Who is pregnant? Defining real-world data-based pregnancy episodes in the National COVID Cohort Collaborative (N3C).

Objectives: To define pregnancy episodes and estimate gestational age within electronic health record (EHR) data from the National COVID Cohort Collaborative (N3C). Materials and Methods: We developed a comprehensive approach, named Hierarchy and rule-based pregnancy episode Inference integrated with Pregnancy Progression Signatures (HIPPS), and applied it to EHR data in the N3C (January 1, 2018-April 7, 2022). HIPPS combines: (1) an extension of a previously published pregnancy episode algorithm, (2) a novel algorithm to detect gestational age-specific signatures of a progressing pregnancy for further episode support, and (3) pregnancy start date inference. Clinicians performed validation of HIPPS on a subset of episodes. We then generated pregnancy cohorts based on gestational age precision and pregnancy outcomes for assessment of accuracy and comparison of COVID-19 and other characteristics. Results: We identified 628 165 pregnant persons with 816 471 pregnancy episodes, of which 52.3% were live births, 24.4% were other outcomes (stillbirth, ectopic pregnancy, abortions), and 23.3% had unknown outcomes. Clinician validation agreed 98.8% with HIPPS-identified episodes. We were able to estimate start dates within 1 week of precision for 475 433 (58.2%) episodes. 62 540 (7.7%) episodes had incident COVID-19 during pregnancy. Discussion: HIPPS provides measures of support for pregnancy-related variables such as gestational age and pregnancy outcomes based on N3C data. Gestational age precision allows researchers to find time to events with reasonable confidence. Conclusion: We have developed a novel and robust approach for inferring pregnancy episodes and gestational age that addresses data inconsistency and missingness in EHR data.

cTULIP: application of a human-based RNA-seq primary tumor classification tool for cross-species primary tumor classification in canine.

Introduction: The domestic dog, Canis familiaris, is quickly gaining traction as an advantageous model for use in the study of cancer, one of the leading causes of death worldwide. Naturally occurring canine cancers share clinical, histological, and molecular characteristics with the corresponding human diseases. Methods: In this study, we take a deep-learning approach to test how similar the gene expression profile of canine glioma and bladder cancer (BLCA) tumors are to the corresponding human tumors. We likewise develop a tool for identifying misclassified or outlier samples in large canine oncological datasets, analogous to that which was developed for human datasets. Results: We test a number of machine learning algorithms and found that a convolutional neural network outperformed logistic regression and random forest approaches. We use a recently developed RNA-seq-based convolutional neural network, TULIP, to test the robustness of a human-data-trained primary tumor classification tool on cross-species primary tumor prediction. Our study ultimately highlights the molecular similarities between canine and human BLCA and glioma tumors, showing that protein-coding one-to-one homologs shared between humans and canines, are sufficient to distinguish between BLCA and gliomas. Discussion: The results of this study indicate that using protein-coding one-to-one homologs as the features in the input layer of TULIP performs good primary tumor prediction in both humans and canines. Furthermore, our analysis shows that our selected features also contain the majority of features with known clinical relevance in BLCA and gliomas. Our success in using a human-data-trained model for cross-species primary tumor prediction also sheds light on the conservation of oncological pathways in humans and canines, further underscoring the importance of the canine model system in the study of human disease.

Cross-diagnostic determinants of cognitive functioning: the muscarinic cholinergic receptor as a model system.

Cognitive impairment is a predictor of disability across different neuropsychiatric conditions, and cognitive abilities are also strongly related to educational attainment and indices of life success in the general population. Previous attempts at drug development for cognitive enhancement have commonly attempted to remedy defects in transmitters systems putatively associated with the conditions of interest such as the glutamate system in schizophrenia. Recent studies of the genomics of cognitive performance have suggested influences that are common in the general population and in different neuropsychiatric conditions. Thus, it seems possible that transmitter systems that are implicated for cognition across neuropsychiatric conditions and the general population would be a viable treatment target. We review the scientific data on cognition and the muscarinic cholinergic receptor system (M1 and M4) across different diagnoses, in aging, and in the general population. We suggest that there is evidence suggesting potential beneficial impacts of stimulation of critical muscarinic receptors for the enhancement of cognition in a broad manner, as well as the treatment of psychotic symptoms. Recent developments make stimulation of the M1 receptor more tolerable, and we identify the potential benefits of M1 and M4 receptor stimulation as a trans-diagnostic treatment model.

