters, they can generate chimeric transcripts and readthrough transcription10. However, many studies on the regulatory effects of LTRs are based on bioinformatic prediction from functional genomic studies or on CRISPR-mediated modification of (cancer) cell lines. There are few studies on the effects of LTRs in their natural in-vivo context9.In 2015, a genetic defect termed Cholesterol Deficiency . But although Gross et al.12 found differences in cholesterol and lipoprotein concentrations between CDC and homozygous wild type (CDF) animals, CDC and CDF cows did not differ in other metabolic parameters, milk yield or fertility. Furthermore, the question arose, whether cholesterol synthesis and turnover are indeed impaired as a consequence of the CD mutation or whether rather the pathophysiological effect of the mutation is only induced via malabsorption and impaired transportation of lipid components due to a lack of APOB to form lipoprotein complexes12.In addition to its molecular genetic background, also pathophysiological consequences of the CD mutation are controversially discussedAPOB expression and subsequent effects on lipid metabolism, we selected an experimental design, which presented a substantial challenge to APOB regulation and lipid metabolism. The lipid metabolism of cows is particularly critical in the first weeks after parturition due to a severe negative energy balance associated with a substantial lipomobilization15, and even more so in situations of pathogen-challenges16. Thus, to explore the molecular regulatory consequences of the ERV-LTR insertion on APOB expression and subsequent effects on lipid metabolism, our study monitored hepatic expression pattern and biochemical phenotypes of CDC and CDF early lactation cows after an intramammary challenge with relevant mastitis pathogens (Staphylococcus (S.) aureus or Escherichia (E.) coli).Thus, to explore the molecular consequences of the LTR insertion on 20. The final cohort comprised 35 animals, seven heterozygous CDC carriers and 28 homozygous wild type CDF animals. Homozygous mutant CDS cows were not available, presumably due to the well-described fatal effects of the mutation in early life. The selected heifers were purchased from dairy cow farms across Germany prior to first calving and were brought to the Clinic for Cattle at the University of Veterinary Medicine Hannover (TiHo) for a highly standardized challenge experiment. Only CDC cows were recruited that did not show manifest clinical symptoms of the CD defect before first calving .The cows investigated in this study were part of a large network project to evaluate genetic predisposition for disease susceptibility in Holstein Friesian cows. Details on the selection and management of the animals have been previously described21. The reporting in the manuscript follows the recommendations in the ARRIVE guidelines. This includes a comprehensive description of experimental animals and experimental procedures as provided in20.For the cow cohort, the experiment was performed under the reference number 33.12-42502-04-15/2024 approved by the Lower Saxony Federal State Office for Consumer Protection and Food Safety. This study was also submitted to and approved by the ethics committees of the Leibniz Institute for Farm Animal Biology and the University of Veterinary Medicine Hanover, respectively. An independent previous calf experiment had been conducted at the Educational and Research Centre for Animal Husbandry, Hofgut Neumuehle, Germany. It had been permitted by the local department for animal welfare affairs  (23 177-07/G 13-20-069). All ethical evaluations were performed as required by the German Animal Care law and associated legislative regulations20. At 36 ± 3 days after parturition, 24 healthy cows  were challenged with 10,000 CFU (colony forming units) S. aureus1027 each in both hindquarters of the mammary gland. Eleven healthy cows  were challenged with 500 CFU E. coli1303 in one hindquarter of the mammary gland22. A control udder quarter of each cow was infused with sterile sodium chloride solution.The 35 animals were challenged in an intramammary infection model essentially as described in detail by Rohmeier et al.20. Particular attention was paid to signs of inflammation as well as to biochemical indicators of impaired energy and fat metabolism. To this end, blood serum concentration of beta-hydroxybutyrate (BHB) (mmol/L) and nonesterified fatty acids  and blood plasma concentrations of insulin and IGF-1 were determined in weekly intervals starting 3 weeks before parturition until the end of the experiment with commercial kits as described by Meyerholz et al. 19 in 3 days intervals starting 3 weeks before parturition until the end of the experiment. Measuring plasma GH concentration was performed with an enzyme-linked immunosorbent assay23 with modifications and adaptations according to Kawashima et al.24 and Meyerholz et al.25.Before and after the experimental challenge, cows were comprehensively monitored for clinical and subclinical diseases as described previously19. All statistical analyses were performed using SAS 9.4.1. The general linear mixed model implemented in the GLIMMIX procedure included fixed effects for day and group, the interaction of day and group and a random sire effect G: y = β0 + β1∙day + β2∙group + β3∙day x group + G. For the effect of day, we had eight categories for days relative to parturition: − 18 ± 3; − 11 ± 3; − 4 ± 3; 1; 4 ± 3; 11 ± 3; 18 ± 3; 25 ± 3, 32 ± 3, the group comprised either CDC or CDF. Furthermore, covariance structure comprised the repeated subject cow in compound symmetry. For those data measured before and after calving, separate models were calculated.For statistical data analyses, the GLIMMIX procedure of SAS 9.4.1  was applied. Prior to evaluation, we log10-transformed NEFA, BHB, insulin, GH and IGF-1 data. For milk yield, we considered energy-corrected milk as described by Meyerholz et al.S. aureus: 96 h after infection; E. coli: 24 h after infection), the animals were stunned with a penetrating captive bolt pistol, immediately followed by exsanguination via longitudinal section of the jugular veins and carotid arteries. Liver tissue from the Lobus caudatus and mammary gland tissue were immediately shock frozen in liquid nitrogen and subsequently stored at − 80 °C.At the end of the experiment  and a Qubit 2.0 fluorometer . RNA integrity was quantified on the Bioanalyzer 2100 . Finally, a strand-specific RNAseq library preparation protocol with poly(A) selection  was applied to the samples. The RNAseq libraries were again monitored for quality on the Bioanalyzer 2100 and were paired-end sequenced on the Illumina HiSeq 2500 system  for two times 90 base pairs.RNA isolation from liver was conducted as described by Heimes et al.28 and SAMtools29 were used. Quality of the raw reads was checked with FastQC version 0.11.530 and MultiQC version 1.431. Subsequently, adapters were clipped with Cutadapt version 1.1232, and low qualities bases were eliminated with QualityTrim33. The reads were aligned to the bovine reference genome ARS1.2 with Ensembl 95 reference annotation with Hisat2 version 2.1.034. The featureCounts option of the subread package version 1.6.235 was used for strand-aware read counting. For differential expression analysis in liver samples, we applied DESeq2 version 1.26.036 in a model fitting the pathogen used for the challenge and the carrier status at the CD locus as fixed