REMAP Periop: a randomised, embedded, multifactorial adaptive platform trial protocol for perioperative medicine to determine the optimal enhanced recovery pathway components in complex abdominal surgery patients within a US healthcare system.

INTRODUCTION: Implementation of enhanced recovery pathways (ERPs) has resulted in improved patient-centred outcomes and decreased costs. However, there is a lack of high-level evidence for many ERP elements. We have designed a randomised, embedded, multifactorial, adaptive platform perioperative medicine (REMAP Periop) trial to evaluate the effectiveness of several perioperative therapies for patients undergoing complex abdominal surgery as part of an ERP. This trial will begin with two domains: postoperative nausea/vomiting (PONV) prophylaxis and regional/neuraxial analgesia. Patients enrolled in the trial will be randomised to arms within both domains, with the possibility of adding additional domains in the future. METHODS AND ANALYSIS: In the PONV domain, patients are randomised to optimal versus supraoptimal prophylactic regimens. In the regional/neuraxial domain, patients are randomised to one of five different single-injection techniques/combination of techniques. The primary study endpoint is hospital-free days at 30 days, with additional domain-specific secondary endpoints of PONV incidence and postoperative opioid consumption. The efficacy of an intervention arm within a given domain will be evaluated at regular interim analyses using Bayesian statistical analysis. At the beginning of the trial, participants will have an equal probability of being allocated to any given intervention within a domain (ie, simple 1:1 randomisation), with response adaptive randomisation guiding changes to allocation ratios after interim analyses when applicable based on prespecified statistical triggers. Triggers met at interim analysis may also result in intervention dropping. ETHICS AND DISSEMINATION: The core protocol and domain-specific appendices were approved by the University of Pittsburgh Institutional Review Board. A waiver of informed consent was obtained for this trial. Trial results will be announced to the public and healthcare providers once prespecified statistical triggers of interest are reached as described in the core protocol, and the most favourable interventions will then be implemented as a standardised institutional protocol. TRIAL REGISTRATION NUMBER: NCT04606264.

Evolving Real-World Effectiveness of Monoclonal Antibodies for Treatment of COVID-19 : A Cohort Study.

BACKGROUND: Treatment guidelines and U.S. Food and Drug Administration emergency use authorizations (EUAs) of monoclonal antibodies (mAbs) for treatment of high-risk outpatients with mild to moderate COVID-19 changed frequently as different SARS-CoV-2 variants emerged. OBJECTIVE: To evaluate whether early outpatient treatment with mAbs, overall and by mAb product, presumed SARS-CoV-2 variant, and immunocompromised status, is associated with reduced risk for hospitalization or death at 28 days. DESIGN: Hypothetical pragmatic randomized trial from observational data comparing mAb-treated patients with a propensity score-matched, nontreated control group. SETTING: Large U.S. health care system. PARTICIPANTS: High-risk outpatients eligible for mAb treatment under any EUA with a positive SARS-CoV-2 test result from 8 December 2020 to 31 August 2022. INTERVENTION: Single-dose intravenous mAb treatment with bamlanivimab, bamlanivimab-etesevimab, sotrovimab, bebtelovimab, or intravenous or subcutaneous casirivimab-imdevimab administered within 2 days of a positive SARS-CoV-2 test result. MEASUREMENTS: The primary outcome was hospitalization or death at 28 days among treated patients versus a nontreated control group (no treatment or treatment ≥3 days after SARS-CoV-2 test date). RESULTS: The risk for hospitalization or death at 28 days was 4.6% in 2571 treated patients and 7.6% in 5135 nontreated control patients (risk ratio [RR], 0.61 [95% CI, 0.50 to 0.74]). In sensitivity analyses, the corresponding RRs for 1- and 3-day treatment grace periods were 0.59 and 0.49, respectively. In subgroup analyses, those receiving mAbs when the Alpha and Delta variants were presumed to be predominant had estimated RRs of 0.55 and 0.53, respectively, compared with 0.71 for the Omicron variant period. Relative risk estimates for individual mAb products all suggested lower risk for hospitalization or death. Among immunocompromised patients, the RR was 0.45 (CI, 0.28 to 0.71). LIMITATIONS: Observational study design, SARS-CoV-2 variant presumed by date rather than genotyping, no data on symptom severity, and partial data on vaccination status. CONCLUSION: Early mAb treatment among outpatients with COVID-19 is associated with lower risk for hospitalization or death for various mAb products and SARS-CoV-2 variants. PRIMARY FUNDING SOURCE: None.