A cross-diagnostic study of Adherence to Ecological Momentary Assessment: Comparisons across study length and daily survey frequency find that early adherence is a potent predictor of study-long adherence.

BACKGROUND: Ecological momentary assessment (EMA) offers a highly valid strategy to assess everyday functioning in people with severe mental illness. Adherence is generally good, but several questions regarding the impact of study length, daily density of sampling, and symptom severity on adherence remain. METHODS: EMA adherence in two separate studies was examined. One sampled participants with schizophrenia (n=106) and healthy controls (n=76) 7 times per day for 7 days and the other sampled participants with schizophrenia (n=104) and participants with bipolar illness (n=76) 3 times per day for 30 days. Participants were asked where they were, who they were with, what they were doing and how they were feeling in both studies. The impact of rates of very early adherence on eventual adherence was investigated across the samples, and adherence rates were examined for associations with mood state and most common location when answering surveys. RESULTS: Median levels of adherence were over 80% across the samples, and the 10th percentile for adherence was approximately 45% of surveys answered. Early adherence predicted study-long adherence quite substantially in every sample. Mood states did not correlate with adherence in the patient samples and being home correlated with adherence in only the bipolar sample. IMPLICATIONS: Adherence was quite high and was not correlated with the length of the study or the density of sampling per study day. There was a tendency for bipolar participants who were more commonly away from home to answer fewer surveys but overall adherence for the bipolar patients was quite high. These data suggest that early nonadherence is a potential predictor of eventual nonadherence and study noncompletion.

Daily Ecological Momentary Assessments of happy and sad moods in people with schizophrenia and bipolar disorders: What do participants who are never sad think about their activities and abilities?

OBJECTIVES: People with schizophrenia have challenges in their self-assessments of everyday functioning and those who report no sadness also tend to overestimate their everyday functional abilities. While previous studies were cross-sectional, this study related longitudinal assessments of sadness to self-reports of abilities in domains of everyday functioning and cognitive abilities. METHODS: 71 people with bipolar illness (BPI) were compared to 102 people with schizophrenia (SCZ). Participants were sampled 3 times per day for 30 days with a smartphone-based Ecological Momentary Assessment (EMA) survey. Each survey asked where they were, with whom they were, what they were doing, and if they were sad. Performance based assessments of executive functioning, social competence, and everyday activities were collected after the EMA period, at which time the participants and observers were asked to provide ratings of three different domains of everyday functioning and neurocognitive ability. RESULTS: 18% of participants with SCZ reported that they were never sad on any one of the 90 EMA surveys. Reports of never being sad were associated with overestimated functioning compared to observers and SCZ participants who reported that they were never sad were more commonly home and alone than both SCZ participants who reported occasional sadness and participants with BPI. These participants reported being significantly happier than all people in the study. IMPLICATIONS: Reporting that you were never sad was associated with overestimation of everyday functioning and cognitive abilities. Although participants who were never sad did not perform more poorly on objective measures than those were occasionally sad, their self-assessed functioning was significantly elevated. These data suggest that negative symptoms constructs such as reduced emotional experience need to consider reduced ability to subjectively evaluate emotional experience as a feature of negative symptoms.

Antigenic Restimulation of Virus-Specific Memory CD8+ T Cells Requires Days of Lytic Protein Accumulation for Maximal Cytotoxic Capacity.