effects. Finally, lists of differentially expressed genes (DEGs) were used for enrichment analyses in DAVID37 and Ingenuity Pathway Analysis  for the identification of significantly enriched GO terms, KEGG and IPA pathways and predicted IPA upstream regulators with a threshold for significance of padj < 0.05.For data management, Linux, and R scriptsAPOB gene exons separately as well as combined for exons 1–4 (exons upstream of the LTR insertion site) and exons 5–29 (within and downstream of the LTR insertion site) using the featureCounts option of the subread package (version 1.6.2)35. The resulting counts were normalized for transcriptome-wide total read counts for each animal and the length of the respective exons. The resulting normalized read counts were screened for differences between CDC and CDF animals in a linear model using the lm function of the R packages lme4 (v. 1.1-25) and lsmeans (v.2.30-0). The fixed effect of pathogen type used for the challenge  as well as the fixed effect of the APOB genotype  were fitted into the model.For all individuals, read counts were determined for all APOB haplotypes contributed equally to the increased APOB exon 1–4 expression in CDC animals, or whether there was an allelic imbalance, we took advantage of SNPs in exon 3 at position 77,887,611 and exon 13 at position 77,899,208 on BTA11 (NC_037338.1) identified after visual inspection of aligned reads via the Integrative Genomics Viewer . To enable unbiased genotyping at the variant positions, genomic DNA was isolated from liver and mammary gland samples. From those samples, a genomic fragment including the entire exon 3 as well as a genomic fragment spanning exon 13 were amplified by PCR for genotyping the SNPs at positions 77,887,611 and 77,899,208, respectively. PCR amplification from genomic DNA was conducted with primers listed in Supplementary Table APOB mRNA (XM_024999521.1) and genomic (NC_037338.1) reference sequences and were visually inspected with Bioedit v7.2.5.0 to obtain the animals’ genotypes for the respective positions.To evaluate, whether both APOB locus. SNPs in the target region were filtered for minor allele frequency (MAF > 0.05), deviation from Hardy–Weinberg equilibrium (p > 0.05) and GenTrain Score (GT > 0.7). Due to the fact, that some CDF and CDC heifers had a common sire, which had a confirmed CDC carrier status, we were able to conclude on the haplotype phases of the sire and the inherited haplotypes of the respective offspring within the APOB gene  comprising 777,692 SNPs to establish genotypes for the genomic target region on BTA11 upstream, downstream and within the 38 (version 1.8) with subsequent Linux scripts. An analogous analysis was done for position 11:77,899,208 by calculating the reads covering the non-reference allele “T” or the reference allele “C”. We calculated the ratio of reads with the two alternative alleles  for both positions. Across all animals with a heterozygous genotype C/T at 77,887,611 or 77,899,208, we tested, if this ratio differed between CDC and CDF animals with a simple linear model fitting the CD genotype as a fixed effect. Furthermore, a t-test conducted in R (version 3.6.2) was applied to test whether the ratio differed significantly from the expected value of 1.This information was used for an allele biased differential expression analysis for the positions 11:77,887,611 and 11:77,899,208. To this end, we established the total number of reads spanning position 11:77,887,611, which carried either the non-reference allele “C” or the reference allele “T” by extracting read coverage information from samtools mpileupAPOB region, genomic DNA from two CDC and CDF animals from the pathogen challenge experiment as well as two CDC calves from a previous independent experiment unrelated for at least three generations39 were used for whole genome resequencing  after PCR free library preparation on a NovaSeq flowcell with 2 × 150 bp sequencing. The reads obtained were processed according to the protocol for the 1000 bulls genome project40. This comprised quality trimming with Trimmomatic (v. Trimmomatic-0.38) and alignment to the bovine genome assembly ARS1.241 with bwa42 and read sorting via samtools (version 1.8). PCR duplicates were marked and removed via Picard tools . GATK43  was used for base recalibration  and variant calling . The respective pipeline code is provided as Supplementary File Text 44), bcftools (29 version 1.14) and in-house Linux scripts. In addition, we had available a further Holstein x Charolais F2 individual with hepatic transcriptome data45  and whole genome sequence data , which also had a CDF wild type status.To obtain further sequence information for the APOB gene alternative to the LTR retrotransposon insertion, we screened the region 500 kb upstream of the APOB gene LTR insertion. To this end, we filtered SNPs and small indels on BTA11 from position 11:77,385,988 to 77,891,739 bp (NC_037338.1) in the six resequenced animals using bcftools version 1.929. We postulated that all CDC animals should be heterozygous for the variant causal for generating the haplotype-biased gene expression, whereas the variant should be homozygous for the CDF animals of the dataset because there was no indication of an allele-biased APOB expression in CDF animals.To exclude other variants on the affected haplotype that could be causative for the allele-biased expression of the https://www.ebi.ac.uk/ena, Project number PRJEB33849, accession numbers ERR3466640-ERR3466680) at EMBL-EBI. Across all samples, on average 92.1% of reads mapped at least once to the reference genome ARS-UCD1.2 with Ensembl genome annotation release 9547. The data analysis revealed a total of 13,213 annotated genes expressed with an FPKM > 1 in at least four samples, which were subjected to subsequent differential expression analysis.The hepatic transcriptome analysis by RNAseq generated 3.8 billion reads (Ø 109 million reads per sample). RNAseq datasets are submitted to the ENA repository (adj < 0.05) differentially expressed genes (DEGs) between CDC cows and CDF cows . The expression analysis revealed 146 significantly  of the expression in the CDF cows. Other gene members of the APO lipoprotein family, namely APOA2 and APOA4, were also statistically significantly lower expressed in CDC heterozygous carriers than in wild-type animals , FADS1, FADS2 (Fatty Acid Desaturase 1 and 2), and SCD (Stearyl CoA desaturase)  investigating the collection of genes differentially expressed between CDC and CDF animals indicated seven enriched GO terms, 16 enriched KEGG pathways and seven REACTOME pathways .The Ingenuity pathway analysis identified 80 pathways significantly enriched with genes differentially expressed between CDC and CDF cows  with the Integrative Genomics Viewer version 2.7.248. As shown in Fig. APOB gene drops abruptly and substantially in CDC cows.To gain further insight into the consequences of structural alterations of the APOB gene upstream of the LTR insertion site in exon 5 and the region downstream of this site. Across exons 1–4, the expression was more than tenfold higher (FPKM 258.7 (se 15.89)) across CDC heterozygous carriers compared to wild-type animals (FPKM 21.4 (se 8.28), p = 7.113 e−15, Supplementary Figure APOB exons upstream of the LTR retrotransposon insertion (exon 1–4) was significantly higher in CDC heterozygous carriers compared to wild-type animals  upstream of the insertion and in exon 13  downstream of the