Morphological evidences indicate that the interference of cimetidine on the peritubular components is responsible for detachment and apoptosis of Sertoli cells

Cimetidine, referred as antiandrogenic agent, has caused alterations in the seminiferous tubules, including alterations in the peritubular tissue and death of myoid cells by apoptosis. Regarding the structural and functional importance of the peritubular tissue for the maintenance of Sertoli cells (SC), we purpose to investigate the SC-basement membrane interface, focusing the morphological features of SC and their interaction with the basement membrane in the affected tubules by cimetidine. Ten animals were distributed into two groups, control (CG) and cimetidine (CmG) which received saline solution and 50 mg of cimetidine per kg of body weight, respectively, for 52 days. The testes were fixed, dehydrated and embedded for analyses under light and transmission electron microscopy. Paraffin sections were submitted to the TUNEL method; sections of testes embedded in glycol methacrylate were submitted to PAS method and stained by H&E for morphological and quantitative analyses of Sertoli Cells. In the CmG, the SC nuclei were positive to the TUNEL method and showed typical morphological alterations of cell death by apoptosis (from early to advanced stages). A significant reduction in the number of Sertoli Cells was probably due to death of these cells by apoptosis. A close relationship between SC nuclear alterations (including a high frequency of dislocated nuclei from the basal portion) and damage in the peritubular tissue was observed. The ultrastructural analysis showed a parallelism between the gradual advancement of apoptotic process in SC and detachment of the anchoring sites (hemidesmosomes) of SC plasma membrane from the lamina densa. The presence of portions of lamina densa underlying the detached hemidesmosomes indicates a continuous deposition of lamina densa, resulting in the thickening of the basal lamina. The results indicate a possible disarrangement of the SC cytoskeleton, including the focal adhesion structure. These alterations are related to SC apoptosis and probably result from disturbs induced by cimetidine on the peritubular tissue

Carotid Body AT4 Receptor Expression and its Upregulation in Chronic Hypoxia

Hypoxia regulates the local expression of angiotensin-generating system in the rat carotid body and the me-tabolite angiotensin IV (Ang IV) may be involved in the modulation of carotid body function. We tested the hypothesis that Ang IV-binding angiotensin AT4 receptors play a role in the adaptive change of the carotid body in hypoxia. The expression and localization of Ang IV-binding sites and AT4 receptors in the rat carotid bodies were studied with histochemistry. Specific fluorescein-labeled Ang IV binding sites and positive staining of AT4 immunoreactivity were mainly found in lobules in the carotid body. Double-labeling study showed the AT4 receptor was localized in glomus cells containing tyrosine hydroxylase, suggesting the expression in the chemosensitive cells. Intriguingly, the Ang IV-binding and AT4 immunoreactivity were more intense in the carotid body of chronically hypoxic (CH) rats (breathing 10% oxygen for 4 weeks) than the normoxic (Nx) control. Also, the protein level of AT4 receptor was doubled in the CH comparing with the Nx group, supporting an upregulation of the expression in hypoxia. To examine if Ang IV induces intracellular Ca2+ response in the carotid body, cytosolic calcium ([Ca2+]i) was measured by spectrofluorimetry in fura-2-loaded glomus cells dissociated from CH and Nx carotid bodies. Exogenous Ang IV elevated [Ca2+]i in the glomus cells and the Ang IV response was significantly greater in the CH than the Nx group. Hence, hypoxia induces an upregulation of the expression of AT4 receptors in the glomus cells of the carotid body with an increase in the Ang IV-induced [Ca2+]i elevation. This may be an additional pathway enhancing the Ang II action for the activation of chemoreflex in the hypoxic response during chronic hypoxia

Isolation and characterisation of human gingival margin-derived STRO-1/MACS+ and MACS− cell populations