In various infections or vaccinations of mice or humans, reports of the persistence and the requirements for restimulation of the cytotoxic mediators granzyme B (GrB) and perforin (PRF) in CD8+ T cells have yielded disparate results. In this study, we examined the kinetics of PRF and GrB mRNA and protein expression after stimulation and associated changes in cytotoxic capacity in virus-specific memory cells in detail. In patients with controlled HIV or cleared respiratory syncytial virus (RSV) or influenza virus infections, all virus-specific CD8+ T cells expressed low PRF levels without restimulation. Following stimulation, they displayed similarly delayed kinetics for lytic protein expression, with significant increases occurring by days 1 to 3 before peaking on days 4 to 6. These increases were strongly correlated with, but were not dependent upon, proliferation. Incremental changes in PRF and GrB percent expression and mean fluorescence intensity (MFI) were highly correlated with increases in HIV-specific cytotoxicity. mRNA levels in HIV-specific CD8+ T-cells exhibited delayed kinetics after stimulation as with protein expression, peaking on day 5. In contrast to GrB, PRF mRNA transcripts were little changed over 5 days of stimulation (94-fold versus 2.8-fold, respectively), consistent with posttranscriptional regulation. Changes in expression of some microRNAs, including miR-17, miR-150, and miR-155, suggested that microRNAs might play a significant role in regulation of PRF expression. Therefore, under conditions of extremely low or absent antigen levels, memory virus-specific CD8+ T cells require prolonged stimulation over days to achieve maximal lytic protein expression and cytotoxic capacity.IMPORTANCE Antigen-specific CD8+ T cells play a major role in controlling most virus infections, primarily by perforin (PRF)- and granzyme B (GrB)-mediated apoptosis. There is considerable controversy regarding whether PRF is constitutively expressed, rapidly increased similarly to a cytokine, or delayed in its expression with more prolonged stimulation in virus-specific memory CD8+ T cells. In this study, the degree of cytotoxic capacity of virus-specific memory CD8+ T cells was directly proportional to the content of lytic molecules, which required antigenic stimulation over several days for maximal levels. This appeared to be modulated by increases in GrB transcription and microRNA-mediated posttranscriptional regulation of PRF expression. Clarifying the requirements for maximal cytotoxic capacity is critical to understanding how viral clearance might be mediated by memory cells and what functions should be induced by vaccines and immunotherapies.

Effect of Antiretroviral Therapy on Bone and Renal Health in Young Adults Infected With HIV in Early Life.

Context: HIV antiretroviral (ARV) therapy is associated with renal and bone toxicity, but little is known about the potential cumulative effects in adults exposed to ARVs from birth. Objective: To prospectively evaluate renal and bone health in young adults with lifelong HIV and extensive ARV exposure. Design: Cross-sectional comparison of bone mineral density (BMD) by dual-energy X-ray absorptiometry, bone turnover, and renal function in young adults infected with HIV in early life (n = 65) to matched healthy controls (n = 23) and longitudinal evaluation (mean follow-up = 4.4 years) within a subset of the HIV cohort (n = 33). Setting: Government outpatient research clinic. Results: Albumin/creatinine ratio, protein/creatinine ratio, anion gap, N-terminal telopeptides, and osteocalcin were significantly increased in persons with HIV compared with controls, whereas whole-body BMD and BMD z scores were lower. Within the HIV group, duration of tenofovir disoproxil fumarate (TDF) correlated with higher anion gap but did not correlate with bone parameters. Longer duration of didanosine and stavudine use correlated with lower BMD and BMD z scores. Longitudinal analyses revealed that BMD and bone metabolism significantly improved over time. No subject had an estimated glomerular filtration rate (eGFR) <60, but decline in eGFR correlated with increasing years of TDF exposure. Conclusions: Subclinical markers of renal dysfunction were increased in HIV-infected young adults and associated with TDF exposure, whereas lower bone density was associated with didanosine and stavudine exposure. The tendency for improvement in markers of bone health over time and the availability of less toxic ARV alternatives may herald improvements in renal and bone health for perinatally infected patients in adulthood.

FolC2-mediated folate metabolism contributes to suppression of inflammation by probiotic Lactobacillus reuteri.

