Cardiac Imaging in Myocarditis: Current Evidence and Future Directions.

Myocarditis is defined as a non-ischemic inflammatory disease of the myocardium. It remains a challenge to diagnose given non-specific symptoms and lack of specific blood biomarkers. Cardiac imaging plays an important role in the evaluation of myocarditis with unique strengths and limitations of different imaging modalities, including cardiac magnetic resonance imaging, echocardiography, cardiac computed tomography, and positron emission tomography. The purpose of this review is to discuss the strengths and limitations of various cardiac imaging techniques in the evaluation of myocarditis, review imaging findings in specific causes of myocarditis including COVID-19 and after vaccination, evaluate the role of imaging in differentiating myocarditis from potential mimics and differential considerations, identify current gaps in knowledge, and propose future directions.

Evaluation of left atrial remodeling using cardiovascular magnetic resonance imaging in breast cancer patients treated with adjuvant trastuzumab.

OBJECTIVES: We evaluated left atrial (LA) remodeling using cardiac MRI (CMR) in patients with human epidermal growth factor receptor 2 (HER2)-positive breast cancer during and after trastuzumab therapy. METHODS: In this prospective 2-center longitudinal study, 41 women with HER2-positive breast cancer received adjuvant trastuzumab for 12 months, in addition to standard chemotherapy. Serial CMRs were performed at baseline, 6, 12, and 18 months after initiation of trastuzumab. LA volumes were measured by a blinded reader. Linear mixed model was used to evaluate longitudinal changes. RESULTS: Of 41 women (mean age 52 ± 11 [SD] years; 56% received anthracycline), one patient experienced trastuzumab-induced cardiotoxicity (TIC) for which trastuzumab was interrupted for one cycle. Mean baseline left ventricular ejection fraction (LVEF) was 68.0 ± 5.9% and LA ejection fraction (LAEF) was 66.0 ± 6.6%. Compared to baseline, LAEF decreased significantly at 6 months (62.7 ± 5.7%, p = 0.027) and 12 months (62.2 ± 6.1%, p = 0.003), while indexed LA minimum volume (LAmin) significantly increased at 12 months (11.6 ± 4.9 ml/m2 vs 13.8 ± 4.5 ml/m2, p = 0.002). At 18 months, all changes from baseline were no longer significant. From baseline to 6 months, change in LAEF correlated with change in LVEF (Spearman's r = 0.41, p = 0.014). No significant interactions (all p > 0.10) were detected between time and anthracycline use for LA parameters. CONCLUSIONS: Among trastuzumab-treated patients with low incidence of TIC, we observed a small but significant decline in LAEF and increase in LAmin that persisted for the duration of therapy and recovered 6 months after therapy cessation. These findings suggest that trastuzumab has concurrent detrimental effects on atrial and ventricular remodeling. KEY POINTS: • In trastuzumab-treated breast cancer patients evaluated by cardiac MRI, left atrial ejection fraction declined and minimum volume increased during treatment and recovered to baseline after trastuzumab cessation. • Changes in left atrial ejection fraction correlated with changes in left ventricular ejection fraction in the first 6 months of trastuzumab treatment. • Trastuzumab therapy is associated with concurrent detrimental effects on left atrial and ventricular remodeling.

Evaluation of Tyro3 expression, Gas6-mediated Akt phosphorylation, and the impact of anti-Tyro3 antibodies in melanoma cell lines.

Tyro3, a member of the Tyro3/Axl/Mer (TAM) family of receptor tyrosine kinases, has emerged as a potential oncogene in melanoma. Here, we confirm that Tyro3 is specifically overexpressed in primary melanoma samples and show that Tyro3 is expressed at varying levels in numerous melanoma cell lines. Short hairpin RNA-mediated knockdown of Tyro3 led to significant cell death via apoptotic mechanisms in nearly all melanoma cell lines tested, regardless of the BRAF or NRAS mutation status or co-expression of Axl and/or Mer. We generated soluble and monomeric versions of the human Tyro3 extracellular domain and human Gas6 for affinity measurements and correlated these values with the level of Gas6 required to induce Tyro3 signaling in cellular assays. Calcium was critical for the correct folding of Gas6 and its binding to Tyro3. In melanoma cell lines, Gas6 induced Tyro3 phosphorylation and downstream Akt phosphorylation without apparent effects on Erk. We generated monoclonal antibodies (mAbs) against Tyro3 to examine their effect on survival signaling in melanoma cell lines. The mAbs generated against Tyro3 included nonligand blockers, partial blockers, and competitive ligand blockers. A number of weak and partial ligand blockers (all recognizing the Tyro3 Ig domains) were the most effective at blocking ligand-mediated downstream signaling of Tyro3. Overall, these data indicate that Tyro3 may confer increased survival signals in melanoma cells and can be stymied using inhibitory mAbs. These mAbs may be useful for further investigations of the role of Tyro3 in melanoma.

