Recirculating Intestinal IgA-Producing Cells Regulate Neuroinflammation via IL-10.

Plasma cells (PC) are found in the CNS of multiple sclerosis (MS) patients, yet their source and role in MS remains unclear. We find that some PC in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) originate in the gut and produce immunoglobulin A (IgA). Moreover, we show that IgA+ PC are dramatically reduced in the gut during EAE, and likewise, a reduction in IgA-bound fecal bacteria is seen in MS patients during disease relapse. Removal of plasmablast (PB) plus PC resulted in exacerbated EAE that was normalized by the introduction of gut-derived IgA+ PC. Furthermore, mice with an over-abundance of IgA+ PB and/or PC were specifically resistant to the effector stage of EAE, and expression of interleukin (IL)-10 by PB plus PC was necessary and sufficient to confer resistance. Our data show that IgA+ PB and/or PC mobilized from the gut play an unexpected role in suppressing neuroinflammation.

A Hematogenous Route for Medulloblastoma Leptomeningeal Metastases.

While the preponderance of morbidity and mortality in medulloblastoma patients are due to metastatic disease, most research focuses on the primary tumor due to a dearth of metastatic tissue samples and model systems. Medulloblastoma metastases are found almost exclusively on the leptomeningeal surface of the brain and spinal cord; dissemination is therefore thought to occur through shedding of primary tumor cells into the cerebrospinal fluid followed by distal re-implantation on the leptomeninges. We present evidence for medulloblastoma circulating tumor cells (CTCs) in therapy-naive patients and demonstrate in vivo, through flank xenografting and parabiosis, that medulloblastoma CTCs can spread through the blood to the leptomeningeal space to form leptomeningeal metastases. Medulloblastoma leptomeningeal metastases express high levels of the chemokine CCL2, and expression of CCL2 in medulloblastoma in vivo is sufficient to drive leptomeningeal dissemination. Hematogenous dissemination of medulloblastoma offers a new opportunity to diagnose and treat lethal disseminated medulloblastoma.

Colony stimulating factor-1 producing endothelial cells and mesenchymal stromal cells maintain monocytes within a perivascular bone marrow niche.

Macrophage colony stimulating factor-1 (CSF-1) plays a critical role in maintaining myeloid lineage cells. However, congenital global deficiency of CSF-1 (Csf1op/op) causes severe musculoskeletal defects that may indirectly affect hematopoiesis. Indeed, we show here that osteolineage-derived Csf1 prevented developmental abnormalities but had no effect on monopoiesis in adulthood. However, ubiquitous deletion of Csf1 conditionally in adulthood decreased monocyte survival, differentiation, and migration, independent of its effects on bone development. Bone histology revealed that monocytes reside near sinusoidal endothelial cells (ECs) and leptin receptor (Lepr)-expressing perivascular mesenchymal stromal cells (MSCs). Targeted deletion of Csf1 from sinusoidal ECs selectively reduced Ly6C- monocytes, whereas combined depletion of Csf1 from ECs and MSCs further decreased Ly6Chi cells. Moreover, EC-derived CSF-1 facilitated recovery of Ly6C- monocytes and protected mice from weight loss following induction of polymicrobial sepsis. Thus, monocytes are supported by distinct cellular sources of CSF-1 within a perivascular BM niche.

CCL19-CCR7-dependent reverse transendothelial migration of myeloid cells clears Chlamydia muridarum from the arterial intima.

Regions of the normal arterial intima predisposed to atherosclerosis are sites of ongoing monocyte trafficking and also contain resident myeloid cells with features of dendritic cells. However, the pathophysiological roles of these cells are poorly understood. Here we found that intimal myeloid cells underwent reverse transendothelial migration (RTM) into the arterial circulation after systemic stimulation of pattern-recognition receptors (PRRs). This process was dependent on expression of the chemokine receptor CCR7 and its ligand CCL19 by intimal myeloid cells. In mice infected with the intracellular pathogen Chlamydia muridarum, blood monocytes disseminated infection to the intima. Subsequent CCL19-CCR7-dependent RTM was critical for the clearance of intimal C. muridarum. This process was inhibited by hypercholesterolemia. Thus, RTM protects the normal arterial intima, and compromised RTM during atherogenesis might contribute to the intracellular retention of pathogens in atherosclerotic lesions.

