Effectively utilizing the Sponsor Contract Research Organization interaction for successful implementation of critical flow cytometry in the clinic.

As the number of therapeutic modalities expand, and the field of scientific research evolves toward finding treatment solutions for complex and rare disease, an ability to demonstrate efficacy through biomarker end points in clinical development studies is becoming increasingly important. Implementing flow cytometry in a clinical setting is challenging and many sponsor organizations take a hybrid approach, developing complex analytical methods internally before identifying and forming partnerships with contract research organizations to conduct the formal analytical method validation and sample bioanalysis. Ensuring that these interactions are effective is critical to the delivery of high-quality, impactful clinical data. This paper provides a review of the recommendations, challenges and solutions for the implementation of decision-making flow cytometry end points effectively utilizing the Sponsor Contract Research Organization interaction.

Protease-activated receptor-2 activation contributes to house dust mite-induced IgE responses in mice.

Aeroallergens such as house dust mite (HDM), cockroach, and grass or tree pollen are innocuous substances that can induce allergic sensitization upon inhalation. The serine proteases present in these allergens are thought to activate the protease-activated receptor (PAR)-2, on the airway epithelium, thereby potentially inducing allergic sensitization at the expense of inhalation tolerance. We hypothesized that the proteolytic activity of allergens may play an important factor in the allergenicity to house dust mite and is essential to overcome airway tolerance. Here, we aimed to investigate the role of PAR-2 activation in allergic sensitization and HDM-induced allergic airway inflammation. In our study, Par-2 deficient mice were treated with two different HDM extracts containing high and low serine protease activities twice a week for a period of 5 weeks. We determined airway inflammation through quantification of percentages of mononuclear cells, eosinophils and neutrophils in the bronchial alveolar lavage fluid and measured total IgE and HDM-specific IgE and IgG1 levels in serum. Furthermore, Th2 and pro-inflammatory cytokines including IL-5, IL-13, Eotaxin-1, IL-17, KC, Chemokine (C-C motif) ligand 17 (CCL17) and thymic stromal lymphopoietin (TSLP), were measured in lung tissue homogenates. We observed that independent of the serine protease content, HDM was able to induce elevated levels of eosinophils and neutrophils in the airways of both wild-type (WT) and Par-2 deficient mice. Furthermore, we show that induction of pro-inflammatory cytokines by HDM exposure is independent of Par-2 activation. In contrast, serine protease activity of HDM does contribute to enhanced levels of total IgE, but not HDM-specific IgE. We conclude that, while Par-2 activation contributes to the development of IgE responses, it is largely dispensable for the HDM-induced induction of pro-inflammatory cytokines and airway inflammation in an experimental mouse model of HDM-driven allergic airway disease.

Alveolar macrophages develop from fetal monocytes that differentiate into long-lived cells in the first week of life via GM-CSF.

Tissue-resident macrophages can develop from circulating adult monocytes or from primitive yolk sac-derived macrophages. The precise ontogeny of alveolar macrophages (AMFs) is unknown. By performing BrdU labeling and parabiosis experiments in adult mice, we found that circulating monocytes contributed minimally to the steady-state AMF pool. Mature AMFs were undetectable before birth and only fully colonized the alveolar space by 3 d after birth. Before birth, F4/80(hi)CD11b(lo) primitive macrophages and Ly6C(hi)CD11b(hi) fetal monocytes sequentially colonized the developing lung around E12.5 and E16.5, respectively. The first signs of AMF differentiation appeared around the saccular stage of lung development (E18.5). Adoptive transfer identified fetal monocytes, and not primitive macrophages, as the main precursors of AMFs. Fetal monocytes transferred to the lung of neonatal mice acquired an AMF phenotype via defined developmental stages over the course of one week, and persisted for at least three months. Early AMF commitment from fetal monocytes was absent in GM-CSF-deficient mice, whereas short-term perinatal intrapulmonary GM-CSF therapy rescued AMF development for weeks, although the resulting AMFs displayed an immature phenotype. This demonstrates that tissue-resident macrophages can also develop from fetal monocytes that adopt a stable phenotype shortly after birth in response to instructive cytokines, and then self-maintain throughout life.