Cx3cr1 controls kidney resident macrophage heterogeneity.

Kidney macrophages are comprised of both monocyte-derived and tissue resident populations; however, the heterogeneity of kidney macrophages and factors that regulate their heterogeneity are poorly understood. Herein, we performed single cell RNA sequencing (scRNAseq), fate mapping, and parabiosis to define the cellular heterogeneity of kidney macrophages in healthy mice. Our data indicate that healthy mouse kidneys contain four major subsets of monocytes and two major subsets of kidney resident macrophages (KRM) including a population with enriched Ccr2 expression, suggesting monocyte origin. Surprisingly, fate mapping data using the newly developed Ms4a3Cre Rosa Stopf/f TdT model indicate that less than 50% of Ccr2+ KRM are derived from Ly6chi monocytes. Instead, we find that Ccr2 expression in KRM reflects their spatial distribution as this cell population is almost exclusively found in the kidney cortex. We also identified Cx3cr1 as a gene that governs cortex specific accumulation of Ccr2+ KRM and show that loss of Ccr2+ KRM reduces the severity of cystic kidney disease in a mouse model where cysts are mainly localized to the kidney cortex. Collectively, our data indicate that Cx3cr1 regulates KRM heterogeneity and niche-specific disease progression.

Analysis of Student Reflections on "What Matters Most" to Palliative Care Patients: A Narrative Medicine Exercise.

Background: Effective communication with ill patients requires practice, yet, the traditional history overlooks patients' personal stories. This information is vital to determining goals of care and facilitates partnership by building trust. Objective: We implemented a narrative medicine exercise for students during their palliative medicine rotation to highlight humanism. Impact was determined through thematic analysis of students' reflections. Design: Students elicited "what matters most" to patients, transcribing this plus a personal reflection. Using an inductive and iterative approach, 100 reflections were analyzed, developing codes then broader themes. Results: Four main themes (Getting to know the patient, Student Reaction, Building-blocks of patient-physician relationship, Student Personal Insights) were identified, with 15 subthemes. Conclusion: The power of uncovering patients' backgrounds and values was demonstrated, reinforcing a palliative medicine approach. Analysis showed a positive impact and the possibility of change to future practice. The intervention was feasible, well received, and encouraged reflection on the physician-patient relationship beyond the medical domain.

Developing Awareness and Allies: Simulating Difficult Conversations about Microaggressions for Faculty and Students.

OBJECTIVES: Discrimination and bias in clinical training often take the form of microaggressions, which, albeit unintentional, are detrimental to the learning environment and well-being of students. Although there are a few reports of medical schools training students to respond to microaggressions, none have included a complementery student-led faculty training module. The aim of this study was to develop and evaluate a case-based approach to improving student resilience and increasing faculty awareness of microaggressions in the clinical setting. METHODS: We created four realistic cases of microaggressions and uncomfortable conversations, based on students' experiences on the wards, to implement training for incoming third-year students and their core faculty. Standardized patients were trained to effectively portray discriminatory faculty, residents, and patients. Institutional review board-approved surveys were administered and statistically analyzed to evaluate for efficacy. RESULTS: Students had greater mean confidence scores for responding to microaggressions immediately and at 6 months after the sessions (P < 0.05). Faculty showed improved mean confidence and understanding of the definition of a microaggression (P < 0.05). CONCLUSIONS: This approach had results similar to other studies, with the additional benefit of training faculty with the same scenarios. We believe that this method helped bridge the gap between students' notions of discrimination and faculty understanding of microaggressions.

Association of Pathogenic Variants in Hereditary Cancer Genes With Multiple Diseases.