insertion. From genotypes in variant positions that are located upstream, within and downstream of the APOB gene, which had been determined from Illumina HD genotype data in a half-sib offspring cohort from a CDC carrier sire, we could conclude that the C allele  at position 11:77,887,611 was on the same haplotype as the LTR insertion in exon 5 of the APOB gene in CDC animals. Comparing the ratio of read counts for the C and the T allele in heterozygous animals at position 11:77,887,611, we found that the ratio for the CDF animals was close to the expected ratio of 1 ranging from 0.74 to 1.73  higher ratio ranging from 35.0 to 82.27 , also in the genomic region downstream of the LTR insertion for position 11:77,998,208 (SNP in exon 13). It has to be noted that the haplotype carrying the LTR retrotransposon insertion has a T  in position 11:77,998,208. Compared to position 11:77,887,611, for 11:77,998,208, the ratio of reads from the two alternative haplotypes was in opposite direction in the heterozygous CDC animals Fig. B. In theAPOB gene. Any potential variant would have to be heterozygous in all CDC animals of our study, and the CDF animals, which did not show an allele biased expression, would have to be homozygous. In the genomic region upstream of the APOB transcription start site, no variant fulfilled this requirement until the boundaries of the next upstream neighbouring gene, TDRD15. The closest position to the APOB gene, which was heterozygous in all four CDC cows and homozygous in both CDF animals, was at 11:77,792,966, which is almost 100 kb distant from the APOB gene and upstream to the transcription start site of TDRD15.Whole genome resequencing obtained a coverage between 12.2 and 16.8 fold per sample. In the interval 11:77,385,988–77,891,739, we detected 3530 heterozygous or homozygous alternative allele positions for the six samples in our dataset. We wanted to explore if other variants than the LTR retrotransposon insertion could potentially be causal for the observed allele biased expression in exon 1–4 of the 49, https://cgtex.roslin.ed.ac.uk/downloads), which were described as cis-acting eQTL for the APOB gene. In liver tissue, no variant within 1 Mb upstream or downstream of the APOB gene showed a nominal p value < 10–4, and even those three with a p-value < 10–3 were located more than 200 kb downstream to the 3′ end of the APOB gene.Finally, we also inspected loci in the Farm Animal GTex dataset . When challenged with S. aureus, no difference was seen in the incidence of clinical mastitis between the CDC (one out of four) and CDF group .All three CDC cows challenged intramammary with APOB gene carrying the LTR retrotransposon insertion. Across all exons of the APOB gene, we observed about 50% decreased expression in CDC carriers compared to the CDF homozygous wild type animals. This is in line with results from an abrogated APOB expression in homozygous CDS carriers8 and would be expected due to the lack of one copy of a fully functional gene. However, our data highlighted a strong discrepancy between the expression level for APOB exons upstream and downstream to the LTR retrotransposon insertion. The lower expression level for exons 6–29 downstream of the insertion compared to exons 1–5 could well be explained by the termination of transcription via a polyadenylation signal in the inserted LTR retrotransposon leading to the premature attachment of a poly(A) tail as described by Harland8. The first obvious explanation for the increased expression of exons 1–5 in the APOB gene would be a regulatory compensatory loop for the lack of an intact APOB protein resulting from the impaired expression of exons 6–29, which encode more than 95% of the APOB protein. However, a respective general compensatory mechanism should affect the expression regulation for both APOB copies regardless of the LTR retrotransposon insertion, e.g. via increased transcription factor activity or recruitment in the promotor region of the APOB gene51. To evaluate this hypothesis, we tested the expression output from both haplotypes separately. For the allele-biased expression analysis, we took benefit from variant positions in the coding part of the APOB gene as well as SNPs upstream and downstream of the gene body. After establishing the APOB haplotype carrying the LTR retrotransposon insertion, we were subsequently able to determine the haplotype origin of reads spanning variant positions in APOB exons 3 and 13.The phenotypic data on the heterozygous carrier CDC animals as well as the results from the transcriptome analysis indicated a phenotypic effect from a single copy of the APOB exons 1–5 in CDC animals seems to be attributed to the haplotype carrying the APOB LTR retrotransposon insertion, which created a very distinct allelic expression bias. Our data were obtained via preparation of a stranded RNAseq library with poly(A) selection from RNA meticulously cleaned of any DNA contamination. This should avoid the issues with transposable element transcription detection due to pervasive transcription and provide reliable information on structure and source of transcripts, particularly with respect to transposable element molecules10.The respective data in our study demonstrated that the enhanced expression of APOB transcription data might be interpreted as a specific cis-regulatory enhancer effect exerted by the LTR retrotransposon insertion in the APOB gene  family K. ERVKs comprise a subset of bovine retroviruses and seem to be currently active in the bovine genome52. For the human genome, HERV-K transposable elements have been found to be enriched with proximal and distal enhancer element motifs compared to the whole genome sequence53. Transposable elements, and LTR retrotransposons in particular, are known for their regulatory activities as reviewed by Chuong et al.9. The ERV-K LTR insertion in the bovine APOB gene comprises two full-length LTR flanking regions. Within LTRs, their U3 region is known to contain enhancer elements, which activate and drive viral transcription54 and can also modulate expression of nearby cellular genes55. LTRs are involved, e.g. in a regulatory dysfunction leading to leukaemia56 and are postulated for being responsible for ectopic CYP19A1 expression associated with the henny feathering phenotype in chicken57. Recently, the advances in CRISPR technology-enabled progress in experimental validation of the regulatory function of LTR retrotransposons9, which previously relied mainly on, e.g. selection signatures and chromatin features. Deniz et al.56 demonstrated via CRISPR-mediated targeted transposable element deletion in cancer cells that an LTR located proximal to the APOC1 gene has an enhancer function for APOC1 expression and also seems to exert a regulatory function on the downstream APOE gene. However, there are still very few studies demonstrating a regulatory function of LTR retrotransposons in vivo, particularly in natural models and non-model organisms. Our data suggest that the bovine APOB gene LTR retrotransposon insertion indeed is an in vivo model for a remarkable enhancer activity of ERV elements. Due to the widespread occurrence of LTR retrotransposon insertions, as reviewed by Thompson et al.58 and particularly due to the biological activity of the ERV-K type retroviruses59, they might be a major source of diversity in gene regulation in the bovine genome.According to HarlandAPOB gene. No gene in the region comprising 2 Mb