National HIV programme testing recommendations.

In recognition of the morbidity and mortality associated with human immunodeficiency virus (HIV), the Joint United Nations Programme on HIV/acquired immunodeficiency syndrome (AIDS) (UNAIDS) aims to end the epidemic by setting and striving to achieve the ambitious 95-95-95 targets. However, Singapore is still not performing well in the first UNAIDS target. The National HIV Programme (NHIVP) developed this set of recommendations based on an adaptation of major international guidelines from the World Health Organization and the US Centers for Disease Control and Prevention. The goals of this recommendation are: (1) to increase the uptake of HIV testing; (2) to allow earlier detection and identification of individuals with unrecognised HIV infection; (3) to facilitate linkage to clinical services; and (4) reduce further transmission of HIV infection in Singapore.

Statins to prevent early cardiac dysfunction in cancer patients at increased cardiotoxicity risk receiving anthracyclines.

BACKGROUND AND AIMS: Anthracyclines can cause cancer therapy-related cardiac dysfunction (CTRCD). We aimed to assess whether statins prevent decline in left ventricular ejection fraction (LVEF) in anthracycline-treated patients at increased risk for CTRCD. METHODS: In this multicenter double-blinded, placebo-controlled trial, patients with cancer at increased risk of anthracycline-related CTRCD (per ASCO guidelines) were randomly assigned to atorvastatin 40 mg or placebo once-daily. Cardiovascular magnetic resonance (CMR) imaging was performed before and within 4 weeks after anthracyclines. Blood biomarkers were measured at every cycle. The primary outcome was post-anthracycline LVEF, adjusted for baseline. CTRCD was defined as a fall in LVEF by >10% to <53%. Secondary endpoints included left ventricular (LV) volumes, CTRCD, CMR tissue characterization, high sensitivity troponin I (hsTnI), and B-type natriuretic peptide (BNP). RESULTS: We randomized 112 patients (56.9 ± 13.6 years, 87 female, and 73 with breast cancer): 54 to atorvastatin and 58 to placebo. Post-anthracycline CMR was performed 22 (13-27) days from last anthracycline dose. Post-anthracycline LVEF did not differ between the atorvastatin and placebo groups (57.3 ± 5.8% and 55.9 ± 7.4%, respectively) when adjusted for baseline LVEF (P = 0.34). There were no significant between-group differences in post-anthracycline LV end-diastolic (P = 0.20) or end-systolic volume (P = 0.12), CMR myocardial edema and/or fibrosis (P = 0.06-0.47), or peak hsTnI (P ≥ 0.99) and BNP (P = 0.23). CTRCD incidence was similar (4% versus 4%, P ≥ 0.99). There was no difference in adverse events. CONCLUSIONS: In patients at increased risk of CTRCD, primary prevention with atorvastatin during anthracycline therapy did not ameliorate early LVEF decline, LV remodeling, CTRCD, change in serum cardiac biomarkers, or CMR myocardial tissue changes. TRIAL REGISTRATION: NCT03186404.

Stable IgG-like bispecific antibodies directed toward the type I insulin-like growth factor receptor demonstrate enhanced ligand blockade and anti-tumor activity.