Importance: Knowledge about the spectrum of diseases associated with hereditary cancer syndromes may improve disease diagnosis and management for patients and help to identify high-risk individuals. Objective: To identify phenotypes associated with hereditary cancer genes through a phenome-wide association study. Design, Setting, and Participants: This phenome-wide association study used health data from participants in 3 cohorts. The Electronic Medical Records and Genomics Sequencing (eMERGEseq) data set recruited predominantly healthy individuals from 10 US medical centers from July 16, 2016, through February 18, 2018, with a mean follow-up through electronic health records (EHRs) of 12.7 (7.4) years. The UK Biobank (UKB) cohort recruited participants from March 15, 2006, through August 1, 2010, with a mean (SD) follow-up of 12.4 (1.0) years. The Hereditary Cancer Registry (HCR) recruited patients undergoing clinical genetic testing at Vanderbilt University Medical Center from May 1, 2012, through December 31, 2019, with a mean (SD) follow-up through EHRs of 8.8 (6.5) years. Exposures: Germline variants in 23 hereditary cancer genes. Pathogenic and likely pathogenic variants for each gene were aggregated for association analyses. Main Outcomes and Measures: Phenotypes in the eMERGEseq and HCR cohorts were derived from the linked EHRs. Phenotypes in UKB were from multiple sources of health-related data. Results: A total of 214 020 participants were identified, including 23 544 in eMERGEseq cohort (mean [SD] age, 47.8 [23.7] years; 12 611 women [53.6%]), 187 234 in the UKB cohort (mean [SD] age, 56.7 [8.1] years; 104 055 [55.6%] women), and 3242 in the HCR cohort (mean [SD] age, 52.5 [15.5] years; 2851 [87.9%] women). All 38 established gene-cancer associations were replicated, and 19 new associations were identified. These included the following 7 associations with neoplasms: CHEK2 with leukemia (odds ratio [OR], 3.81 [95% CI, 2.64-5.48]) and plasma cell neoplasms (OR, 3.12 [95% CI, 1.84-5.28]), ATM with gastric cancer (OR, 4.27 [95% CI, 2.35-7.44]) and pancreatic cancer (OR, 4.44 [95% CI, 2.66-7.40]), MUTYH (biallelic) with kidney cancer (OR, 32.28 [95% CI, 6.40-162.73]), MSH6 with bladder cancer (OR, 5.63 [95% CI, 2.75-11.49]), and APC with benign liver/intrahepatic bile duct tumors (OR, 52.01 [95% CI, 14.29-189.29]). The remaining 12 associations with nonneoplastic diseases included BRCA1/2 with ovarian cysts (OR, 3.15 [95% CI, 2.22-4.46] and 3.12 [95% CI, 2.36-4.12], respectively), MEN1 with acute pancreatitis (OR, 33.45 [95% CI, 9.25-121.02]), APC with gastritis and duodenitis (OR, 4.66 [95% CI, 2.61-8.33]), and PTEN with chronic gastritis (OR, 15.68 [95% CI, 6.01-40.92]). Conclusions and Relevance: The findings of this genetic association study analyzing the EHRs of 3 large cohorts suggest that these new phenotypes associated with hereditary cancer genes may facilitate early detection and better management of cancers. This study highlights the potential benefits of using EHR data in genomic medicine.

A Comprehensive Immune Cell Atlas of Cystic Kidney Disease Reveals the Involvement of Adaptive Immune Cells in Injury-Mediated Cyst Progression in Mice.

BACKGROUND: Inducible disruption of cilia-related genes in adult mice results in slowly progressive cystic disease, which can be greatly accelerated by renal injury. METHODS: To identify in an unbiased manner modifier cells that may be influencing the differential rate of cyst growth in injured versus non-injured cilia mutant kidneys at a time of similar cyst severity, we generated a single-cell atlas of cystic kidney disease. We conducted RNA-seq on 79,355 cells from control mice and adult-induced conditional Ift88 mice (hereafter referred to as cilia mutant mice) that were harvested approximately 7 months post-induction or 8 weeks post 30-minute unilateral ischemia reperfusion injury. RESULTS: Analyses of single-cell RNA-seq data of CD45+ immune cells revealed that adaptive immune cells differed more in cluster composition, cell proportion, and gene expression than cells of myeloid origin when comparing cystic models with one another and with non-cystic controls. Surprisingly, genetic deletion of adaptive immune cells significantly reduced injury-accelerated cystic disease but had no effect on cyst growth in non-injured cilia mutant mice, independent of the rate of cyst growth or underlying genetic mutation. Using NicheNet, we identified a list of candidate cell types and ligands that were enriched in injured cilia mutant mice compared with aged cilia mutant mice and non-cystic controls that may be responsible for the observed dependence on adaptive immune cells during injury-accelerated cystic disease. CONCLUSIONS: Collectively, these data highlight the diversity of immune cell involvement in cystic kidney disease.

Intimate partner violence: analysis of current screening practices in the primary care setting.