Neighborhood disadvantage and the racial disparity in postpartum hypertension.

BACKGROUND: Postpartum hypertension is the leading cause of postpartum readmission and has long-lasting cardiovascular effects. Black patients have higher incidence rates of hypertensive disorders after delivery and subsequent severe maternal morbidity. Neighborhood advantage, a marker of social determinants of health, has not been studied concerning postpartum hypertension. Moreover, the interplay between race and neighborhood advantage and their effect on postpartum hypertension have not been previously explored. OBJECTIVE: This study aimed to evaluate the association between neighborhood-level social determinants of health and postpartum hypertension and explore whether these factors explain previously documented racial disparities. STUDY DESIGN: This study included a retrospective cohort of people enrolled in a remote monitoring program of postpartum hypertension at the time of delivery within 1 health network from March 2019 to September 2021. Patients were eligible for enrollment after a diagnosis of hypertensive disorder during pregnancy or delivery. We further limited the cohort to self-reported Black and White patients with blood pressures recorded at 3 weeks and 6 weeks postpartum. The neighborhood advantage for each person at the time of delivery was classified using the area deprivation index, an accepted surrogate of social determinants of health and our primary exposure. The secondary exposure was self-reported race. Study outcomes of interest were hypertensive status (stage 1 hypertension: ≥130 to 139/80 to 89 mm Hg; stage 2 hypertension: ≥140/90 mm Hg) at 3 and 6 weeks after delivery. In addition, hypertensive status by neighborhood area deprivation index using logistic regression was molded. In secondary analyses, a case-control cohort matched on the area deprivation index was created, and conditional logistic regression was used to evaluate race. Finally, mixed-effects models modeling hypertension by race and clustering within the area deprivation index were used. RESULTS: Of 4193 people enrolled, 2722 were Black or White and had blood pressure data recorded at 3 weeks after delivery, and 1126 had blood pressure data recorded at 6 weeks after delivery. After accounting for prenatal body mass index, smoking status, type of hypertension, and antihypertensives prescribed at discharge, persons living in the most disadvantaged neighborhoods were twice as likely (adjusted odds ratio, 2.03; 95% confidence interval, 1.53-2.69) to develop stage 2 hypertension at 21 days after delivery and 1.67 times more likely (95% confidence interval, 1.06-2.64) to develop stage 2 hypertension at 6 weeks after delivery than persons living in the most advantaged neighborhoods. Both associations were attenuated after adjusting for race. When people with stage 2 hypertension were matched on area deprivation index with normotensive counterparts, Black patients were still 3 to 4 times more likely to develop stage 2 hypertension at 3 (adjusted odds ratio, 3.00; 95% confidence interval, 1.95-4.63) and 6 (adjusted odds ratio, 4.61; 95% confidence interval, 2.05-10.36) weeks after delivery. This association remained after clustering within a neighborhood at 3 (adjusted odds ratio, 3.12; 95% confidence interval, 2.41-4.06) and 6 (adjusted odds ratio, 2.99; 95% confidence interval, 1.96-4.54) weeks after delivery. There was no significant difference in stage 1 hypertension. CONCLUSION: Neighborhood advantage was associated with the development of persistent hypertension at 3 and 6 weeks after delivery. This association did not explain the racial disparity in sustained high blood pressure.

Effectiveness of Casirivimab-Imdevimab and Sotrovimab During a SARS-CoV-2 Delta Variant Surge: A Cohort Study and Randomized Comparative Effectiveness Trial.