Recently, gingival margin-derived stem/progenitor cells isolated via STRO-1/magnetic activated cell sorting (MACS) showed remarkable periodontal regenerative potential in vivo. As a second-stage investigation, the present study's aim was to perform in vitro characterisation and comparison of the stem/progenitor cell characteristics of sorted STRO-1-positive (MACS+) and STRO-1-negative (MACS−) cell populations from the human free gingival margin. Cells were isolated from the free gingiva using a minimally invasive technique and were magnetically sorted using anti-STRO-1 antibodies. Subsequently, the MACS+ and MACS− cell fractions were characterized by flow cytometry for expression of CD14, CD34, CD45, CD73, CD90, CD105, CD146/MUC18 and STRO-1. Colony-forming unit (CFU) and multilineage differentiation potential were assayed for both cell fractions. Mineralisation marker expression was examined using real-time polymerase chain reaction (PCR). MACS+ and MACS− cell fractions showed plastic adherence. MACS+ cells, in contrast to MACS− cells, showed all of the predefined mesenchymal stem/progenitor cell characteristics and a significantly higher number of CFUs (P<0.01). More than 95% of MACS+ cells expressed CD105, CD90 and CD73; lacked the haematopoietic markers CD45, CD34 and CD14, and expressed STRO-1 and CD146/MUC18. MACS− cells showed a different surface marker expression profile, with almost no expression of CD14 or STRO-1, and more than 95% of these cells expressed CD73, CD90 and CD146/MUC18, as well as the haematopoietic markers CD34 and CD45 and CD105. MACS+ cells could be differentiated along osteoblastic, adipocytic and chondroblastic lineages. In contrast, MACS− cells demonstrated slight osteogenic potential. Unstimulated MACS+ cells showed significantly higher expression of collagen I (P<0.05) and collagen III (P<0.01), whereas MACS− cells demonstrated higher expression of osteonectin (P<0.05; Mann–Whitney). The present study is the first to compare gingival MACS+ and MACS− cell populations demonstrating that MACS+ cells, in contrast to MACS− cells, harbour stem/progenitor cell characteristics. This study also validates the effectiveness of the STRO-1/MACS+ technique for the isolation of gingival stem/progenitor cells. Human free gingival margin-derived STRO-1/MACS+ cells are a unique renewable source of multipotent stem/progenitor cells

Radiofrequency-based treatment in therapy-related clinical practice – a narrative review. Part I : acute conditions

This is an Accepted Manuscript of an article published by Taylor & Francis Group in Physical Therapy Reviews on 24 June 2015, available online at: https://www.tandfonline.com/doi/full/10.1179/1743288X15Y.0000000016Background: Radiofrequency electromagnetic field (RFEMF or simply RF)-based electrophysical agents (EPAs) have been employed in therapy-related clinical practice for several decades. They are used to reduce pain and inflammation and enhance tissue healing. Although these agents have generally become less popular in contemporary therapy practice, surveys have shown that some of these modalities are still reasonably widely used. Objective: To review the evidence for the use of non-invasive low frequency RFs (30 kHz–30 MHz) in therapy-related clinical practice. Major findings: All peer reviewed therapy-related clinical studies published in English and concerning low frequency RF were sought. Identified literature was divided into acute and chronic segments based on their clinical area and analysed to assess the volume and scope of current evidence. The studies on acute conditions were reviewed in detail for this paper. One hundred twenty clinical studies were identified, of which 30 related to acute conditions. The majority of studies employed Pulsed Shortwave Therapy (PSWT). Twenty-two studies out of 30 were related to conditions of pain and inflammation, seven to tissue healing and one to acute pneumothorax. No studies were identified on frequencies other than shortwave. Conclusions: Evidence for and against RF-based therapy is available. There is reasonable evidence in support of PSWT to alleviate postoperative pain and promote postoperative wound healing. Evidence for other acute conditions is sparse and conflicting. A general lack of research emphasis in the non-shortwave RF band is evident, with studies on acute conditions almost non-existent. Further and wider research in this area is warranted.Peer reviewe

Malignant phyllodes tumors display mesenchymal stem cell features and aldehyde dehydrogenase/disialoganglioside identify their tumor stem cells

