In situ detection of CD73+ CD90+ CD105+ lineage: Mesenchymal stromal cells in human placenta and bone marrow specimens by chipcytometry.

Mesenchymal stromal cells (MSCs) support endogenous regeneration and present therefore promising opportunities for in situ tissue engineering. They can be isolated and expanded from various tissues, for example, bone marrow, adipose tissue, or placenta. The minimal consensus definition criteria of ex vivo expanded MSCs requires them to be positive for CD73, CD90, and CD105 expression, while being negative for CD34, CD45, CD14, CD19, and HLA-DR. This study aimed to compare the in situ phenotype of MSCs with that of their culture-expanded progeny. We report for the first time in situ detection of cells expressing this marker combination in human placenta cryosections as well as in bone marrow aspirates using multiplex-immunohistology (Chipcytometry), a technique that allows staining of more than 100 biomarkers consecutively on the same cell. © 2018 International Society for Advancement of Cytometry.

Monocyte/macrophage lineage commitment and distribution are affected by the lack of regulatory T cells in scurfy mice.

Foxp3(+) regulatory T (Treg) cells play a pivotal role in maintaining immunological tolerance. Loss-of-function mutations in the Foxp3 gene result in multiorgan inflammation known as immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome in humans and scurfy (Sf) disease in mice. While the impact of missing Treg cells on adaptive immune cells is well documented, their role in regulation of myeloid cells remains unclear. Here we report that Sf mice exhibit an altered composition of stem and progenitor cells, characterized by increased numbers of myeloid precursors and higher efficiency of macrophage generation ex vivo. The proportion of monocytes/macrophages in the bone marrow, blood, and spleen was significantly elevated in Sf mice, which was accompanied with tissue-specific monocyte expression of homing receptor and phagocytic activity. Sf mice displayed high levels of M-CSF and other inflammatory cytokines, including monocyte-recruiting chemokines. Adoptive transfer of WT CD4(+) cells and in vivo neutralization of M-CSF normalized frequencies of monocyte subsets and their progenitors and reduced high levels of monocyte-related cytokines in Sf mice, while Treg cell transfer to RAG2(-/-) mice had no effect on myelopoiesis and monocyte/macrophage counts. Our findings illustrate that deregulated myelopoiesis in Sf mice is mainly caused by the inflammatory reaction resulting from the lack of Treg cells.

α-NAC-Specific Autoreactive CD8+ T Cells in Atopic Dermatitis Are of an Effector Memory Type and Secrete IL-4 and IFN-γ.

Autoreactivity may play a critical role in the chronification of atopic dermatitis (AD). Several studies showed that AD patients produce IgE Abs specific for autoantigens, and we described Th as well as CD8(+) T cells specific for the autoallergen Hom s 2, the α-chain of the nascent polypeptide-associated complex (α-NAC). This study aimed to investigate the frequency and inflammatory phenotype of autoallergen-specific CD8(+) T cells. CD8(+) T cell immunodominant epitopes of α-NAC were mapped by applying prediction softwares, and binding affinity was confirmed by stabilization of empty MHC complexes. MHC class I tetramers were assembled and binding cells were analyzed directly ex vivo by flow cytometry and in terms of single-cell assessment by ChipCytometry. We report significantly elevated numbers of α-NAC-specific peripheral T cells in sensitized patients compared with nonatopic controls. These cells secrete IL-4 and IFN-γ, and surface markers revealed significantly elevated frequencies of circulating terminally differentiated α-NAC-specific CD8(+) T cells in patients with AD compared with nonatopic donors. The observed phenotype of α-NAC-specific CD8(+) T cells indicates a role in the pathogenesis of AD.

Oriented Cell Division in the C. elegans Embryo Is Coordinated by G-Protein Signaling Dependent on the Adhesion GPCR LAT-1.

