Recruitment of monocytes and mature macrophages in mouse pubic symphysis relaxation during pregnancy and postpartum recovery

Appropriate remodeling of the female lower reproductive tract and pelvic floor is essential during normal mammalian pregnancy, labor, and postpartum recovery. During mouse pregnancy, in addition to reproductive tract modifications, the pubic symphysis (PS) is remodeled into a soft interpubic ligament (IpL) to provide safe delivery of the offspring and fast postpartum recovery. Although temporal changes in the phenotypes of myeloid cells, such as mononuclear phagocytes, are crucial to remodeling the lower reproductive tract organs in preparation for a safe delivery, little is known about the involvement of recruited monocytes or macrophages inmouse PS remodeling. We used combined light microscopy, electron microscopy, and qPCR analysis to investigate the profile of recruited monocytes and macrophage polarization markers in C57Bl6 mouse interpubic tissues during pregnancy (D12, D18, and D19) and early days postpartum (1 dpp and 3 dpp) to better identify their presence in proper remodeling of the mouse PS. Our morphological data show that the number of recruited monocytes is increased in interpubic tissues and that recruited monocytes differentiate into proinflammatory or anti-inflammatory macrophage phenotypes from D18 to 3 dpp, which may contribute to dynamic changes in the gene expression of specific inflammatory mediators involved in interpubic tissue remodeling at these time points. Therefore, our morphological and quantitative gene expression data suggest that both differentiated macrophages from recruited monocytes and polarized macrophages may collaborate for IpL relaxation at labor and the appropriate repair of the PS after the first pregnancy. Summary Sentence Recruited monocytes and mature macrophages are present in the mouse pubic symphysis and may contribute to mouse pubic symphysis relaxation during late pregnancy and postpartum recovery1012466477CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP140714/2016-2; 302208/2017-82012/25038-8; 2015/23616-

Delayed phagocytosis and bacterial killing in Chediak-Higashi syndrome neutrophils detected by a fluorochrome assay: ultrastructural aspects

The few studies already published about phagocyte functions in Chediak-Higashi syndrome (CHS) has stated that neutrophils present slow rate of bacterial killing but normally ingest microorganisms. In the present study, both phagocytosis and killing of Staphylococcus aureus were verified to be in neutrophils from two patients with CHS when these functions were simultaneously evaluated by a fluorochrome phagocytosis assay. Electron microscopic examination showed morphologic differences among neutophils from CHS patients and normal neutrophils regarding the cytoplasmic structures and the aspects of the phagolysosomes. It was noteworthy the presence of giant phagolysosomes enclosing bacteria in active proliferation commonly observed in CHS neutrophils after 45 min of phagocytosis, wich corresponded with the impaired bactericidal activity of these leukocytes. The present results suggest that phagocytosis may also be defective in CHS, and point out to the sensitivity of the fluorochrome phagocytosis assay and its application in clinical laboratories

Ultrastructural And Immunohistochemical Analysis Of Proteoglycans In Mouse Pubic Symphysis.

Proteoglycans were accurately localized in mouse pubic symphyseal tissues using the cuprolinic blue method. Specific glycosaminoglycans degradative enzymes, together with chondroitin sulfate and decorin antibodies, allowed the identification of glycosaminoglycans. Chondroitin sulfate proteoglycans were the main proteoglycans observed in hyaline cartilage, fibrocartilage, and dense connective tissue. Ultrastructurally, they were seen as electron-dense granules and filaments. The granules, rich in chondroitin sulfate chains, were exclusively found in hyaline cartilage, whereas filaments were present in cartilage, fibrocartilage, and dense connective tissue. The latter were classified by size and susceptibility to enzyme digestion into F1, F2 and F3 filaments: F1 filaments were small, thin, and collagen fibril-associated; F2 filaments were thick, heavily stained, and localized around individual collagen fibrils and between bundles of collagen fibrils; and F3 filaments were scattered throughout elastic fiber surfaces. Considering their localization, susceptibility to chondroitinase AC and immunohistochemical detection, the symphysial F1 filaments were found to be preferentially decorin substituted with chondroitin sulfate side chains. The F2 filaments were also susceptible to chondroitinase AC treatment, whereas F3 filaments could be digested by heparitinase. The data thus obtained on the localization and identification of pubic symphyseal proteoglycans in virgin mice may be useful in the study of structural modifications that occur throughout pregnancy.27647-5

Growth Factor Stimulation Improves the Structure and Properties of Scaffold-Free Engineered Auricular Cartilage Constructs

<div><p>The reconstruction of the external ear to correct congenital deformities or repair following trauma remains a significant challenge in reconstructive surgery. Previously, we have developed a novel approach to create scaffold-free, tissue engineering elastic cartilage constructs directly from a small population of donor cells. Although the developed constructs appeared to adopt the structural appearance of native auricular cartilage, the constructs displayed limited expression and poor localization of elastin. In the present study, the effect of growth factor supplementation (insulin, IGF-1, or TGF-β1) was investigated to stimulate elastogenesis as well as to improve overall tissue formation. Using rabbit auricular chondrocytes, bioreactor-cultivated constructs supplemented with either insulin or IGF-1 displayed increased deposition of cartilaginous ECM, improved mechanical properties, and thicknesses comparable to native auricular cartilage after 4 weeks of growth. Similarly, growth factor supplementation resulted in increased expression and improved localization of elastin, primarily restricted within the cartilaginous region of the tissue construct. Additional studies were conducted to determine whether scaffold-free engineered auricular cartilage constructs could be developed in the 3D shape of the external ear. Isolated auricular chondrocytes were grown in rapid-prototyped tissue culture molds with additional insulin or IGF-1 supplementation during bioreactor cultivation. Using this approach, the developed tissue constructs were flexible and had a 3D shape in very good agreement to the culture mold (average error <400 µm). While scaffold-free, engineered auricular cartilage constructs can be created with both the appropriate tissue structure and 3D shape of the external ear, future studies will be aimed assessing potential changes in construct shape and properties after subcutaneous implantation.</p></div