upstream or downstream of the APOB gene showed a significantly differential expression between CDC and CDF animals, which excludes an element with general enhancer activity in this region on the LTR insertion carrying haplotype. Since 500 kb upstream of the APOB gene, no other variant fulfilled the requirements of (i) heterozygosity in all resequenced CDC animals and (ii) homozygosity of the resequenced CDF animals, we conclude that other elements with specific cis activity for APOB expression regulation on the LTR insertion carrying haplotype are very unlikely.Based on the gene expression data of our study alone, it could not be formally excluded that other variants on the LTR retrotransposon carrying haplotype could be responsible for the enhancer-like effects in our data set. To explore this option, we analysed transcriptomic and whole genome resequencing data from the neighbourhood of the APOB gene is predominantly expressed in the liver and small intestine with the alternative predominant isoforms in the small intestine (APOB-48) and liver (APOB-100) as reviewed by Whitfield et al.60. This is confirmed by whole transcriptome RNAseq data analyses of blood and mammary gland samples from CDC and CDF cows in this study, which did not show indication of APOB gene expression . The APOB gene is known for a tissue-specific posttranscriptional mRNA editing in the small intestine62, which creates a premature stop codon and is responsible for producing the truncated APOB-48 protein in intestine tissue. The signature of this regulatory editing process was also observed in the jejunal mucosa samples of our study . Due to its essential role in lipid metabolism, alterations in the APOB gene expression are likely to exert function beyond direct modulation of lipid intake.The ine APOB-8 and livine APOB-8 and livAPOB transcription. Earlier studies identified that CDC animals show a decreased plasma cholesterol level12. Gross et al.12 suggested that this results from a lack of APOB protein with the consequence of building less HDL, LDL and/or VLDL complexes containing cholesterol. Thus, the authors postulated that the decreased blood cholesterol levels would not reflect the true cholesterol abundance in the CDC animals. However, hepatic transcriptomes of CDC animals in our study show that important genes encoding enzymes for the final step of cholesterol synthesis are significantly lower expressed compared to CDF wild type animals. Therefore, the decreased plasma cholesterol level in CDC animals carrying the LTR insertion might also result from a decreased cholesterol synthesis in addition to a lack of cholesterol transport capacity. Reduced cholesterol synthesis, as indicated by our data, could explain the detrimental effects observed in heterozygous CDC individuals as described by Häfliger et al.13. In addition, cholesterol is the starting point of estrogen biosynthesis, and our data indicate that also this biological pathway is significantly affected in CDC carriers, which could result in impaired reproduction of CDC carriers.Our transcriptomic data from the differential expression analysis confirm that the ERV-K LTR insertion indirectly affects the regulation of cholesterol synthesis in addition to its direct effect on APOB gene. This is in line with reports from Häfliger et al.13 and confirms that the CD defect does not follow a fully recessive mode of inheritance. As indicated by the effects on milk yield, body weight loss, body condition score, and NEFA, the CDC animals seem to mobilize more internal abdominal fat, however, at a decreased performance level. In cattle, excess energy is primarily stored as triglycerides, which are stored in the adipose tissue and are mobilized when the energy intake does not meet the energy demand64. NEFA is one of the most important blood indicators of lipomobilization in ruminants and an indicator of negative energy balance in postpartum dairy cattle65. CDC animals have a significantly higher level of NEFA compared to CDF animals, but a significantly lower milk yield, whereas the BCS change is comparable between both groups. This suggests that the animals were possibly not fully capable to utilize the mobilized lipid resources.The phenotypic and the transcriptomic data of our study indicated an effect of a single copy of the LTR retrotransposon carrying the E. coli (N = 3) or S. aureus (N = 4) was relatively small in comparison to the number of challenged CDF animals , which could reduce the statistical power. To our knowledge, there is no study that compared the incidence of clinical mastitis between CDC and CDF animals, although Cole et al.14 reported a small favourable effect on genetic merit of the CD mutation carrying haplotype for somatic cell score, an indicator trait for mastitis incidence.There was no statistically significant difference in the incidence of clinical mastitis in CDC animals compared to CDF animals. However, the number of CDC animals challenged with 66. This is in line with the in-vivo experimental data obtained in this study. The gene regulatory effects of the ERV-K LTR beyond the decreased transcript level downstream of its insertion in the APOB gene demonstrate the strong potential of mobile elements to modify gene expression and complex phenotypes. Even though Scott et al.66 found that mobile elements were only weakly enriched for eQTL signals, future investigations of functional diversity within populations should direct specific attention to this class of genetic variants and their potential regulatory effects, because mobile element catalogues and graph-based genome exploration68 are only starting to be available, particularly for non-model organisms.There is upcoming evidence for structural variants playing a substantial role in gene expression diversityAPOB gene in CDC animals. This expression pattern might be a consequence of enhancer activity induced by the LTR insertion in the APOB gene, in addition to an already reported artificial polyadenylation signal within the insertion. Furthermore, the LTR insertion significantly alters the hepatic expression of genes involved in lipid metabolism and cholesterol biosynthesis. CDC animals showed an indication of impaired lipid utilization compared to CDF animals. In addition, the APOB gene LTR insertion might represent an in vivo model demonstrating the regulatory function of a genomic mobile element insertion on gene expression and subsequent consequences.In conclusion, using transcriptomic and whole-genome sequencing data in combination with clinical data of CDC and CDF cows under pathogen challenge during the early first lactation, we demonstrated a divergent allele-biased expression pattern of the Supplementary Figure 1.Supplementary Figure 2.Supplementary Figure 3.Supplementary Figure 4.Supplementary Figure 5.Supplementary Table 1.Supplementary Table 2.Supplementary Table 3.Supplementary Table 4.Supplementary Table 5.Supplementary Legends.Supplementary Information.


