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AbstractAltered bone morphogenetic protein (BMP) signaling is associated with many musculoskeletal diseases. However, it remains unknown whether BMP dysfunction has direct contribution to debilitating pain reported in many of these disorders. Here, we identified a novel neuropathic pain phenotype in patients with fibrodysplasia ossificans progressiva (FOP), a rare autosomal-dominant musculoskeletal disorder characterized by progressive heterotopic ossification. Ninety-seven percent of these patients carry an R206H gain-of-function point mutation in the BMP type I receptor ACVR1 (ACVR1 R206H ), which causes neofunction to Activin A and constitutively activates signaling through phosphorylated SMAD1/5/8. Although patients with FOP can harbor pathological lesions in the peripheral and central nervous system, their etiology and clinical impact are unclear. Quantitative sensory testing of patients with FOP revealed significant heat and mechanical pain hypersensitivity. Although there was no major effect of ACVR1 R206H on differentiation and maturation of nociceptive sensory neurons (iSNs) derived from FOP induced pluripotent stem cells, both intracellular and extracellular electrophysiology analyses of the ACVR1 R206H iSNs displayed ACVR1-dependent hyperexcitability, a hallmark of neuropathic pain. Consistent with this phenotype, we recorded enhanced responses of ACVR1 R206H iSNs to TRPV1 and TRPA1 agonists. Thus, activated ACVR1 signaling can modulate pain processing in humans and may represent a potential target for pain management in FOP and related BMP pathway diseases.

IntroductionReproductive injustices such as forced sterilization, preventable maternal morbidity and mortality, restricted access to family planning services, and policy-driven environmental violence undermine reproductive autonomy and health outcomes, with disproportionate impact on historically marginalized communities. However, curricula focused on reproductive justice (RJ) are lacking in medical education.MethodsWe designed a novel, interactive, case-based RJ curriculum for postclerkship medical students. This curriculum was created using published guidelines on best practices for incorporating RJ in medical education. The session included a prerecorded video on the history of RJ, an article, and four interactive cases. Students engaged in a 2-hour small-group session, discussing key learning points of each case. We evaluated the curriculum's impact with a pre- and postsurvey and focus group.ResultsSixty-eight students participated in this RJ curriculum in October 2020 and March 2021. Forty-one percent of them completed the presurvey, and 46% completed the postsurvey. Twenty-two percent completed both surveys. Ninety percent of respondents agreed that RJ was relevant to their future practice, and 87% agreed that participating in this session would impact their clinical practice. Most respondents (81%) agreed that more RJ content is needed. Focus group participants appreciated the case-based, interactive format and the intersectionality within the cases.DiscussionThis interactive curriculum is an innovative and effective way to teach medical students about RJ and its relevance to clinical practice. Walking alongside patients as they accessed reproductive health care in a case-based curriculum improved students' comfort and self-reported knowledge on several RJ topics.

BackgroundInflammation helps regulate normal growth and tissue repair. Although bone morphogenetic proteins (BMPs) and inflammation are known contributors to abnormal bone formation, how these pathways interact in ossification remains unclear.MethodsWe examined this potential link in patients with fibrodysplasia ossificans progressiva (FOP), a genetic condition of progressive heterotopic ossification caused by activating mutations in the Activin A type I receptor (ACVR1/ALK2). FOP patients show exquisite sensitivity to trauma, suggesting that BMP pathway activation may alter immune responses. We studied primary blood, monocyte, and macrophage samples from control and FOP subjects using multiplex cytokine, gene expression, and protein analyses; examined CD14+ primary monocyte and macrophage responses to TLR ligands; and assayed BMP, TGF-β activated kinase 1 (TAK1), and NF-κB pathways.ResultsFOP subjects at baseline without clinically evident heterotopic ossification showed increased serum IL-3, IL-7, IL-8, and IL-10. CD14+ primary monocytes treated with the TLR4 activator LPS showed increased CCL5, CCR7, and CXCL10; abnormal cytokine/chemokine secretion; and prolonged activation of the NF-κB pathway. FOP macrophages derived from primary monocytes also showed abnormal cytokine/chemokine secretion, increased TGF-β production, and p38MAPK activation. Surprisingly, SMAD phosphorylation was not significantly changed in the FOP monocytes/macrophages.ConclusionsAbnormal ACVR1 activity causes a proinflammatory state via increased NF-κB and p38MAPK activity. Similar changes may contribute to other types of heterotopic ossification, such as in scleroderma and dermatomyositis; after trauma; or with recombinant BMP-induced bone fusion. Our findings suggest that chronic antiinflammatory treatment may be useful for heterotopic ossification.

