An immunophenotype-coupled transcriptomic atlas of human hematopoietic progenitors.

Analysis of the human hematopoietic progenitor compartment is being transformed by single-cell multimodal approaches. Cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) enables coupled surface protein and transcriptome profiling, thereby revealing genomic programs underlying progenitor states. To perform CITE-seq systematically on primary human bone marrow cells, we used titrations with 266 CITE-seq antibodies (antibody-derived tags) and machine learning to optimize a panel of 132 antibodies. Multimodal analysis resolved >80 stem, progenitor, immune, stromal and transitional cells defined by distinctive surface markers and transcriptomes. This dataset enables flow cytometry solutions for in silico-predicted cell states and identifies dozens of cell surface markers consistently detected across donors spanning race and sex. Finally, aligning annotations from this atlas, we nominate normal marrow equivalents for acute myeloid leukemia stem cell populations that differ in clinical response. This atlas serves as an advanced digital resource for hematopoietic progenitor analyses in human health and disease.

A specialized population of Periostin-expressing cardiac fibroblasts contributes to postnatal cardiomyocyte maturation and innervation.

During the postnatal period in mammals, the cardiac muscle transitions from hyperplasic to hypertrophic growth, the extracellular matrix (ECM) undergoes remodeling, and the heart loses regenerative capacity. While ECM maturation and crosstalk between cardiac fibroblasts (CFs) and cardiomyocytes (CMs) have been implicated in neonatal heart development, not much is known about specialized fibroblast heterogeneity and function in the early postnatal period. In order to better understand CF functions in heart maturation and postnatal cardiomyocyte cell-cycle arrest, we have performed gene expression profiling and ablation of postnatal CF populations. Fibroblast lineages expressing Tcf21 or Periostin were traced in transgenic GFP reporter mice, and their biological functions and transitions during the postnatal period were examined in sorted cells using RNA sequencing. Highly proliferative Periostin (Postn)+ lineage CFs were found from postnatal day 1 (P1) to P11 but were not detected at P30, due to a repression of Postn gene expression. This population was less abundant and transcriptionally different from Tcf21+ resident CFs. The specialized Postn+ population preferentially expresses genes related to cell proliferation and neuronal development, while Tcf21+ CFs differentially express genes related to ECM maturation at P7 and immune crosstalk at P30. Ablation of the Postn+ CFs from P0 to P6 led to altered cardiac sympathetic nerve patterning and a reduction in binucleation and hypertrophic growth with increased fetal troponin (TroponinI1) expression in CM. Thus, postnatal CFs are heterogeneous and include a transient proliferative Postn+ population required for cardiac nerve development and cardiomyocyte maturation soon after birth.

Understanding the microbial basis of body odor in pre-pubescent children and teenagers.

BACKGROUND: Even though human sweat is odorless, bacterial growth and decomposition of specific odor precursors in it is believed to give rise to body odor in humans. While mechanisms of odor generation have been widely studied in adults, little is known for teenagers and pre-pubescent children who have distinct sweat composition from immature apocrine and sebaceous glands, but are arguably more susceptible to the social and psychological impact of malodor. RESULTS: We integrated information from whole microbiome analysis of multiple skin sites (underarm, neck, and head) and multiple time points (1 h and 8 h after bath), analyzing 180 samples in total to perform the largest metagenome-wide association study to date on malodor. Significant positive correlations were observed between odor intensity and the relative abundance of Staphylococcus hominis, Staphylococcus epidermidis, and Cutibacterium avidum, as well as negative correlation with Acinetobacter schindleri and Cutibacterium species. Metabolic pathway analysis highlighted the association of isovaleric and acetic acid production (sour odor) from enriched S. epidermidis (teen underarm) and S. hominis (child neck) enzymes and sulfur production from Staphylococcus species (teen underarm) with odor intensity, in good agreement with observed odor characteristics in pre-pubescent children and teenagers. Experiments with cultures on human and artificial sweat confirmed the ability of S. hominis and S. epidermidis to independently produce malodor with distinct odor characteristics. CONCLUSIONS: These results showcase the power of skin metagenomics to study host-microbial co-metabolic interactions, identifying distinct pathways for odor generation from sweat in pre-pubescent children and teenagers and highlighting key enzymatic targets for intervention.