Self-renewing resident arterial macrophages arise from embryonic CX3CR1(+) precursors and circulating monocytes immediately after birth.

Resident macrophages densely populate the normal arterial wall, yet their origins and the mechanisms that sustain them are poorly understood. Here we use gene-expression profiling to show that arterial macrophages constitute a distinct population among macrophages. Using multiple fate-mapping approaches, we show that arterial macrophages arise embryonically from CX3CR1(+) precursors and postnatally from bone marrow-derived monocytes that colonize the tissue immediately after birth. In adulthood, proliferation (rather than monocyte recruitment) sustains arterial macrophages in the steady state and after severe depletion following sepsis. After infection, arterial macrophages return rapidly to functional homeostasis. Finally, survival of resident arterial macrophages depends on a CX3CR1-CX3CL1 axis within the vascular niche.

Off-target piRNA gene silencing in Drosophila melanogaster rescued by a transposable element insertion.

Transposable elements (TE) are selfish genetic elements that can cause harmful mutations. In Drosophila, it has been estimated that half of all spontaneous visible marker phenotypes are mutations caused by TE insertions. Several factors likely limit the accumulation of exponentially amplifying TEs within genomes. First, synergistic interactions between TEs that amplify their harm with increasing copy number are proposed to limit TE copy number. However, the nature of this synergy is poorly understood. Second, because of the harm posed by TEs, eukaryotes have evolved systems of small RNA-based genome defense to limit transposition. However, as in all immune systems, there is a cost of autoimmunity and small RNA-based systems that silence TEs can inadvertently silence genes flanking TE insertions. In a screen for essential meiotic genes in Drosophila melanogaster, a truncated Doc retrotransposon within a neighboring gene was found to trigger the germline silencing of ald, the Drosophila Mps1 homolog, a gene essential for proper chromosome segregation in meiosis. A subsequent screen for suppressors of this silencing identified a new insertion of a Hobo DNA transposon in the same neighboring gene. Here we describe how the original Doc insertion triggers flanking piRNA biogenesis and local gene silencing. We show that this local gene silencing occurs in cis and is dependent on deadlock, a component of the Rhino-Deadlock-Cutoff (RDC) complex, to trigger dual-strand piRNA biogenesis at TE insertions. We further show how the additional Hobo insertion leads to de-silencing by reducing flanking piRNA biogenesis triggered by the original Doc insertion. These results support a model of TE-mediated gene silencing by piRNA biogenesis in cis that depends on local determinants of transcription. This may explain complex patterns of off-target gene silencing triggered by TEs within populations and in the laboratory. It also provides a mechanism of sign epistasis among TE insertions, illuminates the complex nature of their interactions and supports a model in which off-target gene silencing shapes the evolution of the RDC complex.

Intersectin-1 enhances calcium-dependent replenishment of the readily releasable pool of synaptic vesicles during development.