Importance: The effectiveness of monoclonal antibodies (mAbs), casirivimab-imdevimab and sotrovimab, is unknown in patients with mild to moderate COVID-19 caused by the SARS-CoV-2 Delta variant. Objective: To evaluate the effectiveness of mAb against the Delta variant compared with no mAb treatment and to ascertain the comparative effectiveness of casirivimab-imdevimab and sotrovimab. Design, Setting, and Participants: This study comprised 2 parallel studies: (1) a propensity score-matched cohort study of mAb treatment vs no mAb treatment and (2) a randomized comparative effectiveness trial of casirivimab-imdevimab and sotrovimab. The cohort consisted of patients who received mAb treatment at the University of Pittsburgh Medical Center outpatient infusion centers and emergency departments from July 14 to September 29, 2021. Participants were patients with a positive SARS-CoV-2 test result who were eligible to receive mAbs according to emergency use authorization criteria. Exposure: For the trial, patients were randomized to either intravenous casirivimab-imdevimab or sotrovimab according to a system therapeutic interchange policy. Main Outcomes and Measures: For the cohort study, risk ratio (RR) estimates for the primary outcome of hospitalization or death by 28 days were compared between mAb treatment and no mAb treatment using propensity score-matched models. For the comparative effectiveness trial, the primary outcome was hospital-free days (days alive and free of hospitalization) within 28 days after mAb treatment, where patients who died were assigned -1 day in a bayesian cumulative logistic model adjusted for treatment location, age, sex, and time. Inferiority was defined as a 99% posterior probability of an odds ratio (OR) less than 1. Equivalence was defined as a 95% posterior probability that the OR was within a given bound. Results: A total of 3069 patients (1023 received mAb treatment: mean [SD] age, 53.2 [16.4] years; 569 women [56%]; 2046 had no mAb treatment: mean [SD] age, 52.8 [19.5] years; 1157 women [57%]) were included in the prospective cohort study, and 3558 patients (mean [SD] age, 54 [18] years; 1919 women [54%]) were included in the randomized comparative effectiveness trial. In propensity score-matched models, mAb treatment was associated with reduced risk of hospitalization or death (RR, 0.40; 95% CI, 0.28-0.57) compared with no treatment. Both casirivimab-imdevimab (RR, 0.31; 95% CI, 0.20-0.50) and sotrovimab (RR, 0.60; 95% CI, 0.37-1.00) were associated with reduced hospitalization or death compared with no mAb treatment. In the clinical trial, 2454 patients were randomized to receive casirivimab-imdevimab and 1104 patients were randomized to receive sotrovimab. The median (IQR) hospital-free days were 28 (28-28) for both mAb treatments, the 28-day mortality rate was less than 1% (n = 12) for casirivimab-imdevimab and less than 1% (n = 7) for sotrovimab, and the hospitalization rate by day 28 was 12% (n = 291) for casirivimab-imdevimab and 13% (n = 140) for sotrovimab. Compared with patients who received casirivimab-imdevimab, those who received sotrovimab had a median adjusted OR for hospital-free days of 0.88 (95% credible interval, 0.70-1.11). This OR yielded 86% probability of inferiority for sotrovimab vs casirivimab-imdevimab and 79% probability of equivalence. Conclusions and Relevance: In this propensity score-matched cohort study and randomized comparative effectiveness trial, the effectiveness of casirivimab-imdevimab and sotrovimab against the Delta variant was similar, although the prespecified criteria for statistical inferiority or equivalence were not met. Both mAb treatments were associated with a reduced risk of hospitalization or death in nonhospitalized patients with mild to moderate COVID-19 caused by the Delta variant. Trial Registration: ClinicalTrials.gov Identifier: NCT04790786.

The comparative effectiveness of COVID-19 monoclonal antibodies: A learning health system randomized clinical trial.

BACKGROUND: Monoclonal antibodies (mAb) that neutralize SARS-CoV-2 decrease hospitalization and death compared to placebo in patients with mild to moderate COVID-19; however, comparative effectiveness is unknown. We report the comparative effectiveness of bamlanivimab, bamlanivimab-etesevimab, and casirivimab-imdevimab. METHODS: A learning health system platform trial in a U.S. health system enrolled patients meeting mAb Emergency Use Authorization criteria. An electronic health record-embedded application linked local mAb inventory to patient encounters and provided random mAb allocation. Primary outcome was hospital-free days to day 28. Primary analysis was a Bayesian model adjusting for treatment location, age, sex, and time. Inferiority was defined as 99% posterior probability of an odds ratio < 1. Equivalence was defined as 95% posterior probability the odds ratio is within a given bound. FINDINGS: Between March 10 and June 25, 2021, 1935 patients received treatment. Median hospital-free days were 28 (IQR 28, 28) for each mAb. Mortality was 0.8% (1/128), 0.8% (7/885), and 0.7% (6/922) for bamlanivimab, bamlanivimab-etesevimab, and casirivimab-imdevimab, respectively. Relative to casirivimab-imdevimab (n = 922), median adjusted odds ratios were 0.58 (95% credible interval [CI] 0.30-1.16) and 0.94 (95% CI 0.72-1.24) for bamlanivimab (n = 128) and bamlanivimab-etesevimab (n = 885), respectively. These odds ratios yielded 91% and 94% probabilities of inferiority of bamlanivimab versus bamlanivimab-etesevimab and casirivimab-imdevimab, and an 86% probability of equivalence between bamlanivimab-etesevimab and casirivimab-imdevimab. INTERPRETATION: Among patients with mild to moderate COVID-19, bamlanivimab-etesevimab or casirivimab-imdevimab treatment resulted in 86% probability of equivalence. No treatment met prespecified criteria for statistical equivalence. Median hospital-free days to day 28 were 28 (IQR 28, 28) for each mAb. FUNDING AND REGISTRATION: This work received no external funding. The U.S. government provided the reported mAb. This trial is registered at ClinicalTrials.gov, NCT04790786.