Spike-in genomic DNA for validating performance of metagenomics workflows.

Shotgun metagenomics is a powerful platform to characterize human microbiomes. However, to translate such survey data into consumer-relevant products or services, it is critical to have a robust metagenomics workflow. We present a tool - spike-in DNA - to assess performance of metagenomics workflows. The spike-in is DNA from two organisms - Alivibrio fischeri and Rhodopseudomonas palustris, in a ratio of 4:1 added to samples before DNA extraction. With a valid workflow, the output ratio of relative abundances of these organisms should be close to 4. This expectation was tested in samples of varying diversities (n = 110), and the mean ratio was 4.73 (99% CI [4.0, 5.24]). We anticipate this tool to be a relevant community resource for assessing the quality of shotgun metagenomics workflows and thereby enable robust characterization of microbiomes.

The Impact of Clinical Information on the Assessment of Endoscopic Activity: Characteristics of the Ulcerative Colitis Endoscopic Index Of Severity [UCEIS].

BACKGROUND AND AIMS: To determine whether clinical information influences endoscopic scoring by central readers using the Ulcerative Colitis Endoscopic Index of Severity [UCEIS; comprising 'vascular pattern', 'bleeding', 'erosions and ulcers']. METHODS: Forty central readers performed 28 evaluations, including 2 repeats, from a library of 44 video sigmoidoscopies stratified by Mayo Clinic Score. Following training, readers were randomised to scoring with ['unblinded', n = 20, including 4 control videos with misleading information] or without ['blinded', n 20] clinical information. A total of 21 virtual Central Reader Groups [CRGs], of three blinded readers, were created. Agreement criteria were pre-specified. Kappa [κ] statistics quantified intra- and inter-reader variability. RESULTS: Mean UCEIS scores did not differ between blinded and unblinded readers for any of the 40 main videos. UCEIS standard deviations [SD] were similar [median blinded 0.94, unblinded 0.93; p = 0.97]. Correlation between UCEIS and visual analogue scale [VAS] assessment of overall severity was high [r blinded = 0.90, unblinded = 0.93; p = 0.02]. Scores for control videos were similar [UCEIS: p ≥ 0.55; VAS: p ≥ 0.07]. Intra- [κ 0.47-0.74] and inter-reader [κ 0.40-0.53] variability for items and full UCEIS was 'moderate'-to-'substantial', with no significant differences except for intra-reader variability for erosions and ulcers [κ blinded: 0.47 vs unblinded: 0.74; p 0.047]. The SD of CRGs was lower than for individual central readers [0.54 vs 0.95; p < 0.001]. Correlation between blinded UCEIS and patient-reported symptoms was high [stool frequency: 0.76; rectal bleeding: 0.82; both: 0.81]. CONCLUSIONS: The UCEIS is minimally affected by knowledge of clinical details, strongly correlates with patient-reported symptoms, and is a suitable instrument for trials. CRGs performed better than individuals.

Reliability and initial validation of the ulcerative colitis endoscopic index of severity.

