High-throughput micro-CT analysis identifies sex-dependent biomarkers of erosive arthritis in TNF-Tg mice and differential response to anti-TNF therapy.

BACKGROUND: Development of reliable disease activity biomarkers is critical for diagnostics, prognostics, and novel drug development. Although computed tomography (CT) is the gold-standard for quantification of bone erosions, there are no consensus approaches or rationales for utilization of specific outcome measures of erosive arthritis in complex joints. In the case of preclinical models, such as sexually dimorphic tumor necrosis factor transgenic (TNF-Tg) mice, disease severity is routinely quantified in the ankle through manual segmentation of the talus or small regions of adjacent bones primarily due to the ease in measurement. Herein, we sought to determine the particular hindpaw bones that represent reliable biomarkers of sex-dependent disease progression to guide future investigation and analysis. METHODS: Hindpaw micro-CT was performed on wild-type (n = 4 male, n = 4 female) and TNF-Tg (n = 4 male, n = 7 female) mice at monthly intervals from 2-5 (females) and 2-8-months (males) of age, since female TNF-Tg mice exhibit early mortality from cardiopulmonary disease at approximately 5-6-months. Further, 8-month-old WT (n = 4) and TNF-Tg males treated with anti-TNF monoclonal antibodies (n = 5) or IgG placebo isotype controls (n = 6) for 6-weeks were imaged with micro-CT every 3-weeks. For image analysis, we utilized our recently developed high-throughput and semi-automated segmentation strategy in Amira software. Synovial and osteoclast histology of ankle joints was quantified using Visiopharm. RESULTS: First, we demonstrated that the accuracy of automated segmentation, determined through analysis of ~9000 individual bones by a single user, was comparable in wild-type and TNF-Tg hindpaws before correction (79.2±8.9% vs 80.1±5.1%, p = 0.52). Compared to other bone compartments, the tarsal region demonstrated a sudden, specific, and significant bone volume reduction in female TNF-Tg mice, but not in males, by 5-months (4-months 4.3± 0.22 vs 5-months 3.4± 0.62 mm3, p<0.05). Specifically, the cuboid showed significantly reduced bone volumes at early timepoints compared to other tarsals (i.e., 4-months: Cuboid -24.1±7.2% vs Talus -9.0±5.9% of 2-month baseline). Additional bones localized to the anterolateral region of the ankle also exhibited dramatic erosions in the tarsal region of females, coinciding with increased synovitis and osteoclasts. In TNF-Tg male mice with severe arthritis, the talus and calcaneus exhibited the most sensitive response to anti-TNF therapy measured by effect size of bone volume change over treatment period. CONCLUSIONS: We demonstrated that sexually dimorphic changes in arthritic hindpaws of TNF-Tg mice are bone-specific, where the cuboid serves as a reliable early biomarker of erosive arthritis in female mice. Adoption of automated segmentation approaches in pre-clinical or clinical models has potential to translate quantitative biomarkers to monitor bone erosions in disease and evaluate therapeutic efficacy.

Randomized Trial of Patient Outreach Approaches to De-implement Outdated Colonoscopy Surveillance Intervals.

BACKGROUND AND AIMS: Guidelines now recommend patients with low-risk adenomas receive colonoscopy surveillance in 7-10 years and those with the previously recommended 5-year interval be re-evaluated. We tested 3 outreach approaches for transitioning patients to the 10-year interval recommendation. METHODS: This was a 3-arm pragmatic randomized trial comparing telephone, secure messaging, and mailed letter outreach. The setting was Kaiser Permanente Northern California, a large integrated healthcare system. Participants were patients 54-70 years of age with 1-2 small (<10 mm) tubular adenomas at baseline colonoscopy, due for 5-year surveillance in 2022, without high-risk conditions, and with access to all 3 outreach modalities. Patients were randomly assigned to the outreach arm (telephone [n = 200], secure message [n = 203], and mailed letter [n = 201]) stratified by age, sex, and race/ethnicity. Outreach in each arm was performed by trained medical assistants (unblinded) communicating in English with 1 reminder attempt at 2-4 weeks. Participants could change their assigned interval to 10 years or continue their planned 5-year interval. RESULTS: Sixty-day response rates were higher for telephone (64.5%) and secure messaging outreach (51.7%) vs mailed letter (31.3%). Also, more patients adopted the 10-year surveillance interval in the telephone (37.0%) and secure messaging arms (32.0%) compared with mailed letter (18.9%) and rate differences were significant for telephone (18.1%; 97.5% confidence interval: 8.3%-27.9%) and secure message outreach (13.1%; 97.5% confidence interval: 3.5%-22.7%) vs mailed letter outreach. CONCLUSIONS: Telephone and secure messaging were more effective than mailed letter outreach for de-implementing outdated colonoscopy surveillance recommendations among individuals with a history of low-risk adenomas in an integrated healthcare setting. (ClinicalTrials.gov, Number: NCT05389397).