Area Deprivation Index and Oral Anticoagulation in New Onset Atrial Fibrillation.

Objective: Oral anticoagulation is a standard of care for thromboembolic stroke prevention in individuals with atrial fibrillation (AF). Social determinants of health have had limited investigation in AF and particularly in access to anticoagulation. We examined the relation between area deprivation index (ADI) and anticoagulation in individuals at risk of stroke due to AF. Methods: We conducted a retrospective analysis of patients with incident, non-valvular AF from 2015-2020 receiving care at a large, regional health center. We extracted demographics, medications, and problem lists and used administrative coding to identify comorbid conditions and relevant covariates, and individual-level residential address to ascertain ADI. We examined the relation between ADI and receipt of prescribed oral anticoagulation (warfarin or direct-acting oral anticoagulant, or DOAC) at 90 days following AF diagnosis in multivariable-adjusted models. Results: Following exclusions, the dataset included 20,210 individuals (age 74.5±10.9 years; 51% women; 94% white race). In multivariable-adjusted analyses, individuals in the highest quartile of ADI had a 16% lower likelihood of receiving anticoagulation prescription than those in the lowest ADI quartile (Odds Ratio [OR] 0.84; 95% Confidence Interval [CI], 0.75-0.95) at 90 days following AF diagnosis. In those receiving anticoagulation, individuals in the highest ADI quartile had a 24% lower likelihood of receiving a DOAC prescription as opposed to warfarin prescription than those in the lowest quartile (OR 0.76; 95% CI, 0.60-0.96) at 90 days following AF diagnosis. Conclusions: We demonstrate the association of higher neighborhood deprivation as determined by ADI with decreased likelihood of (1) anticoagulation prescribing for stroke prevention in AF and (2) prescription of a DOAC when any oral anticoagulation is prescribed. Our results suggest neighborhood-based health inequities in the receipt of anticoagulation prescription for stroke prevention in AF in a large, regional health care system.

Association of Subcutaneous or Intravenous Administration of Casirivimab and Imdevimab Monoclonal Antibodies With Clinical Outcomes in Adults With COVID-19.

Importance: Monoclonal antibody (mAb) treatment decreases hospitalization and death in high-risk outpatients with mild to moderate COVID-19; however, only intravenous administration has been evaluated in randomized clinical trials of treatment. Subcutaneous administration may expand outpatient treatment capacity and qualified staff available to administer treatment, but the association with patient outcomes is understudied. Objectives: To evaluate whether subcutaneous casirivimab and imdevimab treatment is associated with reduced 28-day hospitalization and death compared with nontreatment among mAb-eligible patients and whether subcutaneous casirivimab and imdevimab treatment is clinically and statistically similar to intravenous casirivimab and imdevimab treatment. Design, Setting, and Participants: This prospective cohort study evaluated high-risk outpatients in a learning health system in the US with mild to moderate COVID-19 symptoms from July 14 to October 26, 2021, who were eligible for mAb treatment under emergency use authorization. A nontreated control group of eligible patients was also studied. Exposures: Subcutaneous injection or intravenous administration of the combined single dose of 600 mg of casirivimab and 600 mg of imdevimab. Main Outcomes and Measures: The primary outcome was the 28-day adjusted risk ratio or adjusted risk difference for hospitalization or death. Secondary outcomes included 28-day adjusted risk ratios and differences in hospitalization, death, a composite end point of emergency department admission and hospitalization, and rates of adverse events. Among 1959 matched adults with mild to moderate COVID-19, 969 patients (mean [SD] age, 53.8 [16.7] years; 547 women [56.4%]) who received casirivimab and imdevimab subcutaneously had a 28-day rate of hospitalization or death of 3.4% (22 of 653 patients) compared with 7.0% (92 of 1306 patients) in nontreated controls (risk ratio, 0.48; 95% CI, 0.30-0.80; P = .002). Among 2185 patients treated with subcutaneous (n = 969) or intravenous (n = 1216; mean [SD] age, 54.3 [16.6] years; 672 women [54.4%]) casirivimab and imdevimab, the 28-day rate of hospitalization or death was 2.8% vs 1.7%, which resulted in an adjusted risk difference of 1.5% (95% CI, -0.6% to 3.5%; P = .16). Among all infusion patients, there was no difference in intensive care unit admission (adjusted risk difference, 0.7%; 95% CI, -3.5% to 5.0%) or need for mechanical ventilation (adjusted risk difference, 0.2%; 95% CI, -5.8% to 5.5%). Conclusions and Relevance: In this cohort study of high-risk outpatients with mild to moderate COVID-19 symptoms, subcutaneously administered casirivimab and imdevimab was associated with reduced hospitalization and death when compared with no treatment. These results provide preliminary evidence of potential expanded use of subcutaneous mAb treatment, particularly in areas that are facing treatment capacity and/or staffing shortages.