Bispecific antibodies (BsAbs) target multiple epitopes on the same molecular target or different targets. Although interest in BsAbs has persisted for decades, production of stable and active BsAbs has hindered their clinical evaluation. Here, we describe the production and characterization of tetravalent IgG-like BsAbs that combine the activities of allosteric and competitive inhibitors of the type-I insulin-like growth factor receptor (IGF-1R). The BsAbs, which were engineered for thermal stability, express well, demonstrate favorable biophysical properties, and recognize both epitopes on IGF-1R. Only one BsAb with a unique geometry, denoted BIIB4-5scFv, was capable of engaging all four of its binding arms simultaneously. All the BsAbs (especially BIIB4-5scFv) demonstrated enhanced ligand blocking over the single monoclonal antibodies (mAbs), particularly at high ligand concentrations. The pharmacokinetic profiles of two IgG-like BsAbs were tested in nude mice and shown to be comparable with that of the parental mAbs. The BsAbs, especially BIIB4-5scFv, demonstrated an improved ability to reduce the growth of multiple tumor cell lines and to inhibit ligand-induced IGF-1R signaling in tumor cells over the parental mAbs. BIIB4-5scFv also led to superior tumor growth inhibition over its parental mAbs in vivo. In summary, BsAbs that bridge multiple inhibitory mechanisms against a single target may generally represent a more effective strategy for intervention in oncology or other indications compared with traditional mAb therapy.

Secretion from bacterial versus mammalian cells yields a recombinant scFv with variable folding properties.

Escherichia coli (E. coli) is the most commonly used organism for expressing antibody fragments such as single chain antibody Fvs (scFvs). Previously, we have utilized E. coli to express well-folded scFvs for characterization and engineering purposes with the goal of using these engineered proteins as building blocks for generating IgG-like bispecific antibodies (BsAbs). In the study, described here, we observed a significant difference in the secondary structure of an scFv produced in E. coli and the same scFv expressed and secreted from chinese hamster ovary (CHO) cells as part of a BsAb. We devised a proteolytic procedure to separate the CHO-derived scFv from its antibody-fusion partner and compared its properties with those of the E. coli-derived scFv. In comparison to the CHO-derived scFv, the E. coli-derived scFv was found trapped in a misfolded, but monomeric state that was stable for months at 4 °C. The misfolded state bound antigen in a heterogeneous fashion that included non-specific binding, which made functional characterization challenging. This odd incidence of obtaining a misfolded scFv from bacteria suggests careful characterization of the folded properties of bacterially expressed scFvs is warranted if anomalous issues with antigen-binding or non-specificity occur during an engineering campaign. Additionally, our proteolytic methodology for obtaining significant levels of intact scFvs from highly expressed IgG-like antibody proteins serves as a robust method for producing scFvs in CHO without the use of designed cleavage motifs.

Generalizable design parameters for soluble T cell receptor-based T cell engagers.

While most biological and cellular immunotherapies recognize extracellular targets, T cell receptor (TCR) therapeutics are unique in their ability to recognize the much larger pool of intracellular antigens found on virus-infected or cancerous cells. Recombinant T cell receptor (rTCR)-based therapeutics are gaining momentum both preclinically and clinically highlighted by recent positive phase III human clinical trial results for a TCR/CD3 bifunctional protein in uveal melanoma. Unlike antibody-based T cell engagers whose molecular formats have been widely and extensively evaluated, little data exist describing the putative activities of varied bifunctional formats using rTCRs. Here we generate rTCR/anti-CD3 bifunctionals directed toward NY-ESO-1 or MAGE-A3 with a variety of molecular formats. We show that inducing strong redirected lysis activity against tumors displaying either NY-ESO-1 or MAGE-A3 is highly restricted to small, tandem binding formats with an rTCR/antiCD3 Fab demonstrating the highest potency, rTCR/anti-CD3 single chain variable domain fragment showing similar but consistently weaker potency, and IgG-like or IgG-Fc-containing molecules demonstrating poor activity. We believe this is a universal trait of rTCR bifunctionals, given the canonical TCR/human leukocyte antigen structural paradigm.

Dichotomous impact of affinity on the function of T cell engaging bispecific antibodies.