INTRODUCTION: Although breast phyllodes tumors are rare, there is no effective therapy other than surgery. Little is known about their tumor biology. A malignant phyllodes tumor contains heterologous stromal elements, and can transform into rhabdomyosarcoma, liposarcoma and osteosarcoma. These versatile properties prompted us to explore their possible relationship to mesenchymal stem cells (MSCs) and to search for the presence of cancer stem cells (CSCs) in phyllodes tumors. METHODS: Paraffin sections of malignant phyllodes tumors were examined for various markers by immunohistochemical staining. Xenografts of human primary phyllodes tumors were established by injecting freshly isolated tumor cells into the mammary fat pad of non-obese diabetic-severe combined immunodeficient (NOD-SCID) mice. To search for CSCs, xenografted tumor cells were sorted into various subpopulations by flow cytometry and examined for their in vitro mammosphere forming capacity, in vivo tumorigenicity in NOD-SCID mice and their ability to undergo differentiation. RESULTS: Immunohistochemical analysis revealed the expression of the following 10 markers: CD44, CD29, CD106, CD166, CD105, CD90, disialoganglioside (GD2), CD117, Aldehyde dehydrogenase 1 (ALDH), and Oct-4, and 7 clinically relevant markers (CD10, CD34, p53, p63, Ki-67, Bcl-2, vimentin, and Globo H) in all 51 malignant phyllodes tumors examined, albeit to different extents. Four xenografts were successfully established from human primary phyllodes tumors. In vitro, ALDH(+) cells sorted from xenografts displayed approximately 10-fold greater mammosphere-forming capacity than ALDH(-) cells. GD2(+) cells showed a 3.9-fold greater capacity than GD2(-) cells. ALDH(+)/GD2(+)cells displayed 12.8-fold greater mammosphere forming ability than ALDH(-)/GD2(-) cells. In vivo, the tumor-initiating frequency of ALDH(+)/GD2(+) cells were up to 33-fold higher than that of ALDH(+) cells, with as few as 50 ALDH(+)/GD2(+) cells being sufficient for engraftment. Moreover, we provided the first evidence for the induction of ALDH(+)/GD2(+) cells to differentiate into neural cells of various lineages, along with the observation of neural differentiation in clinical specimens and xenografts of malignant phyllodes tumors. ALDH(+) or ALDH(+)/GD2(+) cells could also be induced to differentiate into adipocytes, osteocytes or chondrocytes. CONCLUSIONS: Our findings revealed that malignant phyllodes tumors possessed many characteristics of MSC, and their CSCs were enriched in ALDH(+) and ALDH(+)/GD2(+) subpopulations

Microfluidic Endothelium for Studying the Intravascular Adhesion of Metastatic Breast Cancer Cells

BACKGROUND:The ability to properly model intravascular steps in metastasis is essential in identifying key physical, cellular, and molecular determinants that can be targeted therapeutically to prevent metastatic disease. Research on the vascular microenvironment has been hindered by challenges in studying this compartment in metastasis under conditions that reproduce in vivo physiology while allowing facile experimental manipulation. METHODOLOGY/PRINCIPAL FINDINGS:We present a microfluidic vasculature system to model interactions between circulating breast cancer cells with microvascular endothelium at potential sites of metastasis. The microfluidic vasculature produces spatially-restricted stimulation from the basal side of the endothelium that models both organ-specific localization and polarization of chemokines and many other signaling molecules under variable flow conditions. We used this microfluidic system to produce site-specific stimulation of microvascular endothelium with CXCL12, a chemokine strongly implicated in metastasis. CONCLUSIONS/SIGNIFICANCE:When added from the basal side, CXCL12 acts through receptor CXCR4 on endothelium to promote adhesion of circulating breast cancer cells, independent of CXCL12 receptors CXCR4 or CXCR7 on tumor cells. These studies suggest that targeting CXCL12-CXCR4 signaling in endothelium may limit metastases in breast and other cancers and highlight the unique capabilities of our microfluidic device to advance studies of the intravascular microenvironment in metastasis