Launching a comparative effectiveness adaptive platform trial of monoclonal antibodies for COVID-19 in 21 days.

Outpatient treatments that limit progression to severe coronavirus disease 2019 (COVID-19) are of vital importance to optimise patient outcomes and public health. Monoclonal antibodies (mAb) demonstrated ability to decrease hospitalizations in randomized, clinical trials. However, there are many barriers to mAb treatment such as patient access and clinician education. There are no data comparing efficacy or safety of available mAbs. We sought to rapidly launch an adaptive platform trial with the goals of enhancing access to treatment, regardless of geography and socioeconomic status, and evaluating comparative efficacy and safety of available mAbs. Within 21 days from idea genesis, we allocated mAb treatment to all patients within the context of this clinical trial. Within 2 months, we closed the gap of the likelihood of receiving mAb, conditional on background positivity rate, between Black and White patients (Black patients 0.238; White patients 0.241). We describe trial infrastructure, lessons learned, and future directions for a culture of learning while doing.

Area Deprivation Index and Cardiac Readmissions: Evaluating Risk-Prediction in an Electronic Health Record.

Background Assessment of the social determinants of post-hospital cardiac care is needed. We examined the association and predictive ability of neighborhood-level determinants (area deprivation index, ADI), readmission risk, and mortality for heart failure, myocardial ischemia, and atrial fibrillation. Methods and Results Using a retrospective (January 1, 2011-December 31, 2018) analysis of a large healthcare system, we assess the predictive ability of ADI on 30-day and 1-year readmission and mortality following hospitalization. Cox proportional hazards models analyzed time-to-event. Log rank analyses determined survival. C-statistic and net reclassification index determined the model's discriminative power. Covariates included age, sex, race, comorbidity, number of medications, length of stay, and insurance. The cohort (n=27 694) had a median follow-up of 46.5 months. There were 14 469 (52.2%) men and 25 219 White (91.1%) patients. Patients in the highest ADI quintile (versus lowest) were more likely to be admitted within 1 year of index heart failure admission (hazard ratio [HR], 1.25; 95% CI, 1.03‒1.51). Patients with myocardial ischemia in the highest ADI quintile were twice as likely to be readmitted at 1 year (HR, 2.04; 95% CI, 1.44‒2.91]). Patients with atrial fibrillation living in areas with highest ADI were less likely to be admitted within 1 year (HR, 0.79; 95% CI, 0.65‒0.95). As ADI increased, risk of readmission increased, and risk reclassification was improved with ADI in the models. Patients in the highest ADI quintile were 25% more likely to die within a year (HR, 1.25 1.08‒1.44). Conclusions Residence in socioeconomically disadvantaged communities predicts rehospitalization and mortality. Measuring neighborhood deprivation can identify individuals at risk following cardiac hospitalization.

The impact of socioeconomic status in patients with left ventricular assist devices (LVADs).