Multi-omics analysis identifies IgG2b class-switching with ALCAM-CD6 co-stimulation in joint-draining lymph nodes during advanced inflammatory-erosive arthritis.

Introduction: Defective lymphatic drainage and translocation of B-cells in inflamed (Bin) joint-draining lymph node sinuses are pathogenic phenomena in patients with severe rheumatoid arthritis (RA). However, the molecular mechanisms underlying this lymphatic dysfunction remain poorly understood. Herein, we utilized multi-omic spatial and single-cell transcriptomics to evaluate altered cellular composition (including lymphatic endothelial cells, macrophages, B-cells, and T-cells) in the joint-draining lymph node sinuses and their associated phenotypic changes and cell-cell interactions during RA development using the tumor necrosis factor transgenic (TNF-Tg) mouse model. Methods: Popliteal lymph nodes (PLNs) from wild-type (n=10) and TNF-Tg male mice with "Early" (5 to 6-months of age; n=6) and "Advanced" (>8-months of age; n=12) arthritis were harvested and processed for spatial transcriptomics. Single-cell RNA sequencing (scRNAseq) was performed in PLNs from the TNF-Tg cohorts (n=6 PLNs pooled/cohort). PLN histopathology and ELISPOT along with ankle histology and micro-CT were evaluated. Histopathology of human lymph nodes and synovia was performed for clinical correlation. Results: Advanced PLN sinuses exhibited an increased Ighg2b/Ighm expression ratio (Early 0.5 ± 0.1 vs Advanced 1.4 ± 0.5 counts/counts; p<0.001) that significantly correlated with reduced talus bone volumes in the afferent ankle (R2 = 0.54, p<0.001). Integration of single-cell and spatial transcriptomics revealed the increased IgG2b+ plasma cells localized in MARCO+ peri-follicular medullary sinuses. A concomitant decreased Fth1 expression (Early 2.5 ± 0.74 vs Advanced 1.0 ± 0.50 counts, p<0.001) within Advanced PLN sinuses was associated with accumulation of iron-laden Prussian blue positive macrophages in lymph nodes and synovium of Advanced TNF-Tg mice, and further validated in RA clinical samples. T-cells were increased 8-fold in Advanced PLNs, and bioinformatic pathway assessment identified the interaction between ALCAM+ macrophages and CD6+ T-cells as a plausible co-stimulatory mechanism to promote IgG2b class-switching. Discussion: Collectively, these data support a model of flare in chronic TNF-induced arthritis in which loss of lymphatic flow through affected joint-draining lymph nodes facilitates the interaction between effluxing macrophages and T-cells via ALCAM-CD6 co-stimulation, initiating IgG2b class-switching and plasma cell differentiation of the expanded Bin population. Future work is warranted to investigate immunoglobulin clonality and potential autoimmune consequences, as well as the efficacy of anti-CD6 therapy to prevent these pathogenic events.

Declining Colectomy Rates for Nonmalignant Colorectal Polyps in a Large, Ethnically Diverse, Community-Based Population.

INTRODUCTION: Despite studies showing improved safety, efficacy, and cost-effectiveness of endoscopic resection for nonmalignant colorectal polyps, colectomy rates for nonmalignant colorectal polyps have been increasing in the United States and Europe. Given this alarming trend, we aimed to investigate whether colectomy rates for nonmalignant colorectal polyps are increasing or declining in a large, integrated, community-based healthcare system with access to advanced endoscopic resection procedures. METHODS: We identified all individuals aged 50-85 years who underwent a colonoscopy between 2008 and 2018 and were diagnosed with a nonmalignant colorectal polyp(s) at the Kaiser Permanente Northern California integrated healthcare system. Among these individuals, we identified those who underwent a colectomy for nonmalignant colorectal polyps within 12 months after the colonoscopy. We calculated annual colectomy rates for nonmalignant colorectal polyps and stratified rates by age, sex, and race and ethnicity. Changes in rates over time were tested by the Cochran-Armitage test for a linear trend. RESULTS: Among 229,730 patients who were diagnosed with nonmalignant colorectal polyps between 2008 and 2018, 1,611 patients underwent a colectomy. Colectomy rates for nonmalignant colorectal polyps decreased significantly from 125 per 10,000 patients with nonmalignant polyps in 2008 to 12 per 10,000 patients with nonmalignant polyps in 2018 (P < 0.001 for trend). When stratified by age, sex, and race and ethnicity, colectomy rates for nonmalignant colorectal polyps also significantly declined from 2008 to 2018. DISCUSSION: In a large, ethnically diverse, community-based population in the United States, we found that colectomy rates for nonmalignant colorectal polyps declined significantly over the past decade likely because of the establishment of advanced endoscopy centers, improved care coordination, and an organized colorectal cancer screening program.

A novel eukaryotic Na+ methionine selective symporter is essential for mosquito development.