Bacterial-derived compounds from the intestinal microbiome modulate host mucosal immunity. Identification and mechanistic studies of these compounds provide insights into host-microbial mutualism. Specific Lactobacillus reuteri strains suppress production of the proinflammatory cytokine, tumor necrosis factor (TNF), and are protective in a mouse model of colitis. Human-derived L. reuteri strain ATCC PTA 6475 suppresses intestinal inflammation and produces 5,10-methenyltetrahydrofolic acid polyglutamates. Insertional mutagenesis identified the bifunctional dihydrofolate synthase/folylpolyglutamate synthase type 2 (folC2) gene as essential for 5,10-methenyltetrahydrofolic acid polyglutamate biosynthesis, as well as for suppression of TNF production by activated human monocytes, and for the anti-inflammatory effect of L. reuteri 6475 in a trinitrobenzene sulfonic acid-induced mouse model of acute colitis. In contrast, folC encodes the enzyme responsible for folate polyglutamylation but does not impact TNF suppression by L. reuteri. Comparative transcriptomics between wild-type and mutant L. reuteri strains revealed additional genes involved in immunomodulation, including previously identified hdc genes involved in histidine to histamine conversion. The folC2 mutant yielded diminished hdc gene cluster expression and diminished histamine production, suggesting a link between folate and histadine/histamine metabolism. The identification of genes and gene networks regulating production of bacterial-derived immunoregulatory molecules may lead to improved anti-inflammatory strategies for digestive diseases.

Analysis of Bacterial Communities during Clostridium difficile Infection in the Mouse.

Clostridium difficile infection (CDI) is a major cause of health care-associated disease. CDI initiates with ingestion of C. difficile spores, germination in the gastrointestinal (GI) tract, and then colonization of the large intestine. The interactions between C. difficile cells and other bacteria and with host mucosa during CDI remain poorly understood. Here, we addressed the hypothesis that, in a mouse model of CDI, C. difficile resides in multicellular communities (biofilms) in association with host mucosa. To do this, we paraffin embedded and then sectioned the GI tracts of infected mice at various days postinfection (p.i.). We then used fluorescent in situ hybridization (FISH) with 16S rRNA probes targeting most bacteria as well as C. difficile specifically. The results revealed that C. difficile is present as a minority member of communities in the outer (loose) mucus layer, in the cecum and colon, starting at day 1 p.i. To generate FISH probes that identify bacteria within mucus-associated communities harboring C. difficile, we characterized bacterial populations in the infected mouse GI tract using 16S rRNA gene sequence analysis of bacterial DNA prepared from intestinal content. This analysis revealed the presence of genera of several families belonging to Bacteroidetes and Firmicutes. These data suggest that formation of multispecies communities associated with the mucus of the cecum and colon is an important early step in GI tract colonization. They raise the possibility that other bacterial species in these communities modulate the ability of C. difficile to successfully colonize and, thereby, cause disease.

Protection from intestinal inflammation by bacterial exopolysaccharides.

Host inflammatory responses against pathogenic organisms can be abrogated by commensals; however, the molecular mechanisms by which pathogenesis is prevented are still poorly understood. Previous studies demonstrated that administration of a single dose of Bacillus subtilis prevented disease and inflammation by the enteric mouse pathogen Citrobacter rodentium, which causes disease similar to the human pathogen enteropathogenic Escherichia coli. No protection was observed when an exopolysaccharide (EPS)-deficient mutant of B. subtilis was used, suggesting that EPS are the protective factor. In this study, we isolated and characterized EPS and showed that they also prevent C. rodentium-associated intestinal disease after a single injection. Protection is TLR4 dependent because EPS-treated TLR4 knockout mice developed disease. Furthermore, protection could be conveyed to wild-type mice by adoptive transfer of macrophage-rich peritoneal cells from EPS-treated mice. We found that EPS specifically bind peritoneal macrophages, and because mice lacking MyD88 signaling in myeloid cells were not protected by EPS, we conclude that bacterial EPS prevent colitis in a TLR4-dependent manner that requires myeloid cells. These studies provide a simple means of preventing intestinal inflammation caused by enteric pathogens.

Bacillus subtilis-mediated protection from Citrobacter rodentium-associated enteric disease requires espH and functional flagella.