BACKGROUND: Socioeconomic status (SES) can be a powerful predictor of adverse outcomes among heart failure patients but its impact on survival and readmission following left ventricular assist device (LVAD) implantation surgery is poorly understood. We investigated if the LVAD recipients from more deprived neighborhoods experienced higher mortality and readmission rate after device implantation as compared to those from less deprived areas. METHODS: This is a single center, retrospective analysis evaluating adults who received Heartmate III and Heartware HVAD implants between 2009 and 2018. SES indicators were area of deprivation index (ADI), race and income. Our cohort was grouped by ADI quartiles from least deprived (Q1), Q2, Q3 to the most deprived (Q4). Outcomes included overall mortality and readmission following surgery. RESULTS: A total of 191 patients were included in the study. Demographics by SES indicators demonstrated that least deprived (Q1) patients were older than the most deprived (65 vs. 57, p < .01), African-American patients originated from more deprived neighborhoods than Caucasians (ADI 87 vs. 62, p < .001), and high-income patients had higher preoperative BUN and creatinine. Outcome differences included a decreased risk of death in most deprived patients (Q4) compared to the least deprived (Q1), however after adjusting for age, LVAD indication, and INTERMACS profile this was no longer significant. No differences in survival or readmission by race or income was observed CONCLUSION: SES does not independently impact survival and readmission after Heartware HVAD and Heartmate III LVAD implantation. More studies are needed to evaluate if other SES factors affect these outcomes.

Ondansetron use in the first trimester of pregnancy and the risk of neonatal ventricular septal defect.

BACKGROUND: Literature is divided regarding the risk of neonatal ventricular septal defect (VSD) associated with first trimester ondansetron use in pregnancy. METHODS: We evaluated the risk of VSD associated with first trimester exposure to intravenous or oral ondansetron in 33 677 deliveries at Magee-Womens Hospital in Pittsburgh, PA (2006-2014). Using log-binomial regression, we evaluated the risk: (1) in the full cohort, (2) using propensity score designs with both matching and inverse probability weighting and (3) utilizing clustered trajectory analysis evaluating the role of dose. Sensitivity analyses assessed the association between ondansetron and all recorded birth defects in aggregate. RESULTS: A total of 3733 (11%) pregnancies were exposed to ondansetron in the first trimester (dose range: 2.4-1008 mg). Ondansetron was associated with increased risk of VSD with risk ratios ranging from 1.7 [95% confidence interval (CI) 1.0-2.9] to 2.1 (95% CI 1.1-4.0) across methods. Risks correspond to one additional VSD for approximately every 330 pregnancies exposed in the first trimester. The association was dose-dependent with increased risk in women receiving highest cumulative doses compared with lowest doses [adjusted risk ratio: 3.2 (95% CI 1.0-9.9)]. The association between ondansetron and congenital malformations was diluted as the outcome included additional birth defects. CONCLUSIONS: First trimester ondansetron use is associated with an increased risk of neonatal VSD potentially driven by higher doses. This risk should be viewed in the context of risks attributable to severe untreated nausea and vomiting of pregnancy.

A major deletion in the Vκ-Jκ intervening region results in hyperelevated transcription of proximal Vκ genes and a severely restricted repertoire.

Our previous studies have shown that DNase I hypersensitive sites 1 and 2 (HS1-2) and HS3-6 within the mouse Vκ-Jκ intervening region are essential for controlling locus contraction and creating a diverse Ab repertoire. In this article, we demonstrate that a 6.3-kb deletion encompassing HS1-6 altogether not only leads to the predictable sums of these phenotypes, but also results in a novel hyperelevation of transcription of proximal Vκ genes, in both pre-B and splenic B cells. These findings reveal previously unrecognized additional functions for cis-elements within the Vκ-Jκ intervening region, namely, prevention of the production of massive levels of noncoding RNA species by silencing transcription of germline proximal Vκ genes in both developing and mature B cells.

Pronounced cohabitation of active immunoglobulin genes from three different chromosomes in transcription factories during maximal antibody synthesis.

To understand the relationships between nuclear organization and gene expression in a model system, we employed three-dimensional imaging and chromatin immunoprecipitation (ChIP)-chromosome conformation capture (3C) techniques to investigate the topographies of the immunoglobulin (Ig) genes and transcripts during B-cell development. Remarkably, in plasma cells, when antibody synthesis peaks, active Ig genes residing on three different chromosomes exhibit pronounced colocalizations in transcription factories, often near the nuclear periphery, and display trans-chromosomal enhancer interactions, and their transcripts frequently share interchromatin trafficking channels. Conceptually, these features of nuclear organization maximize coordinated transcriptional and transcript trafficking control for potentiating the optimal cytoplasmic assembly of the resulting translation products into protein multimers.

Loss of an Igκ gene enhancer in mature B cells results in rapid gene silencing and partial reversible dedifferentiation.