BACKGROUND: Intimate partner violence (IPV) is a largely undetected occurrence in the United States reported by 36-50% of women in their lifetime and associated with extensive physical and psychological implications. Currently, conflicting recommendations exist regarding screening practices with the United States Preventative Services Task Force (USPSTF) and the World Health Organization (WHO) advocating for and against universal screening, respectively. With research suggesting that women are seldom asked about IPV during clinic visits, further information is needed regarding current screening practices. OBJECTIVES: To identify current IPV screening approaches in the primary care setting and factors that may impact screening completion. METHODS: We retrospectively examined patients presenting for annual examinations at four university-associated primary care clinics in southeast Florida (n = 400). Patient demographics, screener demographics, screening completion, and screening results were collected from the medical record. Results were compared to depression and anxiety screenings due to comparable prevalence and screening recommendations. Pearson chi square and Fisher exact tests were utilized to compare screening rates by demographic characteristics. RESULTS: IPV screening occurred at a much lower frequency (8.5%) compared to screenings for anxiety (37.3%) and depression (71.3%). Among documented IPV screenings, 64.7% of encounters resulted in patient refusal to be screened. Screening rates were found to be marginally impacted by patient ethnicity (P = 0.052). CONCLUSIONS: Findings of both low screening rates and low screening success raise significant concerns for the shortcomings of advocating for universal IPV screening. Therefore, additional studies are necessary to identify covert barriers to screening completion before universal inquiry is advised.

Intimate partner violence (IPV) is a largely undetected occurrence reported by 36­50% of women in their lifetime. Currently, IPV screening recommendations vary across organizations like the USPSTF and WHO. Given its widespread impact, we aimed to identify current IPV screening practices and gaps using patients who presented for annual examinations at four primary care clinics (n = 400). Patient demographics, screener demographics, screening completion, and screening results were collected from the medical record. IPV screening frequency was compared to frequency of depression and anxiety screenings given comparable screening recommendations and prevalence. IPV screening occurred less frequently (8.5%) compared to anxiety (37.3%) and depression (71.3%) screenings. Out of attempted IPV screens, 64.7% resulted in patient refusal to answer related questions. IPV screening rates were marginally impacted by patient ethnicity (P = 0.052). Our findings suggest shortcomings of universal screening and highlight the necessity to further elucidate barriers and improve screening efficacy.

Early infiltrating macrophage subtype correlates with late-stage phenotypic outcome in a mouse model of hepatorenal fibrocystic disease.

Hepatorenal fibrocystic disease (HRFCD) is a genetically inherited disorder related to primary cilia dysfunction in which patients display varying levels of fibrosis, bile duct expansion, and inflammation. In mouse models of HRFCD, the phenotype is greatly impacted by the genetic background in which the mutation is placed. Macrophages are a common factor associated with progression of HRFCD and are also strongly influenced by the genetic background. These data led us to hypothesize that macrophage subtypes that change in relation to the genetic background are responsible for the variable phenotypic outcomes in HRFCD. To test this hypothesis, we utilized a mouse model of HRFCD (Ift88Orpk mice) on the C57BL/6 and BALB/c inbred backgrounds that have well-documented differences in macrophage subtypes. Our analyses of infiltrating macrophage subtypes confirm that genetic strain influences the subtype of infiltrating macrophage present during normal postnatal liver development and in Ift88Orpk livers (Ly6clo in C57BL/6 vs Ly6chi in BALB/c). Each infiltrating macrophage subtype was similarly associated with a unique phenotypic outcome as analysis of liver tissue shows that C57BL/6 Ift88Orpk mice have increased bile duct expansion, but reduced levels of fibrosis compared to BALB/c Ift88Orpk livers. RNA sequencing data suggest that the ability to infiltrate macrophage subtypes to influence the phenotypic outcome may be due to unique ligand-receptor signaling between infiltrating macrophages and cilia dysfunctional biliary epithelium. To evaluate whether specific macrophage subtypes cause the observed phenotypic divergence, we analyzed the liver phenotype in BALB/c Ift88Orpk mice on a CCR2-/- background. Unexpectedly, the loss of Ly6chi macrophages, which were strongly enriched in BALB/c Ift88Orpk mice, did not significantly alter liver fibrosis. These data indicate that macrophage subtypes may correlate with HRFCD phenotypic outcome, but do not directly cause the pathology.

Returning Results in the Genomic Era: Initial Experiences of the eMERGE Network.

A goal of the 3rd phase of the Electronic Medical Records and Genomics (eMERGE3) Network was to examine the return of results (RoR) of actionable variants in more than 100 genes to consenting participants and their healthcare providers. Each of the 10 eMERGE sites developed plans for three essential elements of the RoR process: Disclosure to the participant, notification of the health care provider, and integration of results into the electronic health record (EHR). Procedures and protocols around these three elements were adapted as appropriate to individual site requirements and limitations. Detailed information about the RoR procedures at each site was obtained through structured telephone interviews and follow-up surveys with the clinical investigator leading or participating in the RoR process at each eMERGE3 institution. Because RoR processes at each of the 10 sites allowed for taking into account differences in population, disease focus and institutional requirements, significant heterogeneity of process was identified, including variability in the order in which patients and clinicians were notified and results were placed in the EHR. This heterogeneity in the process flow for eMERGE3 RoR reflects the "real world" of genomic medicine in which RoR procedures must be shaped by the needs of the patients and institutional environments.