AeNAT5 (NCBI, ABZ81822), an orphan member of the insect-specific Nutrient Amino acid Transporter subfamily of SoLute Carrier family 6 (NAT-SLC6) and the first representative of a novel eukaryotic methionine-selective transport system (M), was cloned from cDNA of the vector mosquito, Aedes aegypti. It has orphan orthologs throughout several mosquito genomes, but not in Drosophila or outside Diptera. It shows the highest apparent affinity to L-Met (K(0.5) = 0.021 mM) and its metabolites Homocysteine and Cysteine (K(0.5) = 0.89 and 2.16 mM), but weakly interact with other substrates. It has a Na(+) - coupled mechanism (K(0.5) Na(+) ∼ 46 mM) with 1AA:1Na(+) stoichiometry that maintains ∼60% activity in Cl(-) - free media. In situ hybridization showed accumof AeNAT5 transcript in the absorptive and secretory epithelia, as well as in specific peripheral neurons and the central ganglia of mosquito larvae. The labeling pattern is distinct from that of the previously characterized AeNAT1. RNAi of AeNAT5 increases larval mortality during ecdysis and dramatically suppresses adult emergence. Our results showed that in addition to previously characterized broad spectra and aromatic amino acid selective transport systems, the mosquito NAT-SLC6 subfamily evolved a unique mechanism for selective absorption of sulfur-containing substrates. We demonstrated specific patterns of alimentary and neuronal transcription of AeNAT5 in mosquito larvae that is collateral with the indispensable function of this transporter in mosquito development.

Cross-talk between signaling pathways can generate robust oscillations in calcium and cAMP.

BACKGROUND: To control and manipulate cellular signaling, we need to understand cellular strategies for information transfer, integration, and decision-making. A key feature of signal transduction is the generation of only a few intracellular messengers by many extracellular stimuli. METHODOLOGY/PRINCIPAL FINDINGS: Here we model molecular cross-talk between two classic second messengers, cyclic AMP (cAMP) and calcium, and show that the dynamical complexity of the response of both messengers increases substantially through their interaction. In our model of a non-excitable cell, both cAMP and calcium concentrations can oscillate. If mutually inhibitory, cross-talk between the two second messengers can increase the range of agonist concentrations for which oscillations occur. If mutually activating, cross-talk decreases the oscillation range, but can generate 'bursting' oscillations of calcium and may enable better filtering of noise. CONCLUSION: We postulate that this increased dynamical complexity allows the cell to encode more information, particularly if both second messengers encode signals. In their native environments, it is unlikely that cells are exposed to one stimulus at a time, and cross-talk may help generate sufficiently complex responses to allow the cell to discriminate between different combinations and concentrations of extracellular agonists.

Identification of Ikr kinetics and drug binding in native myocytes.

Determining the effect of a compound on I (Kr) is a standard screen for drug safety. Often the effect is described using a single IC(50) value, which is unable to capture complex effects of a drug. Using verapamil as an example, we present a method for using recordings from native myocytes at several drug doses along with qualitative features of I (Kr) from published studies of HERG current to estimate parameters in a mathematical model of the drug effect on I (Kr). I (Kr) was recorded from canine left ventricular myocytes using ruptured patch techniques. A voltage command protocol was used to record tail currents at voltages from -70 to -20 mV, following activating pulses over a wide range of voltages and pulse durations. Model equations were taken from a published I (Kr) Markov model and the drug was modeled as binding to the open state. Parameters were estimated using a combined global and local optimization algorithm based on collected data with two additional constraints on I (Kr) I-V relation and I (Kr) inactivation. The method produced models that quantitatively reproduce both the control I (Kr) kinetics and dose dependent changes in the current. In addition, the model exhibited use and rate dependence. The results suggest that: (1) the technique proposed here has the practical potential to develop data-driven models that quantitatively reproduce channel behavior in native myocytes; (2) the method can capture important drug effects that cannot be reproduced by the IC(50) method. Although the method was developed for I (Kr), the same strategy can be applied to other ion channels, once appropriate channel-specific voltage protocols and qualitative features are identified.

Computationally efficient strategy for modeling the effect of ion current modifiers.

Electrophysiological studies often seek to relate changes in ion current properties caused by a chemical modifier to changes in cellular properties. Therefore, quantifying concentration-dependent effects of modifiers on ion currents is a topic of importance. In this paper, we sought a mathematical method for using ion current data to predict the effect of several theoretical ion current modifiers on cellular and tissue properties that is computationally efficient without compromising predictive power. We focused on the K+ current I(K,r) as an example case due to its link to long QT syndrome and arrhythmias, but these methods should be generally applicable to other electrophysiological studies. We compared predictions using a Markov model with mass action binding of the modifiers to specific conformational states of the channel to predictions generated by two simplified models. We investigated scaling I(K,r) conductance, and found that although this method produced predictions that agreed qualitatively with the more complicated model, it did not generate quantitatively consistent predictions for all modifiers tested. Our simulations showed that a more computationally efficient Hodgkin-Huxley model that incorporates the effect of modifiers through functional changes in the current produced quantitatively consistent predictions of concentration-dependent changes in cell and tissue properties for all modifiers tested.