Hyaluronan involvement in the changes of mouse interpubic tissue during late pregnancy and post-partum

The present work quantifies hyaluronan (HA) during the late pregnancy and post-partum in order to provide a better understanding of the role of HA in the adaptations that occur in the pubic symphysis during this period. HA was quantified in situ (histochemically) and in interpubic tissue extracts by fluorimetric assay. Samples were taken from virgin mice and from pregnant animals at various stages of pregnancy: 12th- 18th days into pregnancy, the day of delivery (D19) and the 3rd and 5th day post-partum. the quantitative fluorimetric analysis indicated a gradual increase of HA in the interpubic tissue throughout late pregnancy (2.4-4.6 mu g/mg dry weight). This was followed by a decrease beginning on D19 (12.4 mu g/mg), reaching close to virgin levels (2.2 mu g/mg) on the 5th day post-partum. the same optical density changes could be seen in the HA staining. Furthermore, the histochemical analysis demonstrated the presence of HA both in the extracellular matrix of the tissue and within its cells. Such results indicate that the extracellular presence of HA may contribute to the transformation of the symphysis into a flexible structure. in addition, HA's intracellular presence (until the 18th day of pregnancy) may contribute to cellular proliferation. Finally, during parturition and on the 5th day post-partum, HA may contribute to the maintenance of the myofibroblastic phenotype of ligament cells, aiding the ligament involution after parturition. (c) 2008 International Federation for Cell Biology. Published by Elsevier B.V. All rights reserved.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Discipline Histol & Struct Biol, Dept Morphol, BR-04023062 São Paulo, BrazilUniv Estadual Campinas, Inst Biol, Dept Histol & Embryol, São Paulo, BrazilUniversidade Federal de São Paulo, Div Mol Biol, Dept Biochem, São Paulo, BrazilUniversidade Federal de São Paulo, Discipline Histol & Struct Biol, Dept Morphol, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, Div Mol Biol, Dept Biochem, São Paulo, BrazilWeb of Scienc

Biochemical properties of scaffold-free, engineered tissues compared to native auricular cartilage.

<p>Biochemical properties of engineered elastic cartilaginous tissue and native auricular tissue normalized to dry weight or DNA content. Data presented as mean ±SEM.</p><p>PG: Proteoglycans.</p>a<p>Significantly different from all other groups (<i>p</i><0.05).</p>b<p>Significantly different from control (<i>p</i><0.05).</p

Immunohistochemical localization of elastin and collagen X in the scaffold-free, engineered tissue constructs.

<p>Immunohistochemical localization of elastin (red) (A, C, E, G) and collagen X (green) (B, D, F, H) in the engineered elastic cartilaginous constructs generated from monolayer preparations after 4 weeks of bioreactor culture compared to native auricular cartilage. Native auricular cartilage (A, B), Control (C, D), IGF-1 (E, F), and Insulin (G, H). Letters “P” and “C” refer to the perichondrial-like and cartilaginous regions, respectively. Scale bar: 30 µm.</p

Transmission microscopy of cellular ultrastructural and extracellular matrix organization.

<p>Transmission electron micrographs, within the cartilaginous region, of the engineered elastic cartilaginous constructs after 4 weeks of bioreactor culture compared to native auricular cartilage. Native auricular cartilage (A, B), Control (C, D), IGF-1 (E, F), and Insulin (G, H). Separate images were taken for both the cellular ultrastructure (A, C, E, G) and organization of the extracellular matrix (B, D, F, H). Arrows denote presence of elastic fibres. In native auricular cartilage, the elastic fibres (B and insert) appear to be more organized, dense and weakly contrasted compared to the elastic fibres present in the engineered constructs (D, F, H). Scale bars: A, C, E, G: 5 µm; B: 2 µm; D, F, H: 1 µm.</p

Histological and immunohistochemical appearance of generated scaffold-free, engineered tissue constructs.

<p>Histological and immunohistochemical appearance of the tissue generated from the monolayer cell preparations after 4 weeks of bioreactor culture compared to native auricular cartilage. Native auricular cartilage (A, E, I, and M), Control (B, F, J, N), IGF-1 (C, G, K, O), and Insulin (D, H, L, P). Safranin-O (sulphated proteoglycan stain) (A, B, C, D), Resorcin-Fucsin (elastin stain) (E, F, G, H), Collagen I (I, J, K, L), and Collagen II (M, N, O, P). Letters “P” and “C” refer to the perichondrial-like and cartilaginous regions, respectively. Scale bar: 30 µm.</p