Use of medical devices represents a unique opportunity to facilitate scale-up of early infant male circumcision (EIMC) across sub-Saharan Africa. The ShangRing, a circumcision device prequalified by WHO, is approved for use in adults and adolescents and requires topical anaesthesia only. We aimed to investigate the safety and efficacy of the ShangRing versus the Mogen clamp for EIMC in infants across eastern sub-Saharan Africa.NCT03338699, and is complete.In this multicentre, non-inferiority, open-label, randomised controlled trial, we enrolled healthy male infants (aged <60 days), with a gestational age of at least 37 weeks and a birthweight of at least 2·5 kg, from 11 community and referral centres in Kenya, Tanzania, and Uganda. Infants were randomly assigned (1:1) by a computer-generated text message service to undergo EIMC by either the ShangRing or the Mogen clamp. The primary endpoint was safety, defined as the number and severity of adverse events (AEs), analysed in the intention-to-treat population  with a non-inferiority margin of 2% for the difference in moderate and severe AEs. This trial is registered with Clinical. Trials.gov, vs 30 [4·4%] in the Mogen clamp group; difference –0·1%; one-sided 95% CI upper limit of 1·7%; p=0·89). No treatment-related deaths were reported.Between Sept 17, 2018, and Dec 20, 2019, a total of 1420 infants were assessed for eligibility, of whom 1378 (97·0%) were enrolled. 689 (50·0%) infants were randomly assigned to undergo EIMC by ShangRing and 689 (50·0%) by Mogen clamp. 43 (6·2%) adverse events were observed in the ShangRing group and 61 (8·9%) in the Mogen clamp group (p=0·078). The most common treatment-related AE was intraoperative pain , with 19 (2·8%) events in the ShangRing and 23 (3·3%) in the Mogel clamp group. Rates of moderate and severe AEs were similar between both groups (29 [4·2%] in the ShangRing group Use of the ShangRing device for EIMC showed safety, achieved high caregiver satisfaction, and did not differ from the Mogen clamp in other key measures. The ShangRing could be used by health systems and international organisations to further scale up EIMC across sub-Saharan Africa.Bill & Melinda Gates Foundation. Observational studies suggested in 1986 that voluntary medical male circumcision (VMMC) was associated with reduced male HIV acquisition.15 countries in east and southern Africa with low rates of VMMC and a high incidence of HIV have scaled up VMMC programmes, which resulted in nearly 27 million male circumcisions by the end of 2020.Evidence before this studyWe searched PubMed on Sept 1, 2018, before the initiation of the trial, for articles published in English only without date restrictions. Previous studies have shown that voluntary medical male circumcision (VMMC) reduces the incidence of HIV and urinary tract infections in men. Although most VMMC procedures are performed in adolescents and adults, the most long-term coverage and potential population-level impact on HIV transmission might ultimately be achieved through early infant male circumcision (EIMC). Medical devices simplify the performance of VMMC and, therefore, have been proposed to facilitate WHO's goal of 90% access to VMMC in sub-Saharan Africa by 2020, which has not been reached to date. The ShangRing device is the only medical circumcision device prequalified by WHO for use in individuals from age 10 years to adulthood; however, its use in EIMC has not been explored previously.Added value of this studyIn this multicentre, non-inferiority, open-label, adaptive randomised controlled trial in eastern sub-Saharan Africa, we compared the safety and efficacy of the ShangRing versus the Mogen clamp, which is currently considered to be the gold standard for EIMC, in a cohort of 1420 male infants. To our knowledge, this is the first randomised controlled trial to assess the use of ShangRing for EIMC in this age group. Our results show that EIMC by ShangRing is safe and offers excellent outcomes.Implications of all the available evidenceUse of the ShangRing in EIMC showed safety and achieved high caregiver satisfaction. This device could be used by health systems and international organisations to further scale up EIMC efforts across sub-Saharan Africa.Medical devices simplify the performance of VMMC and facilitate task shifting from physicians to non-physicians to augment service delivery. The ShangRing is the only male circumcision device prequalified by WHO for use in individuals from age 10 years to adulthood.We conducted a multicentre, non-inferiority, open-label, randomised controlled trial in 11 community and referral centres across Kenya (Homa Bay and Kisumu), Tanzania , and Uganda . Eligible participants were healthy male infants (aged <60 days) with a gestational age of at least 37 weeks and a birthweight of at least 2·5 kg, who had been brought to the centres to discuss circumcision. Infants with perinatal illnesses requiring treatment, congenital genitourinary abnormality requiring surgical repair , allergies to known anaesthetic components, or a family history of bleeding disorders were excluded.The trial was reviewed and approved by the institutional review board at Weill Cornell Medicine  and institutional review boards or medical councils from each collaborating organisation  and participating country . Written informed consent was obtained from the parents or legally authorised representatives of infants at enrolment. The trial, data collection, and data management adhered to good clinical practice guidelines for clinical trials.Enrolled infants were randomly assigned (1:1) to undergo EIMC by either the ShangRing or the Mogen clamp. Random allocation was achieved with a text message service . The allocation sequence was generated by computer, uploaded to Sealed Envelope, and entered into a secured electronic database. Parents and legally authorised representatives, investigators, physicians and non-physicians, and providers who evaluated infants for pain and other adverse events (AEs) after the procedure were unmasked to the allocated intervention.All participating infants received a rectal suppository of 40 mg acetaminophen 30 mins before the procedure and a 2·0% sucrose solution as a pacifier during the procedure. A mixture of topical 2·5% lidocaine and 2·5% prilocaine cream was applied to the shaft and the glans of the penis before any intervention. Dwell time was measured from agent application to either the absence of response to a noxious stimulus on the foreskin or to the start of the procedure. Supplemental injectable local anaesthetic was available if inadequate analgesia was noted by the provider. The Neonatal Infant Pain Scale (NIPS) score was used to standardise pain assessment. NIPS is a validated pain scale assigning points on the basis of the provider's observation of the infant's facial expression, cry, breathing pattern, arms, legs, and state of arousal.Before trial initiation, all staff performing the procedure completed a training course on EIMC using both devices. The previously described no-flip techniqueFollow-up was performed by an initial telephone call to parents or legally authorised representatives 24 h after the procedure, followed by scheduled clinic visits every 7 days until complete wound healing. Complete healing was defined as complete re-epithelialisation of the circumcision incision. Infants whose parents or legally authorised representatives could not be reached despite multiple telephone attempts were seen in person at the follow-up visits. Interviews assessing satisfaction among parents and legally authorised representatives were conducted at the last follow-up visit.The primary endpoint was safety, defined as the number and severity of AEs. AEs were prespecified as mild, moderate, or severe on the basis of WHO definitions .22 The sSecondary endpoints included time to complete wound healing; the ease of use associated with both circumcision devices and provider preference, determined