Intersectin-1 (Itsn1) is a scaffold protein that plays a key role in coupling exocytosis and endocytosis of synaptic vesicles (SVs). However, it is unclear whether and how Itsn1 regulates these processes to support efficient neurotransmission during development. To address this, we examined the calyx of Held synapse in the auditory brainstem of wild-type and Itsn1 mutant mice before (immature) and after (mature) the onset of hearing. Itsn1 was present in the pre- and postsynaptic compartments at both developmental stages. Loss of function of Itsn1 did not alter presynaptic action potentials, Ca2+ entry via voltage-gated Ca2+ channels (VGCCs), transmitter release or short-term depression (STD) induced by depletion of SVs in the readily releasable pool (RRP) in either age group. Yet, fast Ca2+-dependent recovery from STD was attenuated in mature mutant synapses, while it was unchanged in immature mutant synapses. This deficit at mature synapses was rescued by introducing the DH-PH domains of Itsn1 into the presynaptic terminals. Inhibition of dynamin, which interacts with Itsn1 during endocytosis, had no effect on STD recovery. Interestingly, we found a developmental enrichment of Itsn1 near VGCCs, which may underlie the Itsn1-mediated fast replenishment of the RRP. Consequently, the absence of Itsn1 in mature synapses led to a higher failure rate of postsynaptic spiking during high-frequency synaptic transmission. Taken together, our findings suggest that Itsn1 translocation to the vicinity of VGCCs during development is crucial for accelerating Ca2+-dependent RRP replenishment and sustaining high-fidelity neurotransmission. KEY POINTS: Itsn1 is expressed in the pre- and postsynaptic compartments of the calyx of Held synapse. Developmental upregulation of vesicular glutamate transporter-1 is Itsn1 dependent. Itsn1 does not affect basal synaptic transmission at different developmental stages. Itsn1 is required for Ca2+-dependent recovery from short-term depression in mature synapses. Itsn1 mediates the recovery through its DH-PH domains, independent of its interactive partner dynamin. Itsn1 translocates to the vicinity of presynaptic Ca2+ channels during development. Itsn1 supports high-fidelity neurotransmission by enabling rapid recovery from vesicular depletion during repetitive activity.

Assisted reproductive technology (ART) patient information-seeking behavior: a qualitative study.

BACKGROUND: Approximately 13% of women in the United States of reproductive age seek infertility services. Assisted reproductive technology (ART), including in vitro fertilization, is used to help patients achieve pregnancy. Many people are not familiar with these treatments prior to becoming patients and possess knowledge gaps about care. METHODS: This study employed qualitative methods to investigate how patients interact with information sources during care. Patients who underwent ART including embryo transfer between January 2017 and April 2022 at a large urban healthcare center were eligible. Semi-structured, in-depth interviews were conducted between August and October 2022. Fifteen females with an average age of 39 years participated. Reflexive thematic analysis was performed. RESULTS: Two main themes emerged. Participants (1) utilized clinic-provided information and then turned to outside sources to fill knowledge gaps; (2) struggled to learn about costs, insurance, and mental health resources to support care. Participants preferred clinic-provided resources and then utilized academic sources, the internet, and social media when they had unfulfilled information needs. Knowledge gaps related to cost, insurance, and mental health support were reported. CONCLUSION: ART clinics can consider providing more information about cost, insurance, and mental health support to patients. TRIAL REGISTRATION: The Massachusetts General Hospital Institutional Review Board approved this study (#2022P000474) and informed consent was obtained from each participant.

Building Healthy Babies: A Mixed-Methods Needs Assessment for a Pre-Conception Program in Ontario.

OBJECTIVES: The objective of this study was to gather Ontario clinicians' and public members' views on the design of a pre-conception patient education program. METHODS: In this mixed-methods study, online surveys comprised of rank order, multiple choice, and short answer questions were completed by clinicians and public members. Semi-structured focus groups consisting of 2-6 participants each were then held via videoconference. Demographic variables and survey responses were analyzed quantitatively using descriptive and summary statistics. Descriptive thematic qualitative analysis using the constant comparative method of grounded theory was completed on each transcript to generate themes. RESULTS: A total of 168 public members and 43 clinicians in Ontario completed surveys, while 11 clinicians and 11 public members participated in the focus groups. A pre-conception program in Ontario was felt to be important. An individual appointment with a primary care provider was the favoured program format per survey responses, whereas a virtual format with an interactive component was preferred among focus group participants. Important topics to include were pre-conception health (infertility, genetic screening, folic acid), prenatal and postpartum counselling (diet, activity, substance use, prenatal care, postpartum course), and medical optimization in pregnancy (high-risk medical conditions, medications, mental health). Both groups emphasized the need to consider accommodations for marginalized populations and various cultures and languages. CONCLUSION: A standardized pre-conception patient education program is felt to be of high value by Ontario clinicians and public members. A pre-conception program may help improve obstetrical outcomes and decrease rates of major congenital anomalies in Ontario.

Fetal brain development in pregnancies complicated by gestational diabetes mellitus.

