Multilevel Factors Associated With Participation in Group Well-Child Care.

OBJECTIVE: To identify factors associated with participation in group well-child care (GWCC), wherein families share preventive health care visits. METHODS: We extracted electronic health record data of mother-infant dyads with infants born 2013-18 at Yale New Haven Hospital and followed at the primary care center. Using chi-square analysis and multivariate logistic regression, we examined the extent to which 1) maternal/infant characteristics and recruitment timing were associated with GWCC initiation and continued engagement and 2) initiation was associated with primary care visits. RESULTS: Of 2046 eligible mother-infant dyads, 11.6% initiated GWCC. The odds of initiation were higher among mothers with Spanish versus English primary language (odds ratio: 2.36 [95% confidence interval: 1.52-3.66]), with 1 child versus ≥ 3 children (1.58 [1.13-2.22]), and of non-Hispanic Black versus non-Hispanic White infants (2.72 [1.39-5.32]). Initiation was lower among infants born in 2016 (0.53 [0.32-0.88]) and 2018 (0.29 [0.17-0.52]) versus 2013. Among GWCC initiators with follow-up data (n = 217), continued engagement (n = 132, 60.8%) was positively associated with maternal age of 20-29 years (2.85 [1.10-7.34]) and > 30 years (3.46 [1.15-10.43]) compared with< 20 years, and mothers with 1 child versus ≥ 3 (2.28 [1.04-4.98]). GWCC initiators, versus non-initiators, had 5.06 times higher adjusted odds of attending > 9 primary care appointments in the first 18 months (95% confidence interval: 3.74-6.85). CONCLUSIONS: As evidence builds on health and social benefits of GWCC, recruitment efforts may gain from considering multi-level socio-economic, demographic, and cultural factors associated with GWCC participation. Higher participation among systemically marginalized groups may present unique opportunities for family-based health promotion to mitigate health inequities.

Lymphatic endothelial progenitors originate from plastic myeloid cells activated by toll-like receptor-4.

BACKGROUND: Myeloid-derived lymphatic endothelial cells (M-LECP) are induced by inflammation and play an important role in adult lymphangiogenesis. However, the mechanisms driving M-LECP differentiation are currently unclear. We previously showed that activation of Toll-like receptor-4 (TLR4) induces myeloid-lymphatic transition (MLT) of immortalized mouse myeloid cells. Here the goals were to assess the potential of different TLR4 ligands to induce pro-lymphatic reprogramming in human and mouse primary myeloid cells and to identify transcriptional changes regulating this process. METHODOLOGY/PRINCIPAL FINDINGS: Human and mouse myeloid cells were reprogrammed to the lymphatic phenotype by TLR4 ligands including lipopolysaccharide (LPS), recombinant high mobility group box 1 protein (HMGB1), and paclitaxel. TLR4 induced similar MLT in cells from mice of different strains and immune status. Commonly induced genes were detected by transcriptional profiling in human and mouse myeloid cells from either immunocompetent or immunodeficient mice. Shared trends included: (1) novel expression of lymphatic-specific markers vascular endothelial growth factor receptor-3 (VEGFR-3), lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and podoplanin (PDPN) largely absent prior to induction; (2) lack of notable changes in blood vessel-specific markers; (3) transient expression of VEGFR-3, but sustained increase of vascular endothelial growth factor-C (VEGF-C) and a variety of inflammatory cytokines; (4) dependency of VEGFR-3 upregulation and other LEC genes on NF-κB; and (5) novel expression of lymphatic-specific (e.g., PROX1) and stem/progenitor (e.g., E2F1) transcription factors known for their roles in adult and embryonic vascular formation. M-LECP generated by TLR4 ligands in vitro were functional in vivo as demonstrated by significantly increased lymphatic vessel density and lymphatic metastasis detected in orthotopic breast cancer models. CONCLUSIONS/SIGNIFICANCE: We established a novel TLR4-dependent protocol for in vitro production of functionally competent M-LECP from primary human or mouse myeloid cells and identified many potential regulators of this process. This information can be further exploited for research and therapeutic purposes.

Novel model for basaloid triple-negative breast cancer: behavior in vivo and response to therapy.