BackgroundNon-integrating episomal vectors have become an important tool for induced pluripotent stem cell reprogramming. The episomal vectors carrying the "Yamanaka reprogramming factors" (Oct4, Klf, Sox2, and L-Myc + Lin28) are critical tools for non-integrating reprogramming of cells to a pluripotent state. However, the reprogramming process remains highly stochastic, and is hampered by an inability to easily identify clones that carry the episomal vectors.MethodsWe modified the original set of vectors to express spectrally separable fluorescent proteins to allow for enrichment of transfected cells. The vectors were then tested against the standard original vectors for reprogramming efficiency and for the ability to enrich for stoichiometric ratios of factors.ResultsThe reengineered vectors allow for cell sorting based on reprogramming factor expression. We show that these vectors can assist in tracking episomal expression in individual cells and can select the reprogramming factor dosage.ConclusionsTogether, these modified vectors are a useful tool for understanding the reprogramming process and improving induced pluripotent stem cell isolation efficiency.

Macrophages have a key role in shaping the tumour microenvironment (TME), tumour immunity and response to immunotherapy, which makes them an important target for cancer treatment.sup.1,2. However, modulating macrophages has proved extremely difficult, as we still lack a complete understanding of the molecular and functional diversity of the tumour macrophage compartment. Macrophages arise from two distinct lineages. Tissue-resident macrophages self-renew locally, independent of adult haematopoiesis.sup.3-5, whereas short-lived monocyte-derived macrophages arise from adult haematopoietic stem cells, and accumulate mostly in inflamed lesions.sup.1. How these macrophage lineages contribute to the TME and cancer progression remains unclear. To explore the diversity of the macrophage compartment in human non-small cell lung carcinoma (NSCLC) lesions, here we performed single-cell RNA sequencing of tumour-associated leukocytes. We identified distinct populations of macrophages that were enriched in human and mouse lung tumours. Using lineage tracing, we discovered that these macrophage populations differ in origin and have a distinct temporal and spatial distribution in the TME. Tissue-resident macrophages accumulate close to tumour cells early during tumour formation to promote epithelial-mesenchymal transition and invasiveness in tumour cells, and they also induce a potent regulatory T cell response that protects tumour cells from adaptive immunity. Depletion of tissue-resident macrophages reduced the numbers and altered the phenotype of regulatory T cells, promoted the accumulation of CD8.sup.+ T cells and reduced tumour invasiveness and growth. During tumour growth, tissue-resident macrophages became redistributed at the periphery of the TME, which becomes dominated by monocyte-derived macrophages in both mouse and human NSCLC. This study identifies the contribution of tissue-resident macrophages to early lung cancer and establishes them as a target for the prevention and treatment of early lung cancer lesions. Single-cell RNA sequencing and imaging of macrophages in human non-small cell lung cancer and in a mouse model of lung adenocarcinoma show that tissue-resident macrophages have a key role in early tumour progression., Author(s): María Casanova-Acebes [sup.1] [sup.2] [sup.3] [sup.19] , Erica Dalla [sup.4] [sup.5] [sup.6] [sup.7] , Andrew M. Leader [sup.1] [sup.2] [sup.3] , Jessica LeBerichel [sup.1] [sup.2] [sup.3] , Jovan Nikolic [...]

PurposeTo compare the recovery of erections and potency following the transection of accessory pudendal arteries (APAs) in men undergoing robot-assisted radical prostatectomy (RARP) compared with men with normal vascular anatomy.Materials and methodsA total of 880 consecutive patients who underwent RARP from January 1, 2007 to December 31, 2014 were included with prospectively collected data in cross-sectional analysis. Erectile function (EF) was assessed preoperatively and postoperatively at 3, 6, 12, and 24 months using the International Index of Erectile Function (IIEF)-5, a percent erection fullness compared to preoperative status, and two Expanded Prostate Cancer Index (EPIC) questions: (1) are erections firm enough for penetration and (2) are they satisfactory?ResultsTwo hundred thirty-one (33.1%) men had APAs transected. There were no significant differences in baseline demographics or clinical characteristics in men with or without APAs transected. Multivariate analyses demonstrated that age (confidence interval [95% CI]: 0.94, 0.99) and baseline IIEF-5 (95% CI: 1.15, 1.26) strongly correlated with recovery of erections and potency. Transection of APAs was not a significant predictor of erectile dysfunction (ED).ConclusionGood surgical technique dictates the preservation of APAs. However, when preservation is questioned, we found that APA transection had no measurable effect on recovery of erections or potency regardless of age, preoperative ED, or number of APAs transected.

