Paracrine interactions between primary human macrophages and human fibroblasts enhance murine mammary gland humanization in vivo

Abstract Introduction Macrophages comprise an essential component of the mammary microenvironment necessary for normal gland development. However, there is no viable in vivo model to study their role in normal human breast function. We hypothesized that adding primary human macrophages to the murine mammary gland would enhance and provide a novel approach to examine immune-stromal cell interactions during the humanization process. Methods Primary human macrophages, in the presence or absence of ectopic estrogen stimulation, were used to humanize mouse mammary glands. Mechanisms of enhanced humanization were identified by cytokine/chemokine ELISAs, zymography, western analysis, invasion and proliferation assays; results were confirmed with immunohistological analysis. Results The combined treatment of macrophages and estrogen stimulation significantly enhanced the percentage of the total gland humanized and the engraftment/outgrowth success rate. Timecourse analysis revealed the disappearance of the human macrophages by two weeks post-injection, suggesting that the improved overall growth and invasiveness of the fibroblasts provided a larger stromal bed for epithelial cell proliferation and structure formation. Confirming their promotion of fibroblasts humanization, estrogen-stimulated macrophages significantly enhanced fibroblast proliferation and invasion in vitro, as well as significantly increased proliferating cell nuclear antigen (PCNA) positive cells in humanized glands. Cytokine/chemokine ELISAs, zymography and western analyses identified TNFα and MMP9 as potential mechanisms by which estrogen-stimulated macrophages enhanced humanization. Specific inhibitors to TNFα and MMP9 validated the effects of these molecules on fibroblast behavior in vitro, as well as by immunohistochemical analysis of humanized glands for human-specific MMP9 expression. Lastly, glands humanized with macrophages had enhanced engraftment and tumor growth compared to glands humanized with fibroblasts alone. Conclusions Herein, we demonstrate intricate immune and stromal cell paracrine interactions in a humanized in vivo model system. We confirmed our in vivo results with in vitro analyses, highlighting the value of this model to interchangeably substantiate in vitro and in vivo results. It is critical to understand the signaling networks that drive paracrine cell interactions, for tumor cells exploit these signaling mechanisms to support their growth and invasive properties. This report presents a dynamic in vivo model to study primary human immune/fibroblast/epithelial interactions and to advance our knowledge of the stromal-derived signals that promote tumorigenesis

Lack of Preparedness for Pediatric to Adult-Oriented Health Care Transition in Hospitalized Adolescents and Young Adults

ObjectiveWe examined the self-reported preparedness of hospitalized adolescents and young adults (AYA) for transition from pediatric to adult-oriented health care with regard to: 1) previous health care transition (HCT) preparation, 2) Self-Determination Theory (SDT) constructs of health self-management autonomy and competence, and 3) their perception of medical knowledge, attitudes, and concerns.MethodsFrom 2013 to 2015, 139 hospitalized patients aged 15 to 21 years completed a 40-item survey on HCT preparation, attitudes, concerns, and perception of knowledge adapted in part from validated questionnaires of the Department of Health and Human Services, Maternal and Child Health Bureau, and SDT Treatment Self-Regulation Study.ResultsFewer than 40% of all respondents endorsed previous HCT preparation such as providers discussing taking responsibility for their health, transitioning to adult providers, and only 20% had discussed future health insurance needs. Of our AYA population, 84% had 1 or more special health care needs. Older patients, female patients, and those with increased HCT preparation scores had increased autonomous motivation, positive attitudes toward transition, yet also increased transition concerns. Higher autonomous motivation and perceived competence correlated with increased perception of knowledge (P = .002, \u3c .001 respectively) and more positive attitudes toward transition planning (P \u3c .001, .054 respectively). Multivariate regression analysis revealed those with increased HCT preparation and those with increased perceived competence had increased perception of knowledge (β = .25, P = .005 and β = .35, P \u3c .001).ConclusionsOur findings suggest that hospitalized AYA received limited education and preparation regarding key elements of HCT to adult-oriented health care. Moreover, those previously exposed to transition preparation efforts were more likely to have motivation and a sense of competence in HCT skills

Effectiveness of high-dose methotrexate in T-cell lymphoblastic leukemia and advanced-stage lymphoblastic lymphoma: a randomized study by the Children's Oncology Group (POG 9404)

The Pediatric Oncology Group (POG) phase 3 trial 9404 was designed to determine the effectiveness of high-dose methotrexate (HDM) when added to multi-agent chemotherapy based on the Dana-Farber backbone. Children with T-cell acute lymphoblastic leukemia (T-ALL) or advanced lymphoblastic lymphoma (T-NHL) were randomized at diagnosis to receive/not receive HDM (5 g/m 2 as a 24-hour infusion) at weeks 4, 7, 10, and 13. Between 1996 and 2000, 436 patients were enrolled in the methotrexate randomization. Five-year and 10-year event-free survival (EFS) was 80.2% ± 2.8% and 78.1% ± 4.3% for HDM (n = 219) versus 73.6% ± 3.1% and 72.6% ± 5.0% for no HDM (n = 217; P = .17). For T-ALL, 5-year and 10-year EFS was significantly better with HDM (n = 148, 5 years: 79.5% ± 3.4%, 10 years: 77.3% ± 5.3%) versus no HDM (n = 151, 5 years: 67.5% ± 3.9%, 10 years: 66.0% ± 6.6%; P = .047). The difference in EFS between HDM and no HDM was not significant for T-NHL patients (n = 71, 5 years: 81.7% ± 4.9%, 10 years: 79.9% ± 7.5% vs n = 66, 5 years: 87.8% ± 4.2%, 10 years: 87.8% ± 6.4%; P = .38). The frequency of mucositis was significantly higher in patients treated with HDM ( P = .003). The results support adding HDM to the treatment of children with T-ALL, but not with NHL, despite the increased risk of mucositis