The ubiquitin ligases Cbl and Cbl-b regulate macrophage growth by controlling CSF-1R import into macropinosomes.

The ubiquitination of transmembrane receptors regulates endocytosis, intracellular traffic, and signal transduction. Bone marrow-derived macrophages from myeloid Cbl-/- and Cbl-b-/- double knockout (DKO) mice display sustained proliferation mirroring the myeloproliferative disease that these mice succumb to. Here, we found that the ubiquitin ligases Cbl and Cbl-b have overlapping functions for controlling the endocytosis and intracellular traffic of the CSF-1R. DKO macrophages displayed complete loss of ubiquitination of the CSF-1R whereas partial ubiquitination was observed for either single Cbl-/- or Cbl-b-/- macrophages. Unlike wild type, DKO macrophages were immortal and displayed slower CSF-1R internalization, elevated AKT signaling, and a failure to transport the CSF-1R into the lumen of nascent macropinosomes, leaving its cytoplasmic region available for signaling. CSF-1R degradation depended upon lysosomal vATPase activity in both WT and DKO macrophages, with this degradation confined to macropinosomes in WT but occurring in distributed/tubular lysosomes in DKO cells. RNA-sequencing comparison of Cbl-/-, Cbl-b-/- and DKO macrophages indicated that while the overall macrophage transcriptional program remained intact, DKO macrophages had alterations in gene expression associated with growth factor signaling, cell cycle, inflammation and senescence. Cbl-b-/- had minimal effect on the transcriptional program whereas Cbl-/- led to more alternations but only DKO macrophages demonstrated substantial changes in the transcriptome, suggesting overlapping but unique functions for the two Cbl-family members. Thus, Cbl/Cbl-b-mediated ubiquitination of CSF-1R regulates its endocytic fate, constrains inflammatory gene expression, and regulates signaling for macrophage proliferation.

The structural dynamics of macropinosome formation and PI3-kinase-mediated sealing revealed by lattice light sheet microscopy.

Macropinosomes are formed by shaping actin-rich plasma membrane ruffles into large intracellular organelles in a phosphatidylinositol 3-kinase (PI3K)-coordinated manner. Here, we utilize lattice lightsheet microscopy and image visualization methods to map the three-dimensional structure and dynamics of macropinosome formation relative to PI3K activity. We show that multiple ruffling morphologies produce macropinosomes and that the majority form through collisions of adjacent PI3K-rich ruffles. By combining multiple volumetric representations of the plasma membrane structure and PI3K products, we show that PI3K activity begins early throughout the entire ruffle volume and continues to increase until peak activity concentrates at the base of the ruffle after the macropinosome closes. Additionally, areas of the plasma membrane rich in ruffling had increased PI3K activity and produced many macropinosomes of various sizes. Pharmacologic inhibition of PI3K activity had little effect on the rate and morphology of membrane ruffling, demonstrating that early production of 3'-phosphoinositides within ruffles plays a minor role in regulating their morphology. However, 3'-phosphoinositides are critical for the fusogenic activity that seals ruffles into macropinosomes. Taken together, these data indicate that local PI3K activity is amplified in ruffles and serves as a priming mechanism for closure and sealing of ruffles into macropinosomes.

Seroprevalence of SARS-CoV-2 antibodies among rural healthcare workers.

The objective of this longitudinal cohort study was to determine the seroprevalence of antibodies to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in healthcare workers employed at healthcare settings in three rural counties in eastern South Dakota and western Minnesota from May 13, 2020, through December 22, 2020. Three blood draws were performed at five clinical sites and tested for the presence of antibodies against the SARS-CoV-2. Serum samples were tested for the presence of antibodies using a fluorescent microsphere immunoassay (FMIA), neutralization of SARS-CoV-2 spike-pseudotyped particles (SARS-CoV-2pp) assay, and serum virus neutralization (SVN) assay. The seroprevalence was determined to be 1/336 (0.29%) for samples collected from 5/13/20 to 7/13/20, 5/260 (1.92%) for samples collected from 8/13/20 to 9/25/20, and 35/235 (14.89%) for samples collected from 10/16/20 to 12/22/20. Eight of the 35 (22.8%) seropositive individuals identified in the final draw did not report a previous diagnosis with COVID-19. There was a high correlation (>90%) between the FMIA and virus neutralization assays. Each clinical site's seroprevalence was higher than the cumulative incidence for the general public in the respective county as reported by state public health agencies. As of December 2020, there was a high percentage (85%) of seronegative individuals in the study population.