ObjectiveTo study the combination of thermal magnetic resonance imaging (MRI) and novel hypothermic cooling, via an endorectal cooling balloon (ECB), to assess the effective dispersion and temperature drop in pelvic tissue to potentially reduce inflammatory cascade in surgical applications.MethodsThree male subjects, before undergoing robot-assisted radical prostatectomy, were cooled via an ECB, rendered MRI compatible for patient safety before ECB hypothermia. MRI studies were performed using a 3T scanner and included T2-weighted anatomic scan for the pelvic structures, followed by a temperature mapping scan. The sequence was performed repeatedly during the cooling experiment, whereas the phase data were collected using an integrated MR-high-intensity focused ultrasound workstation in real time. Pelvic cooling was instituted with a cooling console located outside the MRI magnet room.ResultsThe feasibility of pelvic cooling measured a temperature drop of the ECB of 20-25 degrees in real time was achieved after an initial time delay of 10-15 seconds for the ECB to cool. The thermal MRI anatomic images of the prostate and neurovascular bundle demonstrate cooling at this interface to be 10-15 degrees, and also that cooling extends into the prostate itself ~5 degrees, and disperses into the pelvic region as well.ConclusionAn MRI-compatible ECB coupled with thermal MRI is a feasible method to assess effective hypothermic diffusion and saturation to pelvic structures. By inference, hypothermia-induced rectal cooling could potentially reduce inflammation, scarring, and fistula in radical prostatectomy, as well as other urologic tissue procedures of high-intensity focused ultrasound, external beam radiation therapy, radioactive seed implants, transurethral microwave therapy, and transurethral resection of the prostate.

ObjectiveTo assist in preoperative counseling by assessing long-term changes in American Urological Association symptom scores (AUAss) and lower urinary tract symptom (LUTS)-related quality of life (QOL) in patients undergoing robot-assisted radical prostatectomy (RARP).Materials and methodsRARP was performed on 666 men by one surgeon from 2002 to 2007 at a single institution. AUAss and QOL were queried preoperatively and at 3, 9, 15, 24-48, 60-84, and 96+ months postoperatively. LUTS subgroups were compared pre-/postsurgery using univariate and multivariate statistics.ResultsThe mean and median follow-up for all responders was 3.0 and 2.4 years. Pad-free continence at 12 months was 89%. A subset of 174 men reported preoperative and long-term responses; average follow-up was 5.8 years (range 4.0-10.3 years). AUAss for all men declined from baseline to 5 years by 3.7 (8.6 to 4.8) whereas QOL/Bother scores decreased by 0.5 (1.7 to 1.2) (all P

Objectives: To investigate monocyte chemotactic protein-1 (MCP-1) as a novel urinary biomarker to predict prolonged post prostatectomy incontinence. Methods: Men submitted urine samples prior to robotic radical prostatectomy. MCP-1 values were derived using an ELISA test. Pad usage at 7, 30, and 60 days were documented by patient post cards mailed when zero pads was reached. The primary outcome was defined as no incontinence pad usage at 30 days at prostatectomy. Results: After exclusions, 76 patients were included in analyses. Continence was reached by 29% (22/76), 56% (42/76), and (75/76) 98% at 7, 30, and 60 days, respectively. The average MCP-1 (p=0.258) was not different between the continent and incontinent groups. Highest quartile of MCP-1 (MCP > 166 pg/mL) and normalized MCP-1 (MCP-1/TV >0.53) noted a significant delay in continence at 30 days (p=0.050 and p=0.003). Only 26% (5/19) in the highest MCP1/TV quartile were continent, whereas 65% (37/57) of men in the 3 lower quartiles reached zero pad continence (p=0.003). In a logistic regression model the highest quartile of MCP1/TV had a significant chance of being incontinent at 30 days (OR 0.22; 95% CI 0.058-0.80; p=0.022). Conclusion: MCP-1/TV is a urinary biomarker that may predict prolonged urinary incontinence after radical prostatectomy.

ObjectivesWe investigated urinary nerve growth factor (NGF) as a novel urinary biomarker for high-grade prostate cancer (PCa).Methods and materialsAfter institutional review board approval for a prospective pilot study, we enrolled men at the preoperative visit before robotic-assisted radical prostatectomy. Demographics, urinary flow parameters, and urine samples were collected. Urinary NGF and urinary creatinine were obtained in the translational science laboratory. Pathologic and postoperative demographics were collected after surgery. NGF is the primary outcome variable (dependent variable). The pathologic Gleason score (ordinal variable ≤6, 7, and ≤8) served as an independent grouping variable. Multivariate analysis using a general linear model was conducted to investigate associations between independent variables and NGF (dependent variable) after adjusting for urinary concentration and volume.ResultsWe enrolled and analyzed urine samples and pathologic data from 115 subjects. Patient pathology included 24% (n = 28) Gleason score 6 or less, 68% (n = 78) Gleason score 7, and 8% (n = 9) Gleason score 8 or greater. Perineural invasion was more prevalent in higher-grade disease (P