BACKGROUND: Bispecific T cell engagers represent the majority of bispecific antibodies (BsAbs) entering the clinic to treat metastatic cancer. The ability to apply these agents safely and efficaciously in the clinic, particularly for solid tumors, has been challenging. Many preclinical studies have evaluated parameters related to the activity of T cell engaging BsAbs, but many questions remain. MAIN BODY: This study investigates the impact of affinity of T cell engaging BsAbs with regards to potency, efficacy, and induction of immunomodulatory receptors/ligands using HER-2/CD3 BsAbs as a model system. We show that an IgG BsAb can be as efficacious as a smaller BsAb format both in vitro and in vivo. We uncover a dichotomous relationship between tumor-associated antigen (TAA) affinity and CD3 affinity requirements for cells that express high versus low levels of TAA. HER-2 affinity directly correlated with the CD3 engager lysis potency of HER-2/CD3 BsAbs when HER-2 receptor numbers are high (~200 K/cell), while the CD3 affinity did not impact potency until its binding affinity was extremely low (<600 nM). When HER-2 receptor numbers were lower (~20 K/cell), both HER-2 and CD3 affinity impacted potency. The high affinity anti-HER-2/low CD3 affinity BsAb also demonstrated lower cytokine induction levels in vivo and a dosing paradigm atypical of extremely high potency T cell engaging BsAbs reaching peak efficacy at doses >3 mg/kg. This data confirms that low CD3 affinity provides an opportunity for improved safety and dosing for T cell engaging BsAbs. T cell redirection also led to upregulation of Programmed cell death 1 (PD-1) and 4-1BB, but not CTLA-4 on T cells, and to Programmed death-ligand 1 (PD-L1) upregulation on HER-2HI SKOV3 tumor cells, but not on HER-2LO OVCAR3 tumor cells. Using this information, we combined anti-PD-1 or anti-4-1BB monoclonal antibodies with the HER-2/CD3 BsAb in vivo and demonstrated significantly increased efficacy against HER-2HI SKOV3 tumors via both combinations. CONCLUSIONS: Overall, these studies provide an informational dive into the optimization process of CD3 engaging BsAbs for solid tumors indicating that a reduced affinity for CD3 may enable a better therapeutic index with a greater selectivity for the target tumor and a reduced cytokine release syndrome. These studies also provide an additional argument for combining T cell checkpoint inhibition and co-stimulation to achieve optimal efficacy.

Lung Pericytes in Pulmonary Vascular Physiology and Pathophysiology.

Pericytes are mesenchymal-derived mural cells localized within the basement membrane of pulmonary and systemic capillaries. Besides structural support, pericytes control vascular tone, produce extracellular matrix components, and cytokines responsible for promoting vascular homeostasis and angiogenesis. However, pericytes can also contribute to vascular pathology through the production of pro-inflammatory and pro-fibrotic cytokines, differentiation into myofibroblast-like cells, destruction of the extracellular matrix, and dissociation from the vessel wall. In the lung, pericytes are responsible for maintaining the integrity of the alveolar-capillary membrane and coordinating vascular repair in response to injury. Loss of pericyte communication with alveolar capillaries and a switch to a pro-inflammatory/pro-fibrotic phenotype are common features of lung disorders associated with vascular remodeling, inflammation, and fibrosis. In this article, we will address how to differentiate pericytes from other cells, discuss the molecular mechanisms that regulate the interactions of pericytes and endothelial cells in the pulmonary circulation, and the experimental tools currently used to study pericyte biology both in vivo and in vitro. We will also discuss evidence that links pericytes to the pathogenesis of clinically relevant lung disorders such as pulmonary hypertension, idiopathic lung fibrosis, sepsis, and SARS-COVID. Future studies dissecting the complex interactions of pericytes with other pulmonary cell populations will likely reveal critical insights into the origin of pulmonary diseases and offer opportunities to develop novel therapeutics to treat patients afflicted with these devastating disorders. © 2021 American Physiological Society. Compr Physiol 11:2227-2247, 2021.

Evaluation of left atrial remodeling in kidney transplant patients using cardiac magnetic resonance imaging.