Understanding the Return of Genomic Sequencing Results Process: Content Review of Participant Summary Letters in the eMERGE Research Network.

A challenge in returning genomic test results to research participants is how best to communicate complex and clinically nuanced findings to participants in a manner that is scalable to the large numbers of participants enrolled. The purpose of this study was to examine the features of genetic results letters produced at each Electronic Medical Records and Genomics (eMERGE3) Network site to assess their readability and content. Letters were collected from each site, and a qualitative analysis of letter content and a quantitative analysis of readability statistics were performed. Because letters were produced independently at each eMERGE site, significant heterogeneity in readability and content was found. The content of letters varied widely from a baseline of notifying participants that results existed to more detailed information about positive or negative results, as well as materials for sharing with family members. Most letters were significantly above the Centers for Disease Control-suggested reading level for health communication. While continued effort should be applied to make letters easier to understand, the ongoing challenge of explaining complex genomic information, the implications of negative test results, and the uncertainty that comes with some types of test and result makes simplifying letter text challenging.

Deletion of a 19-Amino-Acid Region in Clostridioides difficile TcdB2 Results in Spontaneous Autoprocessing and Reduced Cell Binding and Provides a Nontoxic Immunogen for Vaccination.

Clostridioides difficile toxin B (TcdB) is an intracellular toxin responsible for many of the pathologies of C. difficile infection. The two variant forms of TcdB (TcdB1 and TcdB2) share 92% sequence identity but have reported differences in rates of cell entry, autoprocessing, and overall toxicity. This 2,366-amino-acid, multidomain bacterial toxin glucosylates and inactivates small GTPases in the cytosol of target cells, ultimately leading to cell death. Successful cell entry and intoxication by TcdB are known to involve various conformational changes in the protein, including a proteolytic autoprocessing event. Previous studies found that amino acids 1753 to 1852 influence the conformational states of the proximal carboxy-terminal domain of TcdB and could contribute to differences between TcdB1 and TcdB2. In the current study, a combination of approaches was used to identify sequences within the region from amino acids 1753 to 1852 that influence the conformational integrity and cytotoxicity of TcdB2. Four deletion mutants with reduced cytotoxicity were identified, while one mutant, TcdB2Δ1769-1787, exhibited no detectable cytotoxicity. TcdB2Δ1769-1787 underwent spontaneous autoprocessing and was unable to interact with CHO-K1 or HeLa cells, suggesting a potential change in the conformation of the mutant protein. Despite the putative alteration in structural stability, vaccination with TcdB2Δ1769-1787 induced a TcdB2-neutralizing antibody response and protected against C. difficile disease in a mouse model. These findings indicate that the 19-amino-acid region spanning residues 1769 to 1787 in TcdB2 is crucial to cytotoxicity and the structural regulation of autoprocessing and that TcdB2Δ1769-1787 is a promising candidate for vaccination.

A prospective observational study of the impact of an electronic questionnaire (ePAQ-PO) on the duration of nurse-led pre-operative assessment and patient satisfaction.

OBJECTIVE: Standard pre-operative assessment at our institution involves a comprehensive history and examination by a nurse practitioner. An electronic pre-operative assessment questionnaire, ePAQ-PO® (ePAQ, Sheffield, UK) has previously been developed and validated. This study aimed to determine the impact of ePAQ-PO on nurse consultation times and patient satisfaction in low-risk patients. METHODS: The duration of pre-operative assessment consultation was recorded for American Society of Anesthesiology physical classification 1 and 2 patients undergoing pre-operative assessment by an electronic questionnaire (ePAQ-PO group) and standard face-to-face assessment by a nurse practitioner (standard group). Patients were also asked to complete an eight-item satisfaction questionnaire. Eighty-six patients were included (43 in each group). RESULTS: After adjusting for the duration of physical examination, median (IQR [min-max]) consultation time was longer in the standard compared to the ePAQ-PO group (25 (18-33 [10-49]) min vs. 12 (8-17 [4-45]) min, respectively; p <0.001). Response rate for the satisfaction questionnaire was 93%. There was no significant difference in patient satisfaction scores (38/39 in standard group vs. 39/41 in ePAQ-PO group were fully satisfied with their pre-operative assessment; p = 0.494). CONCLUSION: Pre-operative assessment using ePAQ-PO is associated with a significant reduction of over 50% in the duration of the assessment without impacting on patient satisfaction.