OBJECTIVES: Gestational diabetes mellitus (GDM) carries an increased risk of neurocognitive impairment in offsprings. However, the contribution of maternal hyperglycemia in affecting fetal brain development is not fully elucidated yet. The aim of this study was to evaluate fetal brain and sulci development in pregnancies complicated by GDM. METHODS: Prospective observational study including 100 singleton pregnancies complicated by GDM and 100 matched controls. All fetuses underwent neurosonography at 29-34 weeks of gestation, including the assessment of the length of the corpus callosum (CC), cerebellar vermis (CV), Sylvian (SF), parieto-occipital (POF) and calcarine fissures (CF). Sub-group analysis according to the specific treatment regimen adopted (n 67 diet vs. 33 insulin therapy) was also performed. RESULTS: Fetuses from mothers with GDM under insulin therapy had a smaller CC (35.54 mm) compared to both controls (40 mm; p<0.001) and women with GDM under diet (39.26 mm; p=0.022) while there was no difference in the HC between the groups. Likewise, when corrected for HC, CV depth was smaller in fetuses with GDM both under insulin therapy (7.03 mm) and diet (7.05 mm,) compared to controls (7.36 mm; p=0.013). Finally, when assessing the sulci development of the brain SF (p≤0.0001), POF (p≤0.0001) and CF (p≤0.0001) were significantly smaller in fetuses with maternal GDM. Post-hoc analysis showed that fetuses of GDM mothers requiring insulin therapy had significantly lower values of SF (p=0.032), POF (p=0.016) and CF (p=0.001). CONCLUSIONS: Pregnancies complicated by GDM showed a peculiar pattern of fetal brain growth and cortical development and these changes, which are more evident in those requiring insulin supplementation.

Effects of gender on fetal cortical development: a secondary analysis of a prospective cross-sectional study.

OBJECTIVES: The primary objective was to evaluate the effects of fetal sex on fetal cortical development in low-risk pregnancies. Secondary objective was the evaluate the impact of gestational age. METHODS: This was a secondary analysis of a prospective cross-sectional study on low-risk fetuses undergoing fetal neurosonography between 19 and 34 weeks of gestation. The depth of Sylvian Fissure (SF), Parieto Occipital Fissure (POF) and Calcarine Fissure (CF) were evaluated and related to fetal sex. Neurosonographic variables were normalized for fetal head circumference and expressed as multiple of the median (MoM). RESULTS: A total of 344 fetuses were considered (173 male, 171 female). The baseline characteristic of the two groups were similar except a higher birthweight present in male fetuses (p=0.044). The depth SF (p=0.023) CF (p=0.014) and POF (p=0.046) showed significantly higher values in male fetuses when all the gestational age range was considered. However, when data were controlled for gestational age, these differences resulted significant only after 28 weeks. CONCLUSIONS: Differences in cortical development related to gender occur after 28 weeks of gestation with an increase depth of SF, POF and CF in male fetuses.

Validation of an automated software (Smartpelvic™) in assessing hiatal area from three dimensional transperineal pelvic volumes of pregnant women: comparison with manual analysis.

OBJECTIVES: The aim of this investigation was to evaluate the agreement between a manual and an automatic technique in assessing levator hiatus area (LHA) during pregnancy from three-dimensional (3D) pelvic floor volumes obtained by trans-perineal ultrasound (TPUS). METHODS: 3D volumes were acquired during rest, maximum pelvic floor contraction and Valsalva maneuver from 66 pregnant women. Manual selection of LHA and automatic software (Smart Pelvic™) were applied on TPUS volume starting from a C-plane view. To evaluate intra- and inter-observer variability measurements of LHA were performed twice by the same operator and once by a second sonographer. Reference hiatal contours obtained manually by the first operator were compared with the automated ones. Reproducibility was evaluated by intraclass correlation coefficients (ICC) and Bland-Altman plots. RESULTS: LHA measurement, using automatic software, achieved excellent intra-observer and inter-observer reproducibility in pregnant women both at rest and after dynamic analysis (ICC>0.9). Further, an excellent agreement resulted between manual selection of the LHA and automatic imaging (ICC>0.9). The average time taken to obtain LHA manually was significantly longer when compared to the automatic analysis (p≤0.0001). CONCLUSIONS: Smart pelvic software resulted from a reliable method for automatically measuring the LHA, showing high reproducibility and accuracy.