BACKGROUND: Increased left atrial (LA) size predicts cardiovascular events in patients with end-stage kidney disease. There is a paucity of data on LA changes after kidney transplantation (KT). Accordingly, we used cardiac magnetic resonance imaging (CMR) to evaluate LA remodeling after KT, and examined its relationship with left ventricular (LV) measurements, blood pressure and cardiac biomarkers. METHODS: In this prospective multi-center cohort study, 39 pre-transplant dialysis patients underwent KT and 42 eligible transplant recipients remained on dialysis. CMR, blood pressure and serum measurements for N-terminal pro b-type natriuretic peptide (NT-proBNP), high sensitivity C-reactive protein (hsCRP), and growth differentiation factor-15 (GDF-15) were performed at baseline and 12 months. RESULTS: After 12 months, there were no significant changes in LA end-systolic volume index, LA end-diastolic volume index, or LA ejection fraction (LAEF) within the KT or dialysis group; changes over time did not differ between the 2 groups (all p > 0.25). At baseline and over 12 months, LA volumes and LAEF positively correlated with LV volumes and mass while LAEF positively correlated with LV function. Changes in LA volumes also positively correlated with NT-proBNP and systolic blood pressure (sBP) while LAEF negatively correlated with NT-proBNP. GDF-15 correlated with LA measurements at baseline but not in 12-month changes. hsCRP did not correlate with any LA measurements. CONCLUSIONS: LA volumes and function as measured by CMR did not change significantly over 12 months post-KT. There were significant associations between LA and LV remodeling, NT-proBNP and sBP, suggesting common underlying pathophysiological mechanisms.

Computational stabilization of T cell receptors allows pairing with antibodies to form bispecifics.

Recombinant T cell receptors (TCRs) can be used to redirect naïve T cells to eliminate virally infected or cancerous cells; however, they are plagued by low stability and uneven expression. Here, we use molecular modeling to identify mutations in the TCR constant domains (Cα/Cß) that increase the unfolding temperature of Cα/Cß by 20 °C, improve the expression of four separate α/ß TCRs by 3- to 10-fold, and improve the assembly and stability of TCRs with poor intrinsic stability. The stabilizing mutations rescue the expression of TCRs destabilized through variable domain mutation. The improved stability and folding of the TCRs reduces glycosylation, perhaps through conformational stabilization that restricts access to N-linked glycosylation enzymes. The Cα/Cß mutations enables antibody-like expression and assembly of well-behaved bispecific molecules that combine an anti-CD3 antibody with the stabilized TCR. These TCR/CD3 bispecifics can redirect T cells to kill tumor cells with target HLA/peptide on their surfaces in vitro.

Structural understanding of stabilization patterns in engineered bispecific Ig-like antibody molecules.

Bispecific immunoglobulin-like antibodies capable of engaging multiple antigens represent a promising new class of therapeutic agents. Engineering of these molecules requires optimization of the molecular properties of one of the domain components. Here, we present a detailed crystallographic and computational characterization of the stabilization patterns in the lymphotoxin-beta receptor (LTbetaR) binding Fv domain of an anti-LTbetaR/anti-TNF-related apoptosis inducing ligand receptor-2 (TRAIL-R2) bispecific immunoglobulin-like antibody. We further describe a new hierarchical structure-guided approach toward engineering of antibody-like molecules to enhance their thermal and chemical stability.

Using a HIV registry to develop accurate estimates for the HIV care cascade - the Singapore experience.