BACKGROUND & AIMS: We studied the reliability of the previously described Ulcerative Colitis Endoscopic Index of Severity (UCEIS) and validated it with an independent cohort of investigators. METHODS: We created a new library of 57 videos of flexible sigmoidoscopy and stratified them based on disease severity. Twenty-five investigators were each randomly assigned to assess 28 videos (which included 4 duplicates to assess intraobserver reliability). Investigators were blinded to clinical details except for 2 of 4 duplicated videos (to assess the impact of knowledge of symptoms on assessment). Three descriptors ("vascular pattern", "bleeding", and "erosions and ulcers") comprising the UCEIS were scored with a visual analogue scale (VAS) to assess overall severity. Intrainvestigator and interinvestigator agreement was characterized by κ statistical analysis; reliability ratios were used to compare VAS and UCEIS scores. RESULTS: There was a high level of correlation between UCEIS scores and overall assessment of severity (correlation coefficient, 0.93). Internal consistency (Cronbach α analysis) was 0.86. Intrainvestigator and interinvestigator reliability ratios for UCEIS scores were 0.96 and 0.88, respectively. Intrainvestigator agreement in determination of the UCEIS score was good (κ = 0.72), with individual descriptors ranging from a κ of 0.47 (for bleeding) to 0.87 (for vascular pattern). Interinvestigator agreement in determination of UCEIS scores was moderate (κ = 0.50), with descriptors ranging from a κ of 0.48 (for bleeding) to 0.54 (for vascular pattern). Intrainvestigator variability in determining UCEIS scores did not change appreciably when a video was presented with clinical details. CONCLUSIONS: The UCEIS and its components show satisfactory intrainvestigator and interinvestigator reliability. Among investigators, the UCEIS accounted for a median of 86% of the variability in evaluation of overall severity on the VAS when assessing the endoscopic severity of UC and was unaffected by knowledge of clinical details.

Developing an instrument to assess the endoscopic severity of ulcerative colitis: the Ulcerative Colitis Endoscopic Index of Severity (UCEIS).

BACKGROUND: Variability in endoscopic assessment necessitates rigorous investigation of descriptors for scoring severity of ulcerative colitis (UC). OBJECTIVE: To evaluate variation in the overall endoscopic assessment of severity, the intra- and interindividual variation of descriptive terms and to create an Ulcerative Colitis Endoscopic Index of Severity which could be validated. DESIGN: A two-phase study used a library of 670 video sigmoidoscopies from patients with Mayo Clinic scores 0-11, supplemented by 10 videos from five people without UC and five hospitalised patients with acute severe UC. In phase 1, each of 10 investigators viewed 16/24 videos to assess agreement on the Baron score with a central reader and agreed definitions of 10 endoscopic descriptors. In phase 2, each of 30 different investigators rated 25/60 different videos for the descriptors and assessed overall severity on a 0-100 visual analogue scale. κ Statistics tested inter- and intraobserver variability for each descriptor. A general linear mixed regression model based on logit link and ß distribution of variance was used to predict overall endoscopic severity from descriptors. RESULTS: There was 76% agreement for 'severe', but 27% agreement for 'normal' appearances between phase I investigators and the central reader. In phase 2, weighted κ values ranged from 0.34 to 0.65 and 0.30 to 0.45 within and between observers for the 10 descriptors. The final model incorporated vascular pattern, (normal/patchy/complete obliteration) bleeding (none/mucosal/luminal mild/luminal moderate or severe), erosions and ulcers (none/erosions/superficial/deep), each with precise definitions, which explained 90% of the variance (pR(2), Akaike Information Criterion) in the overall assessment of endoscopic severity, predictions varying from 4 to 93 on a 100-point scale (from normal to worst endoscopic severity). CONCLUSION: The Ulcerative Colitis Endoscopic Index of Severity accurately predicts overall assessment of endoscopic severity of UC. Validity and responsiveness need further testing before it can be applied as an outcome measure in clinical trials or clinical practice.

Testosterone reference ranges in normally cycling healthy premenopausal women.