Comparison of Physical Activity by Lifestyle Between Two Rural Pediatric Population Groups.

BACKGROUND: Obesity prevalence is higher among rural populations than urban, including youth. Reduced physical activity levels are associated with childhood obesity. It could be assumed that the obesity disparity between rural and urban children is attributable, in part, to differences in physical activity levels; however, previous research quantifying and comparing physical activity levels between rural and urban youth are mixed. Lifestyle may be more important than geographic location in determining physical activity levels. Therefore, the objective of this study was to compare sex and lifestyle group (Hutterite vs. non-Hutterite) differences in physical activity in a free-living, rural pediatric population. METHODS: Youth (n=58) were instructed to wear accelerometers for seven days. Mean percent time in light, moderate or vigorous activity during waking hours was calculated. Two-way ANOVAs and multiple regression models were used for analyses. RESULTS: Percent time in vigorous activity was significantly greater for Hutterite males than Hutterite females, and Hutterite males had greater percent time in vigorous activity and moderate plus vigorous activity than non-Hutterite males. CONCLUSIONS: There is evidence to support differences in rural lifestyles to be associated with differences in physical activity levels between children living in the same geographic location, particularly among males. Active transportation and having a safe environment for unstructured outdoor play may account for activity and lifestyle differences between the two rural groups.

Does Exercise Influence Pediatric Bone? A Systematic Review.

BACKGROUND: Periods of growth are thought to be the best time to increase bone mineral content, bone area, and areal bone mineral density (aBMD) through increased loading owing to high rates of bone modeling and remodeling. However, questions remain regarding whether a benefit of exercise is seen at all bone sites, is dependent on pubertal status or sex of the child, or whether other factors such as diet modify the response to exercise. QUESTIONS/PURPOSES: We asked: (1) Does bone-loading exercise in childhood consistently increase bone mineral content, bone area, or aBMD? (2) Do effects of exercise differ depending on pubertal status or sex? (3) Does calcium intake modify the bone response to exercise? METHODS: A literature search identified 22 unique trials for inclusion in this meta-analysis of the effect of exercise on bone changes by bone site, pubertal status, and sex. Sample sizes ranged from 16 to 410 subjects 3 to 18 years old with length of intervention ranging from 3 to 36 months. Fifteen of 22 trials were randomized (child randomized in nine, classroom/school randomized in six) and seven were observational trials. Ten trials were Level 2 and 11 were Level 3 based on the Oxford Centre for Evidence-Based Medicine criteria. Random effects models tested the difference (intervention mean effect-control mean effect) in percent change in bone mineral content, bone area, and aBMD. Meta-regression was used to identify sources of heterogeneity and funnel plots were used to assess publication bias. RESULTS: Children assigned to exercise had greater mean percent changes in bone mineral content and aBMD than children assigned to the control groups. Mean differences (95% CI) in bone mineral content percent change between intervention and control groups at total body (0.8; 95% CI, 0.3-1.3; p = 0.003), femoral neck (1.5; 95% CI, 0.5-2.5; p = 0.003), and spine (1.7; 95% CI, 0.4-3.1; p = 0.01) were significant with no differences in bone area (all p > 0.05). There were greater percent changes in aBMD in intervention than control groups at the femoral neck (0.6; 95% CI, 0.2-1.1; p = 0.006) and spine (1.2; 95% CI, 0.6-1.8; p < 0.001). Benefit of exercise was limited to children who were prepubertal (bone mineral content: total body [0.9; 95% CI, 0.2-1.7; p = 0.01], femoral neck [1.8; 95% CI, 0.0-3.5; p = 0.047], spine [3.7; 95% CI, 0.8-6.6; p = 0.01], and aBMD: femoral neck [0.6; 95% CI, -0.1-1.2; p = 0.07], spine [1.5; 95% CI, 0.7-2.3; p < 0.001]), with no differences among children who were pubertal (all p > 0.05). Changes in aBMD did not differ by sex (all p > 0.05), although the number of studies providing male-specific results was small (six of 22 eligible studies included boys). There was significant heterogeneity in bone mineral content and bone area for which a source could not be identified. Heterogeneity in spine aBMD was reduced by including calcium intake and intervention length as covariates. Three trials designed to determine whether calcium intake modified the bone response to exercise all reported a greater effect of exercise on leg bone mineral content in children randomized to receive supplemental calcium than those receiving placebo. CONCLUSIONS: Exercise interventions during childhood led to 0.6% to 1.7% greater annual increase in bone accrual, with effects predominantly among children who were prepubertal. If this effect were to persist into adulthood, it would have substantial implications for osteoporosis prevention. It is important to identify sources of heterogeneity among studies to determine factors that might influence the bone response to increased exercise during growth. LEVEL OF EVIDENCE: Level II, therapeutic study.