Orientation of spindles and cell division planes during development of many species ensures that correct cell-cell contacts are established, which is vital for proper tissue formation. This is a tightly regulated process involving a complex interplay of various signals. The molecular mechanisms underlying several of these pathways are still incompletely understood. Here, we identify the signaling cascade of the C. elegans latrophilin homolog LAT-1, an essential player in the coordination of anterior-posterior spindle orientation during the fourth round of embryonic cell division. We show that the receptor mediates a G protein-signaling pathway revealing that G-protein signaling in oriented cell division is not solely GPCR-independent. Genetic analyses showed that through the interaction with a Gs protein LAT-1 elevates intracellular cyclic AMP (cAMP) levels in the C. elegans embryo. Stimulation of this G-protein cascade in lat-1 null mutant nematodes is sufficient to orient spindles and cell division planes in the embryo in the correct direction. Finally, we demonstrate that LAT-1 is activated by an intramolecular agonist to trigger this cascade. Our data support a model in which a novel, GPCR-dependent G protein-signaling cascade mediated by LAT-1 controls alignment of cell division planes in an anterior-posterior direction via a metabotropic Gs-protein/adenylyl cyclase pathway by regulating intracellular cAMP levels.

Distinct phenotypic features of neonatal murine macrophages.

Neonates rely on their innate immune system. Resident tissue macrophages are considered to be initiators and regulators of the innate immune response and thus, appear to be especially important to neonates. We hypothesized that the phenotype and function of neonatal tissue macrophages differ from their adult counterparts. Peritoneal macrophages from neonatal (<24 h) and adult (6 weeks old) C57BL/6J mice were isolated and analyzed by high-content chipcytometry. After stimulation for 6 h with LPS (0, 1, 10, 100 ng/mL), macrophage transcriptome was analyzed by microarray and cytokine release was measured using multiparametric bead assays. Antigen presenting capacity was compared by T-cell stimulation assays. We observed that neonatal murine peritoneal macrophages are characterized by selective lack of expression of F4/80, MHC class II, and costimulatory molecules (CD80, CD86). Furthermore, we found differences in the transcriptome between neonatal and adult macrophages, unstimulated and after LPS stimulation. Although neonatal macrophages showed a significantly increased secretion of proinflammatory cytokines upon LPS stimulation, their potential to induce T-cell proliferation was significantly reduced. In conclusion, we observed a distinct phenotype of the neonatal macrophage population. The specific functions of this macrophage population could help to understand the excessive inflammatory reactions observed in the very young.

A posteriori dietary patterns: how many patterns to retain?

Principal component analysis (PCA) and cluster analysis are used frequently to derive dietary patterns. Decisions on how many patterns to extract are primarily based on subjective criteria, whereas different solutions vary in their food-group composition and perhaps association with disease outcome. Literature on reliability of dietary patterns is scarce, and previous studies validated only 1 preselected solution. Therefore, we assessed reliability of different pattern solutions ranging from 2 to 6 patterns, derived from the aforementioned methods. A validated food frequency questionnaire was administered at baseline (1993-1997) to 39,678 participants in the European Prospective Investigation into Cancer and Nutrition-The Netherlands (EPIC-NL) cohort. Food items were grouped into 31 food groups for dietary pattern analysis. The cohort was randomly divided into 2 halves, and dietary pattern solutions derived in 1 sample through PCA were replicated through confirmatory factor analysis in sample 2. For cluster analysis, cluster stability and split-half reproducibility were assessed for various solutions. With PCA, we found the 3-component solution to be best replicated, although all solutions contained ≥1 poorly confirmed component. No quantitative criterion was in agreement with the results. Associations with disease outcome (coronary heart disease) differed between the component solutions. For all cluster solutions, stability was excellent and deviations between samples was negligible, indicating good reproducibility. All quantitative criteria identified the 2-cluster solution as optimal. Associations with disease outcome were comparable for different cluster solutions. In conclusion, reliability of obtained dietary patterns differed considerably for different solutions using PCA, whereas cluster analysis derived generally stable, reproducible clusters across different solutions. Quantitative criteria for determining the number of patterns to retain were valuable for cluster analysis but not for PCA. Associations with disease risk were influenced by the number of patterns that are retained, especially when using PCA. Therefore, studies on associations between dietary patterns and disease risk should report reasons to choose the number of retained patterns.