by procedure time and problems encountered during the procedure provided by in postoperative interviews; neonatal infant pain scale scores, assessed by NIPS; and satisfaction among parents and legally authorised representatives, evaluated by interviews at the final follow-up visit.Sample size was estimated using WHO's requirements for prequalification of adult and adolescent circumcision devices. We expected that the benefits of the ShangRing would allow a non-inferiority margin of 2·0% for the AE rate. This margin was selected because the anticipated rate of moderate and severe AEs is 2·0% for the Mogen clamp based on the literature.t test and Kaplan-Meier survival analysis using a log-rank test to compare the time to complete wound healing between the two intervention groups. In the event that a participant received an intervention that differed from the one randomly assigned, the discrepancy was documented and an intention-to-treat analysis was performed. Continuous variables were analysed with the Student's t test and categorical variables with the χ2 test . Differences in proportions are reported as a point estimate and a 95% Agresti-Caffo CI. Statistical significance was set at p=0·05 and all tests were two-sided for secondary analyses. The p values were not adjusted for the multiple secondary analyses. Statistical analyses were done with R (version 3.6.1). This trial is registered with Clinical. Trials.gov, NCT03338699.The primary endpoint was analysed in the intention-to-treat population, defined as all infants who underwent an EIMC procedure. We established non-inferiority in the rate of moderate and severe AEs for use of ShangRing versus use of the Mogen clamp using a one-sided 95% Agresti-CaffoThe funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report.Between Sept 17, 2018, and Dec 20, 2019, a total of 1420 infants were assessed for eligibility, of whom 1378 (97·0%) were enrolled. 689 (50·0%) infants were randomly assigned to undergo EIMC by ShangRing and 689 (50·0%) by Mogen clamp . The medvs 12·4 mins [9·5] in the Mogen clamp group; mean difference –0·1 mins [95% CI –1·0 to 0·8]). In both groups, the procedures were performed mainly by nurses, followed by clinical officers and doctors  received their assigned procedure while 18 (2·6%) underwent EIMC with the Mogen clamp and one (0·1%) underwent EIMC by dorsal slit alone . Of the  doctors . All proThere were no missing data for the primary endpoint. There were few missing data for other endpoints and the analyses were done on a complete-case basis. A total of 43 (6·2%) AEs were observed in the ShangRing group and 61 (8·9%) in the Mogen clamp group, with no significant difference between both groups (p=0·078). We observed a total of 59 moderate and severe AEs, with no difference between both groups: 29 (4·2%) in the ShangRing group and 30 (4·4%) in the Mogen clamp group  in the ShangRing group and 16·9 days (5·9) in the Mogen clamp group , favouring EIMC by Mogen clamp. By day 35, complete healing was observed in almost all infants . In the A total of 1327  parents and legally authorised representatives completed a satisfaction interview; 665 (96·5%) parents or legally authorised representatives of infants who underwent EIMC by ShangRing and 662 (96·1%) parents or legally authorised representatives of infants who underwent EIMC by Mogen clamp (difference 0·4% [95% CI –1·6% to 2·5%]). When parents and legally authorised representatives were asked what they liked about the procedure, top responses included less pain than expected, improved hygiene, cosmetic appearance, and short procedure time . When asThis is the first multicountry randomised controlled trial comparing the use of ShangRing versus Mogen clamp for EIMC in three eastern sub-Saharan countries. Our results show that the ShangRing was safe and efficacious for EIMC by non-physicians. AEs, postoperative pain, and satisfaction rates were similar between the two techniques. Thus, we believe that ShangRing could be included in the armamentarium of countries implementing EIMC and could help to achieve the goal of increasing the number of circumcisions and decreasing HIV transmission in Africa.Except for pain, no severe or serious AEs related to the ShangRing were observed in this study. Importantly, severe complications, such as complete or partial transection of the glans, wound infection, or necrosis, which have been previously reported with the Mogen clamp and other devices, were not noted.Pain was not only the most frequently reported but also the most challenging AE to assess in infants. We chose to use NIPS to minimise providers’ subjectivity;Both the ShangRing and Mogen clamp techniques showed short and similar procedural times and were easily performed by non-physicians. Notably, all physicians, nurses, and clinical officers safely performed both interventions, similar to rates of successful medical circumcision interventions in older individuals. Several studies have found no association between AEs and provider type, which further facilitates task-shifting initiatives.We also explored the effects of EIMC on parents and legally authorised representatives. The newborn and early infant period is a stressful time for caregivers. Therefore, maximising the satisfaction of parents and legally authorised representatives and understanding their motivations behind opting their infant for EIMC are of paramount importance in all efforts to scale up EIMC. Mavhu and colleaguesThis study has several limitations. First, the use of multiple sites and personnel introduces heterogeneity in terms of interpretation of clinical data, even though it improves the generalisability of our findings. Second, the trial was designed to detect non-inferiority of the ShangRing versus Mogen clamp, a widely used device for EIMC. Our results suggest that the two techniques do not differ in efficacy, usability, or, most importantly, their safety profile. Third, the NIPS was not as effective at sufficiently differentiating between mild, moderate, and severe pain in infants as has been reported in adolescent and adults. Fourth, paternal circumcision status was not obtained when assessing the views of parents and legally authorised representatives on EIMC, despite its previously described influence on perceptions of EIMC.EIMC by the ShangRing is non-inferior to EIMC by the Mogen clamp and represents a safe and efficacious tool with excellent outcomes and a mostly high satisfaction rate among parents and legally authorised representatives. Despite how accurate pain evaluation was challenging in this trial, almost all EIMC procedures by ShangRing were successfully performed with use of local topical anaesthetics only. Thus, this device could be used by health systems and international organisations as an integral part of efforts to scale up future EIMC services across sub-Saharan Africa.Data collected for the study, including individual participant data and a data dictionary defining each field in the set, will be made available to the sponsor and participating researchers. Deidentified patient data, the data dictionary, the study protocol, and the informed consent form will be made available at publication upon request to the corresponding author with investigator approval and a signed data access agreement.We declare no competing interests.