INTRODUCTION: Achieving UNAIDS 90-90-90 targets is a crucial step towards ending the AIDS epidemic. Many countries have published estimates of care cascades, although often with methodological limitations. We describe an approach that used the national HIV registry as a starting-point to determine the HIV care cascade and resulting UNAIDS 90-90-90 estimates for Singapore in 2014. METHODS: HIV is a legally notifiable disease in Singapore. The anonymized HIV registry data provided for a back-calculation model from the European Centre for Disease Prevention and Control to obtain 2014 estimates for the total number of persons living with HIV (PLHIV), and the count in the registry for proportions diagnosed with HIV and linked to care. Using additional data collected for a simple random sample from the registry, outcomes in 2015 and 2016 were ascertained retrospectively to derive proportions for those retained in care, on antiretroviral therapy, and achieved viral suppression. Findings were extrapolated to derive national estimates and UN90-90-90 estimates. Bootstrapped samples from the model and sample were used to derive 95% confidence intervals. RESULTS: An estimated 6900 (95% CI 6650, 7050) persons were living with HIV and AIDS in 2014. Of these, 4948 were diagnosed with HIV, and 4820 had been linked to care. The random sample of 500 persons was further analysed, and of these, 87.2% were retained in care, 84.6% on antiretroviral therapy, and 79.6% had suppressed viral loads. The proportions of HIV-infected individuals on antiretroviral therapy and achieving viral suppression were 60.7% (95% CI 58.4, 63.6) and 57.1% (95% CI 55.0, 60.5) respectively. The corresponding UNAIDS 90-90-90 estimates were 71.7% (95% CI 70.0, 74.2) of all persons diagnosed; 84.6% (95% CI 81.6, 87.4) of diagnosed persons being on antiretroviral therapy, and 94.1% (95% CI 91.6, 96.2) of persons on therapy having achieved viral suppression. CONCLUSIONS: A national HIV registry, alongside back-calculation and additional data from a sample, can be used to estimate attainment of UNAIDS 90-90-90 targets and identify system gaps. The registry had advantages of providing a well-established, comprehensive capture of diagnosed persons and easily accessible data. The same approach can be used elsewhere if similar data are available.

Long-term complications, reoperations and survival following cardioverter-defibrillator implant.

OBJECTIVE: Implantable cardioverter-defibrillators (ICDs) reduce risk of death in select populations, but are also associated with harms. We aimed to characterise long-term complications and reoperation rate. METHODS: We assessed the rate, cumulative incidence and predictors of long-term reoperation and survival using a prospective, multicentre registry serving British Columbia in Canada, a universal single payer healthcare system with 4.5 million residents. 3410 patients (mean 63.3 years, 81.7% male) with new primary (n=1854) or secondary prevention (n=1556) ICD implant from 2003 to 2012 were followed for a median of 34 months (single chamber n=1069, dual chamber n=1905, biventricular n=436). Independent predictors of adverse outcomes were defined using Cox regression models. RESULTS: The overall reoperation rate was 12.0% per patient-year, and less for single vs dual vs biventricular ICDs (9.1% vs 12.5% vs 17.8% per patient-year, respectively). The Kaplan-Meier complication estimates (excluding generator end of life) at 1, 3 and 5 years were respectively: single chamber 10.2%, 16.2% and 21.6%; dual 11.7%, 19.1% and 27.4% and biventricular 15.9%, 22.2% and 24.7%. Cardiac resynchronisation therapy had the highest rate of early lead complications, but lower long-term need for upgrade. Device complexity, age and atrial fibrillation were key determinants of complications. Overall mortality at 1, 3 and 5 years was 5.4%, 17.4% and 32.7%, respectively. In younger patients, observed 5-year survival approached the expected survival in the general population (relative survival ratio=0.96 (0.90-0.98)). With increasing age, observed survival steadily declined relative to expected. CONCLUSIONS: In a prospective registry capturing all procedures, complication and reoperation rates following de novo ICD implantation were high. Shared decision making must carefully consider these factors.

Comparing domain interactions within antibody Fabs with kappa and lambda light chains.

IgG antibodies are multi-domain proteins with complex inter-domain interactions. Human IgG heavy chains (HCs) associate with light chains (LCs) of the κ or λ isotype to form mature antibodies capable of binding antigen. The HC/LC interaction involves 4 domains: VH and CH1 from the HC and VL and CL from the LC. Human Fabs with κ LCs have been well characterized for their unfolding behaviors and demonstrate a significant level of cooperativity and stabilization when all 4 domains are intact. Very little is known regarding the thermodynamic properties of human Fabs with λ LCs. Here, we dissect the domain contributions to Fab stability for both κ and λ LC-containing Fabs. We find the cooperativity of unfolding between the constant domains, CH1/Cλ, and variable domains, VH/Vλ, within λ LC-containing Fabs is significantly weaker than that of κ LC-containing Fabs. The data suggests there may not be an evolutionary necessity for strong variable/constant domain cooperativity within λ LC-containing Fabs. After investigating the biophysical properties of Fabs with mismatched variable and constant domain subunits (e.g., VH/Vκ paired with CH1/Cλ or T cell receptor Cα/Cß), the major role of the constant domains for both κ- and λ-containing Fabs may be to reduce the hydrophobic exposure at the VH/VL interface. Even though Fabs with these non-native pairings were thermodynamically less stable, they secreted well from mammalian cells as well behaved monodisperse proteins, which was in contrast to what was observed with the VH/Vκ and VH/Vλ scFvs that secreted as a mixture of monomer and aggregates.