Identification and quantification of basophils in the airways of asthmatics following segmental allergen challenge.

During asthma attacks, allergens activate sensitized basophils in the lung, thereby aggravating symptoms. Due to the paucity of basophils in bronchial lavage fluid and the lack of specific basophil detection and quantification methods, basophil-directed research in these samples was hampered in the past. This study aimed to establish and validate a flow cytometry-based basophil detection and quantification method for human basophils from bronchoalveolar lavage (BAL) and blood as a prerequisite for a better understanding of their pathogenic contribution and subtyping of asthma phenotypes. BAL and blood leukocytes from seasonal asthmatics were analyzed by flow cytometry. Chipcytometry, a highly sensitive single-cell analysis method, was used to validate the staining panel for basophils. Cell differentials of May-Grünwald-Giemsa-stained cytospins were used to compare basophil percentages. BAL basophils are identifiable as CD123(+) HLA-DR(-) CD3(-) CD14(-) CD19(-) CD20(-) CD56(-) cells in flow cytometrical analysis. Their identity was validated by Chipcytometry. CD203c was highly expressed by BAL basophils, whereas it was expressed at variable levels on blood basophils. The two quantification methods correlated, although more basophils were detected by flow cytometry. Furthermore, the increase in basophil percentages in the lung correlated with the decrease in the basophil percentages in the blood after allergen challenge. We here validated a reliable basophil quantification method, which is independent of the cell's activation and degranulation state. The results obtained with this method indicate that basophils are directly recruited from the blood circulation to the airway lumen.

Lung function and inflammation during murine Pseudomonas aeruginosa airway infection.

BACKGROUND: Following any acute irritation lung function declines rapidly. Reasons for pulmonary deterioration in humans had been attributed to the action of either interleukin-6 or interleukin-8 in the lungs. OBJECTIVES: The present study investigates the association between immune response and decline in lung function in a murine bacterial lung infection model. METHODS: Upon intratracheal inoculation of C57BL/6J mice with a sublethal dose of Pseudomonas aeruginosa lung function, cytokine, chemokine and cytometry in bronchoalveolar lavage fluid, bacterial counts and lung histology was assessed at 2, 4, 6, 8, 10, 12, 18, 24, 48, 72, 96 and 120 h post inoculation. RESULTS: Lung function measured by non-invasive head-out spirometry decreased most strongly between 6 and 10 h post inoculation and required up to 72 h to recover for selected parameters. CFU counts in the lungs peaked at 4h post inoculation with subsequent decline until at 24-48 h post inoculation background levels were reached. Cytokine and chemokine responses could be separated into an early pro-inflammatory phase (2-8h post inoculation; mainly tumor-necrosis factor α (TNFα) and interleukin-1α driven) and a late anti-inflammatory resolution phase (starting at 24h post inoculation; mainly interleukin-10 and interleukin-4 driven). Interleukin-6 levels correlated with the deterioration of lung function. Lung histology showed maximal changes in terms of inflammation and edema between 24 and 48 h post inoculation. CONCLUSIONS: In summary, elevated interleukin-6, high local neutrophil counts and lung edema were found to be the most characteristic signs of the transient period of deterioration of lung function.

Successful treatment of autoimmune and lymphoproliferative complications of patients with intrinsic B-cell immunodeficiencies with Rituximab.

The heterogeneous group of primary immunodeficiencies requires personalized diagnosis and therapy to acheive an optimal outcome for each patient. This was exemplified by two patients with intrinsic B-cell class-switch defects (subclass of Hyper-IgM syndromes), where lymphoproliferation and autoimmunity determined the clinical course for many years due to lack of exact diagnosis. Based on genetics or a novel functional diagnostic approach, a definite individual diagnosis was established for each patient and they started Rituximab therapy. Autoimmune phenomena and generalized lymphadenopathy disappeared and remained well controlled during the observation period (3-4 years) without adverse effects. Quality of life increased remarkably in both patients.