Validation of drinking water disinfection by-product exposure assessment for rural areas in the National Children's Study.

The objective of this study was to provide evidence to evaluate the proposed National Children's Study (NCS) protocol for household water sampling in rural study areas. Day-to-day variability in total trihalomethane (TTHM) concentrations in community water supplies (CWS) in rural areas was determined, and the correlation between TTHM concentrations from household taps and CWS monitoring reports was evaluated. Daily water samples were collected from 7 households serviced by 7 different CWS for 15 days. Coefficients of variation for TTHM concentration over 15 days ranged from 8% to 20% depending on the household. Correlations were tested between TTHM household concentrations and the closest date- and location-matched CWS monitoring reports for the 15-day mean (R=0.85, P<0.01). To simulate the NCS-proposed protocol, correlations were tested for 30 additional NCS household samples (polynomial fit: R=0.74, P=0.04). CWS reported TTHM concentrations >50 µg/l corresponded to measured NCS household concentrations ranging from 2 to 60 µg/l. TTHM concentrations were higher in CWS than NCS samples (11.2±3.2 µg/l, mean difference±SE, P<0.01). These results show that in rural areas there is high variability within households and poor correlation at higher concentrations, suggesting that TTHM concentrations from CWS monitoring reports are not an accurate measure of exposure in the household.

Tissue-specific induction of COX-2 and prostaglandins in lipopolysaccharide-stimulated extraplacental human gestational membranes in a 2-chamber transwell culture system.

The current study investigates tissue-specific prostaglandin secretion and cyclooxygenase 2 (COX-2) induction in full-thickness human gestational membranes. Gestational membranes were collected from healthy, nonlaboring cesarean deliveries at 37 to 39 weeks gestation and cultured in 2-chamber Transwell devices. Lipopolysaccharide exposure (100 ng/mL for 8 hours) elevated prostaglandin E(2) and F(2α) concentrations in the amniotic chamber medium regardless of whether exposure was to the amniotic, decidual, or both sides of the membranes. However, prostaglandin E(2) and F(2 α) concentrations in the decidual chamber medium were elevated compared with controls only if the decidual side was exposed directly to lipopolysaccharide. Whereas prostaglandin F(2α) concentrations increased to similar extents in the amniotic and decidual chambers regardless of lipopolysaccharide exposure modality, prostaglandin E(2) concentrations were 22-fold higher on the amniotic side than the decidual side after lipopolysaccharide stimulation of the amnion. These findings demonstrate the propagation of prostaglandins, prostaglandin precursors, or other factors in the direction of the decidua to the amnion, but the reverse situation was not evident. Immunostaining for COX-2 was related to the side of lipopolysaccharide exposure, that is, exposure to the amnion caused immunostaining in cells of the collagen layers of the amnion and chorion, whereas exposure to the decidual side caused staining in decidual cells. These findings suggest that the inflammatory effect of lipopolysaccharide on COX-2 induction occurs within a localized area of exposure and that prostaglandins or their precursors move across the tissues of the gestational membranes by currently undefined transport mechanisms.

Tissue-specific cytokine release from human extra-placental membranes stimulated by lipopolysaccharide in a two-compartment tissue culture system.