INTRODUCTION: At present, there are no well-accepted reference ranges for serum testosterone concentrations in women. AIM: The aim of this study was to determine the reference ranges for serum testosterone and sex hormone-binding globulin (SHBG) in premenopausal women with normal menstrual cycles. METHODS: We measured serum total, free, and bioavailable testosterone and SHBG concentrations in 161 healthy, normally cycling women (18-49 years). Morning blood samples were collected during follicular, mid-cycle, and luteal phases of the menstrual cycle and analyzed using validated methods. Mean, median, and weighted average hormone levels across menstrual cycle phases as well as percentiles for a typical 30-year-old woman were determined. MAIN OUTCOME MEASURES: Age-related serum levels of total, free, and bioavailable testosterone and SHBG levels in normally cycling premenopausal women. RESULTS: Serum testosterone concentrations exhibited an age-related decline, whereas SHBG remained relatively stable across studied age ranges. Reference ranges for total, free, and bioavailable testosterone and SHBG were established using 5th and 95th percentiles. The estimated 5th and 95th percentiles for a 30-year-old woman were: testosterone, 15-46 ng/dL (520-1595 pmol/L); free testosterone, 1.2-6.4 pg/mL (4.16-22.2 pmol/L); calculated free testosterone, 1.3-5.6 pg/mL (4.5-19.4 pmol/L); bioavailable testosterone, 1.12-7.62 ng/dL (38.8-264.21 pmol/L); and SHBG 18-86 nmol/L. The variations of hormones and SHBG across menstrual cycle were consistent with previous literature. CONCLUSIONS: Reference ranges for free, total, and bioavailable testosterone and SHBG were established in premenopausal women using validated immunoassays and an adequate number of subjects consistent with recommendations by the National Committee for Clinical Laboratory Standards. The increase in testosterone in the mid-cycle period is relatively small compared with the overall variability, so these reference ranges can be applied irrespective of the day in the menstrual cycle the sample has been taken.

Esophageal transit and in vivo disintegration of branded risedronate sodium tablets and two generic formulations of alendronic acid tablets: a single-center, single-blind, six-period crossover study in healthy female subjects.

BACKGROUND: Delayed esophageal transit or disintegration of oral bisphosphonate tablets before they enter the stomach may be of concern with respect to iatrogenic complications among patients receiving longterm treatment. Different formulations of generic bisphosphonate tablets meeting regulatory requirements may have substantial differences in pharmaceutical attributes from the branded product that may result in different characteristics during esophageal transit. OBJECTIVE: The primary objective of this study was to evaluate and compare esophageal transit times and in vivo disintegration of 3 bisphosphonate formulations, one branded and the others generic, that are commercially available in Canada and the United Kingdom. METHODS: This was a single-center, randomized, singleblind, 6-period crossover study in healthy postmenopausal women aged >50 years. Each subject received a single oral dose of a branded risedronate sodium 35-mg tablet and 2 generic formulations of alendronic acid 70-mg tablets (Novopharm Limited, Toronto, Canada, and Teva UK Limited, Morley, United Kingdom) in both the erect and semisupine (45 degrees ) positions. Although the products are labeled to be taken in the erect position, the semisupine position was included to simulate dosing in bedridden patients. Subjects took tablets with 30 mL of water in the morning after an overnight fast. The tablets were radiolabeled with technetium-99m ion-exchange resins to enable visualization and measurement of esophageal transit time and disintegration using a gamma camera. Dynamic scintigraphic images were obtained for a total of 10 minutes: 2 images per second for the first 30 seconds and 1 image every 15 seconds for 9.5 minutes. This was a mechanistic study and tolerability was not assessed. RESULTS: The study was conducted in 20 healthy white female subjects with a mean age of 62 years (range, 51-77 years). The effect of body position was statistically significant (P = 0.043), with the estimated hazard ratio (HR) of 0.74 indicating longer transit time in the semisupine position relative to the erect position. There was a statistically significant difference in transit time among the 3 types of tablets (P = 0.007), with the Novopharm tablet (HR = 0.59; P < 0.001) and Teva tablet (HR = 0.71; P = 0.042) having longer transit times compared with the risedronate tablet. In 4 instances, the Novopharm tablet disintegrated and dispersed in the esophagus, once in the erect position and 3 times in the semisupine position. CONCLUSIONS: In these healthy female subjects, esophageal transit was delayed when the tablets were given in the semisupine position. The branded risedronate tablet had a significantly faster transit time than the 2 generic formulations of alendronate tested.