INTRODUCTION: The basaloid triple-negative breast cancer (B-TNBC) is one of the most aggressive, therapy-resistant, and metastatic tumors. Current models do not recapitulate the basaloid phenotype of TNBC, thus limiting the understanding of its biology and designing new treatments. We identified HCC1806 as a line expressing typical B-TNBC markers, engineered a subline with traceable reporters, and determined growth, drug sensitivity, recurrence, and vascular and metastatic patterns of orthotopic xenografts in immunodeficient mice. METHODS: mRNA and protein analyses showed that HCC1806 expresses basal but not luminal or mesenchymal markers. HCC1806-RR subline stably expressing red fluorescent protein and Renilla luciferase was generated and characterized for sensitivity to chemodrugs, orthotopic growth, vascular properties, recurrence, metastasis, and responsiveness in vivo. RESULTS: The HCC1806 cells were highly sensitive to paclitaxel, but cytotoxicity was accompanied by pro-survival vascular endothelial growth factor-A loop. In vivo, HCC1806-RR tumors display linear growth, induce peritumoral lymphatics, and spontaneously metastasize to lymph nodes (LNs) and lungs. Similarly to human B-TNBC, HCC1806-RR tumors were initially sensitive to taxane therapy but subsequently recur. Bevacizumab significantly suppressed recurrence by 50% and reduced the incidence of LN and pulmonary metastases by, respectively, 50% and 87%. CONCLUSIONS: The HCC1806-RR is a new model that expresses bona fide markers of B-TNBC and traceable markers for quantifying metastases. Combination of bevacizumab with nab-paclitaxel significantly improved the outcome, suggesting that this approach can apply to human patients with B-TNBC. This model can be used for defining the metastatic mechanisms of B-TNBC and testing new therapies.

New model of macrophage acquisition of the lymphatic endothelial phenotype.

BACKGROUND: Macrophage-derived lymphatic endothelial cell progenitors (M-LECPs) contribute to new lymphatic vessel formation, but the mechanisms regulating their differentiation, recruitment, and function are poorly understood. Detailed characterization of M-LECPs is limited by low frequency in vivo and lack of model systems allowing in-depth molecular analyses in vitro. Our goal was to establish a cell culture model to characterize inflammation-induced macrophage-to-LECP differentiation under controlled conditions. METHODOLOGY/PRINCIPAL FINDINGS: Time-course analysis of diaphragms from lipopolysaccharide (LPS)-treated mice revealed rapid mobilization of bone marrow-derived and peritoneal macrophages to the proximity of lymphatic vessels followed by widespread (∼50%) incorporation of M-LECPs into the inflamed lymphatic vasculature. A differentiation shift toward the lymphatic phenotype was found in three LPS-induced subsets of activated macrophages that were positive for VEGFR-3 and many other lymphatic-specific markers. VEGFR-3 was strongly elevated in the early stage of macrophage transition to LECPs but undetectable in M-LECPs prior to vascular integration. Similar transient pattern of VEGFR-3 expression was found in RAW264.7 macrophages activated by LPS in vitro. Activated RAW264.7 cells co-expressed VEGF-C that induced an autocrine signaling loop as indicated by VEGFR-3 phosphorylation inhibited by a soluble receptor. LPS-activated RAW264.7 macrophages also showed a 68% overlap with endogenous CD11b(+)/VEGFR-3(+) LECPs in the expression of lymphatic-specific genes. Moreover, when injected into LPS- but not saline-treated mice, GFP-tagged RAW264.7 cells massively infiltrated the inflamed diaphragm followed by integration into 18% of lymphatic vessels. CONCLUSIONS/SIGNIFICANCE: We present a new model for macrophage-LECP differentiation based on LPS activation of cultured RAW264.7 cells. This system designated here as the "RAW model" mimics fundamental features of endogenous M-LECPs. Unlike native LECPs, this model is unrestricted by cell numbers, heterogeneity of population, and ability to change genetic composition for experimental purposes. As such, this model can provide a valuable tool for understanding the LECP and lymphatic biology.

Inflammation induces lymphangiogenesis through up-regulation of VEGFR-3 mediated by NF-kappaB and Prox1.

The concept of inflammation-induced lymphangiogenesis (ie, formation of new lymphatic vessels) has long been recognized, but the molecular mechanisms remained largely unknown. The 2 primary mediators of lymphangiogenesis are vascular endothelial growth factor receptor-3 (VEGFR-3) and Prox1. The key factors that regulate inflammation-induced transcription are members of the nuclear factor-kappaB (NF-kappaB) family; however, the role of NF-kappaB in regulation of lymphatic-specific genes has not been defined. Here, we identified VEGFR-3 and Prox1 as downstream targets of the NF-kappaB pathway. In vivo time-course analysis of inflammation-induced lymphangiogenesis showed activation of NF-kappaB followed by sequential up-regulation of Prox1 and VEGFR-3 that preceded lymphangiogenesis by 4 and 2 days, respectively. Activation of NF-kappaB by inflammatory stimuli also elevated Prox1 and VEGFR-3 expression in cultured lymphatic endothelial cells, resulting in increased proliferation and migration. We also show that Prox1 synergizes with the p50 of NF-kappaB to control VEGFR-3 expression. Collectively, our findings suggest that induction of the NF-kappaB pathway by inflammatory stimuli activates Prox1, and both NF-kappaB and Prox1 activate the VEGFR-3 promoter leading to increased receptor expression in lymphatic endothelial cells. This, in turn, enhances the responsiveness of preexisting lymphatic endothelium to VEGFR-3 binding factors, VEGF-C and VEGF-D, ultimately resulting in robust lymphangiogenesis.