Generation of Two-color Antigen Microarrays for the Simultaneous Detection of IgG and IgM Autoantibodies.

Autoantibodies, which are antibodies against self-antigens, are present in many disease states and can serve as markers for disease activity. The levels of autoantibodies to specific antigens are typically detected with the enzyme-linked immunosorbent assay (ELISA) technique. However, screening for multiple autoantibodies with ELISA can be time-consuming and requires a large quantity of patient sample. The antigen microarray technique is an alternative method that can be used to screen for autoantibodies in a multiplex fashion. In this technique, antigens are arrayed onto specially coated microscope slides with a robotic microarrayer. The slides are probed with patient serum samples and subsequently fluorescent-labeled secondary antibodies are added to detect binding of serum autoantibodies to the antigens. The autoantibody reactivities are revealed and quantified by scanning the slides with a scanner that can detect fluorescent signals. Here we describe methods to generate custom antigen microarrays. Our current arrays are printed with 9 solid pins and can include up to 162 antigens spotted in duplicate. The arrays can be easily customized by changing the antigens in the source plate that is used by the microarrayer. We have developed a two-color secondary antibody detection scheme that can distinguish IgG and IgM reactivities on the same slide surface. The detection system has been optimized to study binding of human and murine autoantibodies.

Reporting of Lost to Follow-Up and Treatment Discontinuation in Pharmacotherapy and Device Trials in Chronic Heart Failure: A Systematic Review.

BACKGROUND: Premature treatment discontinuation and loss to follow-up (LTFU) with unknown outcomes leave uncertainty about the true efficacy and safety of a treatment and a lack of confidence in the results of any trial. We reviewed the extent of (and trends over time in) reporting LTFU and treatment discontinuation in large studies in chronic heart failure published since 1990. METHODS AND RESULTS: Online databases were systematically reviewed to identify randomized controlled clinical trials (RCTs) in chronic heart failure with >400 participants and utilizing all-cause mortality as a component of the primary or secondary end point. Assessments were made of documentation of treatment discontinuation, LTFU, inclusion of and completeness of a Consolidated Standards Of Reporting Trials (CONSORT) diagram, and whether LTFU was differentiated from withdrawal of consent. Sixty-eight trials were identified, with >154 000 participants. Reasons for treatment discontinuation in pharmacotherapy trials were infrequently reported (35%), particularly in a CONSORT diagram (20%). Eighty-three percent of trials reported LTFU, although only 34% of these differentiated LTFU for vital status from withdrawal of consent. Use of a CONSORT diagram increased over time, although reporting of LTFU in the CONSORT diagram remained low overall at 35%. CONCLUSIONS: Participant flow through RCTs in chronic heart failure has not been uniformly reported, and the use of a complete CONSORT diagram has been low, although it seems to be improving. All study participants should be accounted for within a CONSORT diagram in any RCT to enable the practicing cardiologist to interpret how the results should influence his/her clinical practice.

Computationally Designed Bispecific Antibodies using Negative State Repertoires.

A challenge in the structure-based design of specificity is modeling the negative states, i.e., the complexes that you do not want to form. This is a difficult problem because mutations predicted to destabilize the negative state might be accommodated by small conformational rearrangements. To overcome this challenge, we employ an iterative strategy that cycles between sequence design and protein docking in order to build up an ensemble of alternative negative state conformations for use in specificity prediction. We have applied our technique to the design of heterodimeric CH3 interfaces in the Fc region of antibodies. Combining computationally and rationally designed mutations produced unique designs with heterodimer purities greater than 90%. Asymmetric Fc crystallization was able to resolve the interface mutations; the heterodimer structures confirmed that the interfaces formed as designed. With these CH3 mutations, and those made at the heavy-/light-chain interface, we demonstrate one-step synthesis of four fully IgG-bispecific antibodies.