Citrus latifolia) and King mandarin (Citrus nobilis L.) in addition to natural gums—xanthan gum and latex from the “lechero” plant (Euphorbia laurifolia)—were used to create an eco-friendly corrosion inhibitor. The optimal extraction conditions for pectin were determined from different combinations of pH, temperature, and time in a 23 factorial design and evaluated according to the obtained pectin yield. The highest pectin extraction yields  were reached at pH = 1, 85 °C, and 2 h. Extraction of pectic compounds was confirmed using Fourier-transform infrared spectroscopy, differential scanning calorimetry, and thermogravimetry analyses. Subsequently, a simplex-centroid mixture design was applied to determine the formulation of extracted pectin and natural gums that achieved the highest corrosion inhibitor effect . Impedance spectroscopy analysis showed that the addition of xanthan gum to pectin (formulation 50% pectin–50% xanthan gum) improved the corrosion inhibitor effect from 29.20 to 78.21% at 400 ppm due to higher adsorption of inhibitory molecules on the metal surface.The production and use of eco-friendly corrosion inhibitors allows valuable compounds contained in plant waste to be identified and repurposed while reducing the use of polluting synthetic substances. Pectin extracted from Tahiti limes ( Protecting industrial facilities from corrosion involves high annual expenses; as such, extensive research has been undertaken on how to cope with this widespread problem ,2. The aThe protective action of corrosion inhibitors is very effective on metal surfaces when they are injected into the circulating feed currents; film-forming corrosion inhibitors, which decrease the corrosion rate by being adsorbed on the metal surface, are the most frequently used . HoweverPectin is a high-molecular-weight polysaccharide  present in the cell walls of plants and fruit peels. This biomolecule is a linear, water-soluble polymer comprising approximately 1000 monosaccharide units (degree of esterification) with a molecular weight of about 50,000–180,000 Da, ,8. In aqSolanum licopersicum) as a corrosion inhibitor applied to tin.Pectin contains functional groups (heteroatoms) that can interact with metallic surfaces through a first- or second-order bind through adsorption , which mIn general, the corrosion-inhibiting results from pectin have been positive, but its protective performance is still lower compared to the inhibitor effect of synthetic inhibitors; therefore, options to improve its effectiveness include molecule modification or the use of several compounds in a mixture formulation .Xantomona campestris bacteria and has been studied as a corrosion inhibitor for aluminum [Euphorbia laurifolia), contain terpenes and aromatic compounds [Natural gums are complex substances with interesting structures and properties that promote corrosion inhibition . They alaluminum . Other nompounds  that couompounds  demonstrCitrus latifolia) and King mandarin (Citrus nobilis L.) peels together with xanthan gum and/or latex from the lechero plant to obtain a highly effective eco-friendly corrosion inhibitor for API-5LX52 carbon steel.Thus, this study examines the use of pectin extracted from Tahiti lime (3O+), which allows for a greater degree of breaking of glycosidic bonds by protopectin chains  [Analysis of variance (ANOVA) and Pareto charts from pectin extraction factorial designs are shown in  matrix) . Methaca matrix)  highligh3O+ and matrix molecules occurred; however, temperatures higher than 85 °C promoted pectin destruction at a pH = 1 and an extraction time longer than 2 h. With this combination, glycosidic bonds as well as ester bonds are broken, which form the base of the pectin structure [Temperature had a significant effect both individually and combined with pH. At high temperatures, molecular movement was improved, and better interaction between Htructure .Liew et al.  found thResponse surface plots b show thEquivalent weight (PEq)PEq refers to the number of galacturonic acid units  per pectin molecule .The PEq of pectin according to the pH of the extraction medium is shown in Methoxyl content (%Me) and esterification degree (%DE).%Me represents the amount of esterified methoxyl groups from available carboxyl groups in the pectic chain , which rGalacturonic acid (%AUA) and ash content.Pectins utilized as corrosion inhibitors are not required to be as pure as those destined for food purposes; however, the pectin in this study had characteristics and physicochemical specifications similar to food-grade pectin . AccordiThe results of physicochemical characterization showed that all the products obtained in the experimental design are pectic compounds, of which the polysaccharides extracted at a pH = 1 show the most favorable physicochemical characteristics (PEq and %Me) regarding their application as a corrosion inhibitor and higher extraction yield. Therefore, the best pectin extraction conditions for both types of citrus peel are pH = 1, 85 °C, and a hydrolysis time of 2 h.−1 were assigned to O-H and C-H stretching. Absorption bands of 1732 and 1643 cm−1 are evidenced and belong to the pectin fingerprint since they are attributable to the stretching vibration of esterified C=O and free carboxylic groups (COO=), respectively [The Fourier-transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), and thermogravimetry (TGA) spectra of pectin extracted from Tahiti lime (TL), King mandarin  are shown in ectively . These fectively , Sato etectively , Urías-Oectively , and Garectively , given tThe DSC thermic profile of Cm, TL and MK pectin b shows aConversely, the DSC profile of extracted pectin (TL and MK) does not show steep peaks between 180 and 210 °C, and weight loss according to the TGA was 30% until 250 °C; therefore, these samples present higher thermic resistance than Cm starting at 225 °C.The F8 formulation of Pex 50% and Gx 50% resulted in 78.10% of metallic protection, the highest percentage of corrosion inhibition among the mixtures. According to Ptaszek et al. , Gx and −1, which is related to the presence of carboxylic groups from Gx that, together with those present in Pex and an adequate polydispersity in the saline media, improve pectin’s individual performance.When comparing the FTIR spectra of the Pex 50%–Gx 50% to the effect of Pex and Gx used individually, an intense band can be seen at 1610 cmLc application in the F9 formulation (50:50 ratio with Pex) achieved high efficiency. This mixture protected 51.10% of the metallic surface, but its performance did not exceed that of Pex 50%–Gx 50%.The incompatibility of Gx with Lc was evident in ternary formulations F2, F3, and F4, particularly in F2, in which Lc was added in a greater proportion to Gx (33% Lc and 8% Gx). Further, the addition of Lc in F2 affected the individual performance of Pex, which decreased from 21.00 to 2.10% due to the immiscibility of the mixture in the media. In the other formulations, there was no immiscibility, but the highest efficiency obtained was 39.15% with Pex 8%, Lc 8%, and Gx 8%. As both Gx and Lc are natural gums, compatibility was expected; however, it is possible that the vegetal nature of Lc and microbial properties inhibited interaction in the saline media.Calderón, Rossa, and Esteves  found thAccording to ASTM G59-97 , higher In the Tafel plot c, two diThe corrosion potential (Ecorr) displacement in negative and positive directions is notable. In the case of Pex 50%–Gx 50%, there was a displacement of 80 mV (less than 85 mV), which is why this formulation can be considered a mixed inhibitor with a