BACKGROUND: The extra-placental gestational membranes secrete cytokines in response to bacteria and other infectious agents, with potentially adverse consequences for pregnancy. The present study used lipopolysaccharide (LPS) as a prototype endotoxin to investigate the pattern of stimulated cytokine release from the amniotic and choriodecidual sides of full-thickness human gestational membranes in a two-compartment tissue culture system. METHODS: Gestational membranes were collected from healthy non-laboring caesarean deliveries at term. Full-thickness membranes from each placenta were cut into pieces, mounted on Transwell frames, and placed in culture wells to create a two-compartment culture with the gestational membranes serving as the barrier between compartments. The LPS (100 ng/ml) was added to the amniotic, choriodecidual or both chambers of the culture, and cytokines were assayed in the medium of the amniotic and choriodecidual chambers after 8 h of LPS exposure. Cytokine concentrations were analyzed by two-way analysis of variance for effects of treatment and side specificity of cytokine release from the membranes. RESULTS: LPS exposure on the choriodecidual side of the membranes significantly increased TNF-alpha, IL-6, IL-10 and IL-8 in the choriodecidual compartment, whereas TNF-alpha was the only cytokine observed to increase in the amniotic compartment. When LPS treatment was to the amniotic side of the membranes, there were significant increases in TNF-alpha and IL-6 in the amniotic compartment as well as increased concentrations of TNF-alpha, IL-6 and IL-8 in the choriodecidual compartment; however, there were no statistically significant differences for IL-10 in either compartment. No statistically significant differences were observed for IL-1beta, TGF-beta or IL-4 concentrations in response to LPS, regardless of the exposure modality. CONCLUSION: The amnion and choriodecidua exhibited distinct patterns of response to LPS with evidence of inflammatory signaling across the layers of the gestational membranes. These results suggest a complicated network of signaling within the gestational membranes, in which cytokine- and tissue-specific responses to inflammatory stimulation may have important implications for maintaining pregnancy in the challenge of microbial invasion of the uterine compartment.

Targeting and blocking B7 costimulatory molecules on antigen-presenting cells using CTLA4Ig-conjugated liposomes: in vitro characterization and in vivo factors affecting biodistribution.

PURPOSE: CTLA4Ig, a fusion protein of CTLA-4 and Fc of immunoglobulin (Ig) heavy chain, inhibits the essential costimulatory signal for full T cell activation via blocking the interaction between CD28 and B7 molecules and renders T cell nonresponsiveness. CTLA4Ig has been used to control deleterious T cell activation in many experimental systems. We hypothesized that by conjugating CTLA4Ig to liposomes the efficacy of CTLA4Ig could be enhanced through multivalent ligand effect, superior targetability, and modification of the fate of ligated costimulatory molecules. METHODS AND RESULTS: Consistent with this hypothesis, liposome-conjugated CTLA4Ig bound to B7 and blocked their binding sites more efficiently than free CTLA4Ig, lowering the half maximal dose for B7 blocking by an order of the magnitude. These results were similar both in B7-1 expressing p815 cells and in activated macrophages. Moreover, CTLA4Ig-liposomes underwent rapid internalization upon cell surface binding through B7 molecules. In allogenic mixed lymphocyte reaction assays, the CTLA4Ig-liposomes were tested to show effective inhibition of T cell proliferation. In vivo, however, when CTLA4Ig-liposomes were injected into mice, a significant fraction was localized to the reticuloendothelial system (RES), presumably because of its binding to Fc receptors expressed on tissue macrophages. The Fc receptor-mediated uptake could be alleviated by coinjection of anti-FcR monoclonal antibody. In the mouse engrafted with pancreatic islets of Langerhans underneath the capsule of one kidney, despite the increased localization in RES, enhanced accumulation of CTLA4Ig-conjugated liposome was observed in the engrafted kidney compared to the contralateral kidney. CONCLUSION: We show that the conjugation of CTLA4Ig to liposome could increase the efficiency of the targeting by increasing the binding avidity at cellular level and by increasing the concentration at the target site in in vivo system. The biodistribution and circulation time data suggested that the CTLA4Ig-liposomes could be improved upon minimizing the FcR-mediated uptake by Fc receptor-bearing cells. Thus, the strategy of conjugating CTLA4Ig to liposomes could be exploited for immune intervention in transplantation and autoimmune diseases for the efficient blocking of costimulation.