Commensals limit disease caused by invading pathogens; however, the mechanisms and genes utilized by beneficial microbes to inhibit pathogenesis are poorly understood. The attaching and effacing mouse pathogen Citrobacter rodentium associates intimately with the intestinal epithelium, and infections result in acute colitis. C. rodentium is used to model the human pathogens enterohemorrhagic Escherichia coli and enteropathogenic E. coli. To confirm that Bacillus subtilis, a spore-forming bacterium found in the gut of mammals, could reduce C. rodentium-associated disease, mice received wild-type B. subtilis spores and 24 h later were infected by oral gavage with pathogenic C. rodentium. Disease was assessed by determining the extent of colonic epithelial hyperplasia, goblet cell loss, diarrhea, and pathogen colonization. Mice that received wild-type B. subtilis prior to enteric infection were protected from disease even though C. rodentium colonization was not inhibited. In contrast, espH and hag mutants, defective in exopolysaccharides and flagellum production, respectively, did not protect mice from C. rodentium-associated disease. A motAB mutant also failed to protect mice from disease, suggesting that B. subtilis-mediated protection requires functional flagella. By expanding our current mechanistic knowledge of bacterial protection, we can better utilize beneficial microbes to prevent intestinal disease caused by pathogenic bacteria, ultimately reducing human disease. Our data demonstrate that wild-type B. subtilis reduced disease caused by C. rodentium infection through a mechanism that required espH and functional flagella.

Cyclopropane fatty acid synthase mutants of probiotic human-derived Lactobacillus reuteri are defective in TNF inhibition.

Although commensal microbes have been shown to modulate host immune responses, many of the bacterial factors that mediate immune regulation remain unidentified. Select strains of human-derived Lactobacillus reuteri synthesize immunomodulins that potently inhibit production of the inflammatory cytokine TNF. In this study, genetic and genomic approaches were used to identify and investigate L. reuteri genes required or human TNF immunomodulatory activity. Analysis of membrane fatty acids from multiple L. reuteri strains cultured in MRS medium showed that only TNF inhibitory strains produced the cyclopropane fatty acid (CFA) lactobacillic acid. The enzyme cyclopropane fatty acid synthase is required for synthesis of CFAs such as lactobacillic acid, therefore the cfa gene was inactivated and supernatants from the cfa mutant strain were assayed for TNF inhibitory activity. We found that supernatants from the wild-type strain, but not the cfa mutant, suppressed TNF production by activated THP-1 human monocytoid cells Although this suggested a direct role for lactobacillic acid in immunomodulation, purified lactobacillic acid did not suppress TNF at physiologically relevant concentrations. We further analyzed TNF inhibitory and TNF non-inhibitory strains under different growth conditions and found that lactobacillic acid production did not correlate with TNF inhibition. These results indicate that cfa indirectly contributed to L. reuter immunomodulatory activity and suggest that other mechanisms, such as decreased membrane fluidity or altered expression of immunomodulins, result in the loss of TNF inhibitory activity. By increasing our understanding of immunomodulation by probiotic species, beneficial microbes can be rationally selected to alleviate intestinal inflammation.

Probiotic Lactobacillus reuteri biofilms produce antimicrobial and anti-inflammatory factors.

BACKGROUND: Commensal-derived probiotic bacteria inhibit enteric pathogens and regulate host immune responses in the gastrointestinal tract, but studies examining specific functions of beneficial microbes in the context of biofilms have been limited in scope. RESULTS: Lactobacillus reuteri formed biofilms that retained functions potentially advantageous to the host including modulation of cytokine output and the production of the antimicrobial agent, reuterin. Immunomodulatory activities of biofilms were demonstrated by the abilities of specific L. reuteri strains to suppress human TNF production by LPS-activated monocytoid cells. Quantification of the antimicrobial glycerol derivative, reuterin, was assessed in order to document the antipathogenic potential of probiotic biofilms. L. reuteri biofilms differed in the quantities of reuterin secreted in this physiological state. CONCLUSION: L. reuteri biofilms secreted factors that confer specific health benefits such as immunomodulation and pathogen inhibition. Future probiotic selection strategies should consider a strain's ability to perform beneficial functions as a biofilm.