stronger effect in the anodic reaction modification (the displacement moved in the positive direction from the Ecorr of the blank). Similar results were obtained by Sangeetha et al. , Umoren The corrosion inhibitory effect of pectin, Cm, Pex, and the Pex 50%–Gx 50% formulation (best efficiency) is presented in dl) (Nyquist curves are presented in dl) , which iThe Nyquist curves do not have a perfect form since they represent actual impedance systems, so the parameter n  allows tThere is no official explanation for Nyquist curve deviation, but some authors, such as Umoren et al. , attribuThe highest value of corrosion inhibition was obtained by adding the 400 ppm formulation, resulting in an efficiency of 73.15% . Higher According to the weight loss method results, corrosion inhibition efficiency increased with the Pex 50%–Gx 50% formulation, which is also seen in the electrochemical test results. Additionally, this method confirmed that the highest inhibitory efficiency was obtained at 400 ppm. The electrochemical tests also showed that after 400 ppm, the inhibitory effect decreased due to the lack of active sites on the metallic surface, which could be attributed to the molecule size of the polysaccharides in the formulation.The corrosion inhibition efficiency results from the weight loss tests differ by a higher percentage than those from the LPR and EIS electrochemical results  because The LPR method with Tafel adjustment is the most widely used because it is more practical, requires less execution time, and is not highly affected by interference from outside the system. The impedance spectroscopy method allows the presence of important adsorption phenomena to be verified for the analysis of the corrosion inhibition process, which complements the LPR method by broadening the field of analysis. The weight-loss method, which for a long time was the main method to study corrosion, allows specific results of the oxidation rate of the metal to be obtained, which is very important. The disadvantage of this method is that, for field analysis, it requires months of development to obtain effective results, but when used in the laboratory, it complements the electrochemical analysis of corrosion .2H2O4)  and ethanol (C2H5OH)  were used as pectin extraction and clotting media, respectively. Sodium hydroxide (NaOH)  and hydrochloric acid (HCl)  were used in the pectin characterization, with commercial pectin  as a standard.Pectin was extracted from peels from Tahiti lime and King mandarin purchased in Yaruquí in the province of Pichincha, Ecuador. Oxalic acid (C2), and magnesium chloride (MgCl2·7 H2O)  were used for electrochemical tests.Food-grade xanthan gum  and latex extracted from the lechero plant were added to the mixture formulation with citrus pectin. Sodium chloride (NaCl) , calcium chloride , extraction pH , and hydrolysis time . The response variable was pectin extraction yield (g of pectin/g of dry citrus peel used) [A 2el used) ,19. Citrv/v) was added in a 2:1 ratio to the hydrolyzed product so the pectin would clot and precipitate. After 24 h of precipitation, the coagulated pectin was separated with a nylon sieve and dried at 60 °C for 12 h. Finally, the extracted dried pectin was ground to pass through a 40-mesh sieve.An acid hydrolysis process  for 1 h in a 250 mL conical flask. Then, 100 mL of NaCl (1% w/v) was added, and the mixture was homogenized. This solution was titrated against NaOH 0.1 M (3 drops of phenol red as an indicator) to create the neutralized solution, S1. Equivalent weight (PEq) was determined according to Equation (1):First, 0.5 g of pectin was moistened with 5 mL of ethanol  was titrated against NaOH 0.1 M. Methoxyl content (%Me) was calculated with Equation (2):Galacturonic acid content (%AUA) was determined by Equation (3):Esterification degree (%DE) was determined by Equation (4):Ash content was determined using the ASTM E1755-01 method “Standard Test Method for Ash in Biomass” ; 0.5 g o−1 [Pectin extracted at optimal conditions was characterized using FTIR, DSC, and TGA. FTIR analysis was carried out using a Spectrum One FT-IR spectrometer , with a range of 4000–400 cm−1 ; absorpt−1 . In this−1 . For DSCA simple-centroid three-component mixture design was applied to extracted pectin and added gums (xanthan and latex from the lechero plant) to obtain the formulation with the highest corrosion inhibitory effect. The proposed mixtures were added to the corrosion cell at 500 ppm. The minimum percentage of pectin in each formulation was 50%, and the response variable was the inhibition efficiency obtained by the LPR technique using a BIOLOGIC SP-300 potentiostat/galvanostat, according to ASTM G-59 “Standard Test Method for Conducting Potentiodynamic Polarization Resistance Measurements” .2; a graphite bar and an Ag/AgCl2 electrode were selected as counter and reference electrodes, respectively. The corrosion cell was bubbled with N2 for 5 min before each test.A three-electrode corrosion cell was created by adding 50 mL of saline solution (corrosion medium), prepared according to NACE 1D-196 “Laboratory Test Methods for Evaluating Oil-field Corrosion Inhibitors” , to a gl2), and 2).The carbon steel’s oxidation potential range was determined by cyclic voltammetry (VC), which showed ±110 mV around the steady-state EoC. The corrosion system was stabilized for 20 min, and LPR analysis was performed immediately for each formulation with a potential rate of 1 mV/s. Tafel plots were drawn from test results, and the inhibition efficiency (η%) was calculated according to Equation (5):To obtain the highest inhibitor concentration of the optimal formulation of pectin and gums, the mixture was analyzed using LPR in a range between 100 and 1000 ppm with intervals of 100 ppm. The same process was performed for extracted and commercial pectin, as well.ct is the charge transfer resistance (Ω/cm2), and 2).EIS was carried out using the formulation of pectin and gums with the highest efficiency; the measurements were executed between a frequency range of 10,000.0–0.1 Hz with an amplitude of 10 mV to minimize interference . The forc is corrosion rate (mmpy), K is a constant (8.76 × 104), W is weight loss (g), d is density (g/cm3), a is coupon exposed area (cm2), and t is time (h).Weight loss analysis was performed according to Da Rocha et al.  based onThe optimal pectin extraction conditions for the selected fruits were pH = 1, 85 °C, and hydrolysis time of 120 min; yields of 38.10% and 41.20% (with respect to the dry peel sample mass) were obtained for King mandarin and Tahiti lime, respectively. Pectin with a high degree of methoxy was obtained, with an esterification percentage of 54% and 55% for King mandarin and Tahiti lime, respectively.The addition of xanthan gum to the extracted pectin improved its individual inhibitory effect, from a protection level of 26% to 78%, evaluated at a concentration of 500 ppm at room temperature. The concentration of the Pex 50%–Gx 50% formulation that produced the strongest corrosion-inhibiting effect was 400 ppm with a metal protection percentage of 78%.Future studies should evaluate the corrosion-inhibiting effect produced by pectin’s antioxidant capacity. In addition, it is important to continue the search for a formulation with a more effective inhibition effect compared to that obtained in this study, from pectin modified with compounds such as amines and carboxymethylcellulose, to achieve higher corrosion protection.