We address here whether there is cellular memory of a transcriptional enhancer once it has served its purpose to establish an active chromatin state. We have previously shown that the mouse Igκ gene's downstream enhancers, E3' and Ed, are essential but play redundant roles for establishing transcriptional activity in the locus during B cell development. To determine whether these enhancers are also necessary for the maintenance of transcriptional activity, we conditionally deleted E3' in mature B cells that possessed Ed(-/-) alleles. Upon E3' deletion, the locus became rapidly silenced and lost positive histone epigenetic marks, and the mature B cells partially dedifferentiated, induced RAG-1 and -2 along with certain other pro-B cell makers, and then redifferentiated after triggering Igλ gene rearrangements. We conclude that the Igκ gene's downstream enhancers are essential for both the establishment and maintenance of transcriptional activity and that there is no cellular memory of previous transcriptional activity in this locus. Furthermore, upon enhancer loss, the mature B cells unexpectedly underwent reversible retrograde differentiation. This result establishes that receptor editing can occur in mature B cells and raises the possibility that this may provide a tolerance mechanism for eliminating autoreactive B cells in the periphery.

Vκ gene repertoire and locus contraction are specified by critical DNase I hypersensitive sites within the Vκ-Jκ intervening region.

The processes of Ig gene locus contraction and looping during V(D)J-recombination are essential for creating a diverse Ab repertoire. However, no cis-acting sequence that plays a major role in specifying locus contraction has been uncovered within the Igκ gene locus. In this article, we demonstrate that a 650-bp sequence corresponding to DNase I hypersensitive sites HS1-2 within the mouse Igκ gene V-J intervening region binds CCCTC-binding factor and specifies locus contraction and long-range Vκ gene usage spanning 3.2 Mb in pre-B cells. We call this novel element Cer (for "contracting element for recombination"). Targeted deletion of Cer caused markedly increased proximal and greatly diminished upstream Vκ gene usage, higher allele usage, more splenic Igκ(+) B cells, and nonlineage-specific Igκ rearrangement in T cells. Relative to wild-type mice, Cer-deletion mice exhibited similar levels of Vκ gene germline transcription and H3K4me3 epigenetic marks but displayed a dramatic decrease in locus contraction in pre-B cells. Thus, our studies demonstrate that DNase I hypersensitive sites HS1-2 within the Vκ-Jκ intervening region are essential for controlling locus contraction and creating a diverse Ab repertoire.

A new hypersensitive site, HS10, and the enhancers, E3' and Ed, differentially regulate Igκ gene expression.

The mouse Igκ gene locus has three known transcriptional enhancers: an intronic enhancer (Ei), a 3' enhancer (E3'), and a further downstream enhancer (Ed). We previously discovered, using the chromosome conformation-capture technique, that Ei and E3' interact with a novel DNA sequence near the 3' end of the Igκ locus, specifically in B cells. In the present investigation, we examined the function of this far downstream element. The sequence is evolutionarily conserved and exhibits a plasmacytoma cell-specific DNase I-hypersensitive site in chromatin, henceforth termed HS10 in the locus. HS10 acts as a coactivator of E3' in transient transfection assays. Although HS10(-/-) mice exhibited normal patterns of B cell development, they were tested further along with E3'(-/-) and Ed(-/-) mice for their Igκ expression levels in plasma cells, as well as for both allelic and isotype exclusion in splenic B cells. HS10(-/-) and Ed(-/-), but not E3'(-/-), mice exhibited 2.5-fold lower levels of Igκ expression in antigenically challenged plasma cells. E3'(-/-) mice, but not HS10(-/-) mice, exhibited impaired IgL isotype and allelic exclusion in splenic B cells. We have suggestive results that Ed may also weakly participate in these processes. In addition, HS10(-/-) mice no longer exhibited regional chromosome interactions with E3', and they exhibited modestly reduced somatic hypermutation in the Jκ-Cκ intronic region in germinal center B cells from Peyer's patches. We conclude that the HS10, E3', and Ed differentially regulate Igκ gene dynamics.

A multifunctional element in the mouse Igκ locus that specifies repertoire and Ig loci subnuclear location.