Opportunities for de-escalation of aspirin therapy in patients with atrial fibrillation at high stroke risk receiving direct oral anticoagulants.

OBJECTIVES: Atrial fibrillation (AF) is associated with increased risk of stroke that can be attenuated with newer anticoagulants, called direct oral anticoagulants (DOACs). Before the emergence of DOACs, warfarin or aspirin (ASA) was used for stroke prevention. Owing to the increased risk of bleed with concomitant anticoagulation therapy, populations that may benefit from ASA therapy are becoming limited. The primary objective of this study was to evaluate ASA utilization in an outpatient setting for patients with AF at high risk of stroke receiving a DOAC. The secondary objective was to evaluate what characteristics influence ASA use using a multivariate logistical regression model. DESIGN: This was a retrospective study conducted through electronic health record extraction between June 1, 2021, and May 31, 2022. SETTING AND PARTICIPANTS: Study sites included 219 outpatient Banner Health Facilities. A total of 5716 patients were included in the study. OUTCOME MEASURES: Patient characteristics and demographics, including CHA2DS2-VASc and HAS-BLED scores, were evaluated in adults 18 years and older with AF and an active DOAC prescription. RESULTS: There were 955 patients (16.7%) on ASA and 4761 patients (83.3%) not on ASA. Of the 955 patients on ASA, 33% (n = 315) did not have vascular disease. A total of 2289 patients had at least one vascular disease diagnosis. Of these patients, 28% (n = 640) were on ASA and 72% (n = 1649) were not on ASA. There were 142 patients with vascular disease who experienced a bleeding event with 36% of patients (n = 51) on ASA. Patients on ASA had a higher average CHA2DS2-VASc score (4.02 vs. 3.74) and HAS-BLED score (3.10 vs. 2.35) than patients not on ASA, respectively. CONCLUSION: This study found approximately one-third of patients with documented ASA use had no documentation of vascular disease and an unclear pattern of use in patients with documented vascular disease, suggesting opportunities to de-escalate ASA in patients with AF on a DOAC.

Determination and Characterization of Gold Nanoparticles in Liquor Using Asymmetric Flow Field-Flow Fractionation Hyphenated with Inductively Coupled Plasma Mass Spectrometry.

The EU has approved the usage of gold as a food additive (E175) and it has been applied in numerous foods for coloring and decoration purposes. Different from the general assumption that edible gold is mainly present in the form of flakes or external coating in foods, this work demonstrated that gold nanoparticles (Au NPs) can be released from gold flakes and extracted under optimized conditions. To support future risk assessment associated with the exposure of Au NPs to human health, an effective approach was established in this study for both size characterization and mass determination of Au NPs released in a commercial gold-containing liquor using Asymmetric Flow Field-flow Fractionation (AF4) hyphenated with Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Our results showed that no Au NPs were detected in the original liquor product and only after ultrasonication for several minutes did Au NPs occur in the ultrasound-treated liquor. Particularly, Au NPs released in the liquor can be well extracted after 100-fold enrichment of gold flakes and the subsequent ultrasonication for 25 min. Size characterization of Au NPs was conducted by AF4-ICP-MS under calibration with Au NP standards. The gold particle sizes detected ranged from 8.3-398.0 nm and the dominant size of the released Au NPs was around 123.7 nm in the processed liquor. The mass concentration of gold particles determined in the liquor sample with gold flakes concentrated and subsequently sonicated was 48.1 µg L-1 by pre-channel calibration and the overall detection recoveries ranged over 82-95%. For the comparison control samples without ultrasonication, there was no detection of Au NPs. The established method was demonstrated to be useful for monitoring Au NPs in liquor and is possibly applied to other similar foodstuffs.

Congenic hematopoietic stem cell transplantation promotes survival of heart allografts in murine models of acute and chronic rejection.