Intrinsic Toxin-Derived Peptides Destabilize and Inactivate Clostridium difficile TcdB.

Clostridium difficile infection (CDI) is a major cause of hospital-associated, antibiotic-induced diarrhea, which is largely mediated by the production of two large multidomain clostridial toxins, TcdA and TcdB. Both toxins coordinate the action of specific domains to bind receptors, enter cells, and deliver a catalytic fragment into the cytosol. This results in GTPase inactivation, actin disassembly, and cytotoxicity. TcdB in particular has been shown to encode a region covering amino acids 1753 to 1851 that affects epitope exposure and cytotoxicity. Surprisingly, studies here show that several peptides derived from this region, which share the consensus sequence 1769NVFKGNTISDK1779, protect cells from the action of TcdB. One peptide, PepB2, forms multiple interactions with the carboxy-terminal region of TcdB, destabilizes TcdB structure, and disrupts cell binding. We further show that these effects require PepB2 to form a higher-order polymeric complex, a process that requires the central GN amino acid pair. These data suggest that TcdB1769-1779 interacts with repeat sequences in the proximal carboxy-terminal domain of TcdB (i.e., the CROP domain) to alter the conformation of TcdB. Furthermore, these studies provide insights into TcdB structure and functions that can be exploited to inactivate this critical virulence factor and ameliorate the course of CDI.IMPORTANCEClostridium difficile is a leading cause of hospital-associated illness that is often associated with antibiotic treatment. To cause disease, C. difficile secretes toxins, including TcdB, which is a multidomain intracellular bacterial toxin that undergoes conformational changes during cellular intoxication. This study describes the development of peptide-based inhibitors that target a region of TcdB thought to be critical for structural integrity of the toxin. The results show that peptides derived from a structurally important region of TcdB can be used to destabilize the toxin and prevent cellular intoxication. Importantly, this work provides a novel means of toxin inhibition that could in the future develop into a C. difficile treatment.

Guideline for low-cost antimicrobial use in the outpatient setting.

In an effort to increase appropriate prescribing of low-cost antimicrobials in the outpatient setting, an evidence-based guideline was created to identify situations when low-cost medications can be used. A literature search identified relevant clinical trials describing the efficacy of antimicrobials used in the outpatient setting. These were analyzed to identify low-cost medications defined as $15 or less. The information was put into guideline format that includes the level of evidence for recommending the drug and information about cost. Sixteen common infections and their treatments were included in the guideline. The efficacy data were similar for the low-cost and higher-cost antimicrobials for all infections included. We created a low-cost antimicrobial guideline for common infections treated in the outpatient setting. The treatment options have similar efficacy to higher cost medications. This guideline will serve as an information source for providers to help them rapidly determine the low-cost treatments for common infections. In addition, it can serve as a template for the development of similar guidelines in other therapeutic classes. These guidelines should be customized before implementation at other health care organizations, with consideration of local resistance patterns, drug availability and patient factors. The effect of guideline implementation on future prescribing habits and providers' opinions about availability of cost information and subsequent conversations with patients and prescribers of medications deserves further study.

T-cell stimulating protein A (TspA) of Neisseria meningitidis is required for optimal adhesion to human cells.

T-cell stimulating protein A (TspA) is an immunogenic, T-cell and B-cell stimulating protein of Neisseria meningitidis. Sequence similarity between TspA and FimV, a Pseudomonas aeruginosa protein involved in twitching motility, suggested a link between TspA and type IV pili (Tfp). To determine the role of TspA an isogenic deletion mutant was created. Loss of TspA did not affect twitching motility or piliation indicating that there are functional differences between TspA and FimV. Mutation of tspA led to a significant reduction in adhesion of meningococci to meningothelial and HEp-2 cells, which was not due to a lack of transcription of adjacent genes or pilC1. Other Tfp-mediated phenotypes (i.e. auto-aggregation and transformation competence) were not altered. Our results indicate that the role of TspA in adhesion is unlikely to be directly linked to the function of Tfp. TspA was expressed by all N. meningitidis and Neisseria polysaccharea strains examined but not by Neisseria gonorrhoeae or Neisseria lactamica, although sequences with homology to tspA were present in their genomes. In summary, TspA is a highly conserved antigen that is required for optimal adhesion of meningococci to human cells.