Nonbiased V gene usage for V(D)J joining is essential for providing an optimal immune system, but no cis-acting sequence with this function has been uncovered. We previously identified a recombination silencer and heterochromatin targeting element in the Vκ-Jκ intervening sequence of germline Igκ transgenes, which we termed Sis. We now have generated Sis knockout mice in the endogenous locus. Intriguingly, Sis(-/-) mice exhibit a skewed Igκ repertoire with markedly decreased distal and enhanced proximal Vκ gene usage for primary rearrangement, which is associated with reduced occupancy of Ikaros and CCCTC-binding factor in the Vκ-Jκ intervening sequence in pre-B cells, proteins believed to be responsible for dampening the recombination of nearby Vκ genes and altering higher-order chromatin looping. Furthermore, monoallelic heterochromatin localization is significantly reduced in Sis(-/-) mice for Igκ in cis and IgH loci in trans in pre-B cells. Because Sis(-/-) mice still allelically excluded Igκ and IgH loci and still exhibited IgL isotype exclusion, we concluded that stable localization at pericentromeric heterochromatin is neither necessary nor sufficient for the establishment or maintenance of allelic exclusion. Hence, Sis is a novel multifunctional element that specifies repertoire and heterochromatin localization to Ig genes.

The Igκ gene enhancers, E3' and Ed, are essential for triggering transcription.

The mouse Igκ gene locus has three known transcriptional enhancers: an intronic enhancer (Ei), a 3' enhancer (E3'), and a further downstream enhancer (Ed). Previous studies on B lymphocytes derived from mutant embryonic stem cells have shown that deletion of either Ei or E3' significantly reduces Igκ gene rearrangement, whereas the combined deletion of both Ei and E3' eliminates such recombination. Furthermore, deletion of either E3' or Ed significantly reduces rearranged Igκ gene transcription. To determine whether the combined presence of both E3' and Ed are essential for Igκ gene expression, we generated homozygous double knockout (DKO) mice with targeted deletions in both elements. Significantly, homozygous DKO mice were unable to generate κ(+) B cells both in bone marrow and the periphery and exhibited surface expression almost exclusively of Igλ-chains, despite the fact that they possessed potentially functional rearranged Igκ genes. Compared with their single-enhancer-deleted counterparts, Igκ loci in homozygous DKO mice exhibited dramatically reduced germline and rearranged gene transcription, lower levels of gene rearrangement and histone H3 acetylation, and markedly increased DNA methylation. This contributed to a partial developmental block at the pre-B cell stage of development. We conclude that the two downstream enhancers are essential in Igκ gene expression and that Ei in homozygous DKO mice is incapable of triggering Igκ gene transcription. Furthermore, these results reveal unexpected compensatory roles for Ed in E3' knockout mice in triggering germline transcription and Vκ gene rearrangements to both Jκ and RS elements.

Divergent roles of RelA and c-Rel in establishing chromosomal loops upon activation of the Igkappa gene.

Precise regulation of eukaryotic gene expression requires interactions between distal cis-acting regulatory sequences with the looping out of the intervening DNA, but how trans-acting regulatory proteins work to establish and maintain DNA loops during gene activation remains largely unexplored. LPS-induced transcription of the mouse Igkappa gene in B lymphocytes utilizes three distal enhancers and requires the transcription factor NF-kappaB, whose family members include RelA and c-Rel. Using chromosome conformation capture technology in combination with chromatin immunoprecipitation, here we demonstrate that LPS-induced Igkappa gene activation creates chromosomal loops by bridging together all three pairwise interactions between the distal enhancers and RNA polymerase II, the apparent molecular tie for the bases of these loops. RelA and actin polymerization are essential for triggering these processes, which do not require new transcription, protein synthesis, or c-Rel. We have thus identified both essential and nonessential events that establish higher order chromatin reorganization during Igkappa gene activation.

The Downstream Transcriptional Enhancer, Ed, positively regulates mouse Ig kappa gene expression and somatic hypermutation.

The mouse Igkappa locus has three known transcriptional enhancers: the matrix association region/intronic enhancer, the 3' enhancer (E3'), and the further downstream enhancer (Ed). Previous studies have shown that both matrix association region/intronic and E3' enhancers are required for maximal gene rearrangement of the locus, and that E3' is also required for maximal expression and somatic hypermutation (SHM). To functionally elucidate Ed in vivo, we generated knockout mice with a targeted germline deletion of Ed. Ed deleted homozygous mice (Ed-/-) have moderately reduced numbers of Igkappa expressing B cells and correspondingly increased numbers of Iglambda expressing B cells in spleen. Ed-/- mice also have decreased Igkappa mRNA expression in resting and T cell-dependent activated splenic B cells and reduced Igkappa chains in sera. However, our analysis indicates that Igkappa gene rearrangement is normal in Ed-/- mice. In addition, our results show that Ed-/- mice exhibit reduced SHM in the Igkappa gene J-C intronic region in germinal center B cells from Peyer's patches. We conclude that Ed positively regulates Igkappa gene expression and SHM, but not gene rearrangement.