The ability to induce tolerance would be a major advance in the field of solid organ transplantation. Here, we investigated whether autologous (congenic) hematopoietic stem cell transplantation (HSCT) could promote tolerance to heart allografts in mice. In an acute rejection model, fully MHC-mismatched BALB/c hearts were heterotopically transplanted into C57BL/6 (CD45.2) mice. One week later, recipient mice were lethally irradiated and reconstituted with congenic B6 CD45.1 Lin-Sca1+ckit+ cells. Recipient mice received a 14-day course of rapamycin both to prevent rejection and to expand regulatory T cells (Tregs). Heart allografts in both untreated and rapamycin-treated recipients that did not undergo HSCT were rejected within 33 days (median survival time = 8 days for untreated recipients, median survival time = 32 days for rapamycin-treated recipients), whereas allografts in HSCT-treated recipients had a median survival time of 55 days (P < 0.001 vs. both untreated and rapamycin-treated recipients). Enhanced allograft survival following HSCT was associated with increased intragraft Foxp3+ Tregs, reduced intragraft B cells, and reduced serum donor-specific antibodies. In a chronic rejection model, Bm12 hearts were transplanted into C57BL/6 (CD45.2) mice, and congenic HSCT was performed two weeks following heart transplantation. HSCT led to enhanced survival of allografts (median survival time = 70 days vs. median survival time = 28 days in untreated recipients, P < 0.01). Increased allograft survival post-HSCT was associated with prevention of autoantibody development and absence of vasculopathy. These data support the concept that autologous HSCT can promote immune tolerance in the setting of allotransplantation. Further studies to optimize HSCT protocols should be performed before this procedure is adopted clinically.

Modeling fetal cortical development by quantile regression for gestational age and head circumference: a prospective cross sectional study.

OBJECTIVES: To develop charts for fetal brain cortical structures following a proposed standardized methodology and using quantile regression. METHODS: Prospective cross-sectional study including 344 low-risk singleton pregnancies between 19 and 34 weeks of gestation. The depth of Sylvian (SF), Parieto-occipital (POF) and Calcarine fissures (CF) were measured on ultrasound images using a standardized technique and their changes were evaluated by quantile regression as a function of gestational age (GA) interval or head circumference (HC). RESULTS: The measurements of SF, POF and CF depth significantly increased with gestation. Linear models better described the changes of cortical variables with GA and HC. When the fit of sulci depth with GA and HC were compared, a close relationship was highlighted for the latter variable. CONCLUSIONS: We provided prospective charts of fetal cortical development using quantile regression and following a strict standardized methodology These new charts may help in better identifying cases at higher risk of abnormal cortical neurodevelopment.

Validation of an automatic software in assessing fetal brain volume from three dimensional ultrasonographic volumes: Comparison with manual analysis.

OBJECTIVE: This study was aimed to test the agreement between a manual and an automatic technique in measuring fetal brain volume (FBV) from three-dimensional (3D) fetal head datasets. METHODS: FBV were acquired independently by two operators from low risk singleton pregnancies at a gestational age between 19 and 34 weeks. FBV measurements were obtained using an automatic software (Smart ICV™) and manually by Virtual Organ Computer-aided AnaLysis (VOCAL™). Intraclass correlation coefficient (ICC) were calculated to assess reliability, while bias and agreement were evaluate by examining Bland-Altman plots. The time spent in measuring volumes was calculated and values obtained compared. RESULTS: Sixty-three volumes were considered for the study. In all the included volumes successful volume analysis were obtained with both techniques. Smart ICV™ showed a high intra-observer (0.996; 95% CI 0.994-0.998) and inter-observer (ICC 0.995; 95% CI 0.991-0.997). An excellent degree of reliability was found when the two techniques were compared (ICC 0.995; 95% CI 0.987-0.998). The time required to perform FBV was significantly lower for Smart ICV™ than VOCAL™ (8.2 ± 4.5 vs. 121.3 ± 19.0 s; p < 0.0001). CONCLUSIONS: The measurement of FBV is feasible with both manual and automatic techniques. Smart ICV™ showed an excellent intra- and inter-observer reliability associated with a valuable agreement with volume measurements obtained manually with VOCAL™. Volumes may be measured significantly faster with smart ICV™ than manually and this automatic software has the potential to become the preferred methods for the assessment of FBV.