Characterisation of a New Human Alveolar Macrophage-Like Cell Line (Daisy).

PURPOSE: There is currently no true macrophage cell line and in vitro experiments requiring these cells currently require mitogenic stimulation of a macrophage precursor cell line (THP-1) or ex vivo maturation of circulating primary monocytes. In this study, we characterise a human macrophage cell line, derived from THP-1 cells, and compare its phenotype to the THP-1 cells. METHODS: THP-1 cells with and without mitogenic stimulation were compared to the newly derived macrophage-like cell line (Daisy) using microscopy, flow cytometry, phagocytosis assays, antigen binding assays and gene microarrays. RESULTS: We show that the cell line grows predominantly in an adherent monolayer. A panel of antibodies were chosen to investigate the cell surface phenotype of these cells using flow cytometry. Daisy cells expressed more CD11c, CD80, CD163, CD169 and CD206, but less CD14 and CD11b compared with mitogen-stimulated THP-1 cells. Unlike stimulated THP-1 cells which were barely able to bind immune complexes, Daisy cells showed large amounts of immune complex binding. Finally, although not statistically significant, the phagocytic ability of Daisy cells was greater than mitogen-stimulated THP-1 cells, suggesting that the cell line is more similar to mature macrophages. CONCLUSIONS: The observed phenotype suggests that Daisy cells are a good model of human macrophages with a phenotype similar to human alveolar macrophages.

Diagnostic utility of cell block in fine needle aspiration cytology of thyroid gland.

BACKGROUND: This study was conducted to evaluate the diagnostic utility of cell block material in fine needle aspiration (FNA) of thyroid nodules. DESIGN: A total of 242 thyroid fine need aspirations (FNAs) were performed between January 2015 and December 2015. Of those, all consecutive thyroid FNA cases with cell blocks (n = 140) from 129 patients (age: 58.9 ± 12.8 years) are included in this study. Cytology slides and cell blocks are reviewed for adequacy assessment based on the Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) and then categorizing them into TBSRTC diagnostic categories. These cases are divided into two groups, combined cytology and cell block (C + CB) and cytology without cell block (C). RESULTS: In the first group (C + CB), a total 140 cases are categorized in TBSRTC as follows: I: 13 (9.3%) cases, II: 78 (55.7%) cases, III: 7 (5%), IV: 16 (11.4%), V: 3(2.2%) and VI: 23 (16.4%). In the second group (C), the cases are classified in TBSRTC as follows: I: 23 (16.4%) cases, II: 70 (50%), III: 7 (5%), IV: 16 (11.4%), V: 3 (2.2%) and VI: 21 (15%). Nondiagnostic rate was 7.1% lower in the first group (C + CB) as compared with second group (C) (First group: 9.3% vs second group: 16.4%, P = .0764). CONCLUSIONS: Combined use of cytology slides and cell block decreases the nondiagnostic rate up to 7.1% as compared with cytology without cell block.

Effect of High Glucose on Human Alveolar Macrophage Phenotype and Phagocytosis of Mycobacteria.

PURPOSE: Diabetes mellitus (DBM) reduces immunological activity and increases susceptibility to various infections, including tuberculosis (TB). Human alveolar macrophage (hAM) functions are altered in DBM. METHODS: To mimic hyperglycemic conditions in the lung alveolus, we co-cultured a hAM cell line (Daisy cell line) with human umbilical vein endothelial cells for 48 h in the presence of culture media alone, normal glucose (5 mM), and high glucose (22 mM). Using flow cytometry, immunophenotype characterization included cell surface markers CD 11c, CD14, CD16, CD86, CD163, CD169, CD206, CX3CR-1, CSF-1R, and matrix metalloproteinase-9 (MMP9). Phagocytic function was measured by immunofluorescence microscopy at 24 h after inoculation of cells with GFP-expressing Mycobacterium smegmatis. RESULTS: Direct exposure of AMs to high glucose and exposure in the co-culture system yield different results for the same phenotypic markers. MMP9 expression was increased under both conditions. CD169 and CX3CR1 expressions were decreased when AMs were exposed directly to high glucose but increased under co-culture. Immunofluorescence assay revealed that phagocytosis decreased in AMs when directly exposed to increased glucose levels from 2.5 mM to normal glucose (5 mM), yet AMs under co-culture did not show decreased phagocytosis until concentrations were raised to 25 mM. CONCLUSION: Alteration in the expression of certain receptors may contribute to defective sentinel function of AMs, promoting susceptibility to TB in a diabetic host. Variability in cell surface marker expression under direct glucose exposure compared to exposure via co-culture reveals that cell signaling between endothelial cells and AMs may play a crucial role in the phenotypic expression of AMs.

Management of Multiple Sclerosis in the Breastfeeding Mother.

Multiple Sclerosis (MS) is an autoimmune neurological disease characterized by inflammation of the brain and spinal cord. Relapsing-Remitting MS is characterized by acute attacks followed by remission. Treatment is aimed at halting these attacks; therapy may last for months to years. Because MS disproportionately affects females and commonly begins during the childbearing years, clinicians treat pregnant or nursing MS patients. The intent of this review is to perform an in-depth analysis into the safety of drugs used in breastfeeding women with MS. This paper is composed of several drugs used in the treatment of MS and current research regarding their safety in breastfeeding including immunomodulators, immunosuppressants, monoclonal antibodies, corticosteroids, and drugs used for symptomatic treatment. Typically, some medications are large polar molecules which often do not pass into the milk in clinically relevant amounts. For this reason, interferon beta is likely safe for the infant when given to a breastfeeding mother. However, other drugs with particularly dangerous side effects may not be recommended. While treatment options are available and some data from clinical studies does exist, there continues to be a need for investigation and ongoing review of the medications used in breastfeeding mothers.

The NC2 domain of collagen IX provides chain selection and heterotrimerization.

The mechanism of chain selection and trimerization of fibril-associated collagens with interrupted triple helices (FACITs) differs from that of fibrillar collagens that have special C-propeptides. We recently showed that the second carboxyl-terminal non-collagenous domain (NC2) of homotrimeric collagen XIX forms a stable trimer and substantially stabilizes a collagen triple helix attached to either end. We then hypothesized a general trimerizing role for the NC2 domain in other FACITs. Here we analyzed the NC2 domain of human heterotrimeric collagen IX, the only member of FACITs with all three chains encoded by distinct genes. Upon oxidative folding of equimolar amounts of the alpha1, alpha2, and alpha3 chains of NC2, a stable heterotrimer with a disulfide bridge between alpha1 and alpha3 chains is formed. Our experiments show that this heterotrimerization domain can stabilize a short triple helix attached at the carboxyl-terminal end and allows for the proper oxidation of the cystine knot of type III collagen after the short triple helix.