Expression of CD44 in previously grafted alveolar bone / Expresión del marcador óseo CD44 en hueso alveolar previamente injertado

Objetive: To determine the expressions of the bone surface marker CD44 in samples of alveolar bone previously regenerated with allograft, xenograft, and mixed, using the technique of guided bone regeneration. Material and Methods: This exploratory study was approved by the institutional research and ethics committee. By means of intentional sampling and after obtaining informed consent for tissue donation, 20 samples of alveolar bone previously regenerated with guided bone regeneration therapy with particulate bone graft and membrane were taken during implant placement. The samples were stained with hematoxylin-eosin for histological analysis, and by immunohistochemistry for the detection of CD44. Results: Sections with hematoxylin-eosin showed bone tissue with the presence of osteoid matrix and mature bone matrix of usual appearance. Of the CD44+ samples, 80% were allograft and 20% xenograft. The samples with allograft-xenograft were negative. There were no differences in the intensity of CD44 expression between the positive samples. The marker was expressed in osteocytes, stromal cells, mononuclear infiltrate, and some histiocytes. Eighty percent of the CD44+ samples and 100% of the samples in which 60 or more cells were labelled corresponded to allografts (p=0.000). A total of 67% of the samples from the anterior sector, and 40% from the posterior sector were CD44+ (p=0.689). Conclusion: This study shows for the first time that guided bone regeneration using allografts is more efficient for the generation of mature bone determined by the expression of CD44, compared to the use of xenografts and mixed allograft-xenograft, regardless of the regenerated anatomical area.

Objetivo: Determinar la expresión del marcador de membrana óseo CD44 en muestras de hueso alveolar previamente regenerado con aloinjerto, xenoinjerto y mezcla mediante la técnica de regeneración ósea guiada. Material y Métodos: Con aval del Comité de Investigación y Ética, se realizó un estudio exploratorio. Por muestreo intencional y firma de consentimiento informado de donación, se tomaron durante la colocación del implante, 20 muestras de hueso alveolar previamente regenerado con terapia de regeneración ósea guiada con injerto óseo particulado y membrana. Las muestras fueron teñidas con hematoxilina-eosina para el análisis histológico y por inmunohistoquímica para la detección del CD44. Resultados: : Los cortes con hematoxilina-eosina mostraron tejido óseo con presencia de matriz osteoide y matriz ósea madura de aspecto usual. De las muestras CD44+, 80% fueron de aloinjerto y 20% de xenoinjerto. Las muestras con aloinjerto-xeoninjerto fueron negativas. No hubo diferencias en la intensidad de la expresión del CD44 entre las muestras positivas. El marcador se expresó en osteocitos, células estromales, infiltrado mononuclear y algunos histiocitos. El 80% de las muestras CD44+ y el 100% de las muestras con marcación de 60 o más células correspondían a aloinjertos (p=0,000). El 67% de las muestras del sector anterior y el 40% del sector posterior fueron CD44+ (p=0,689). Conclusión: Este estudio muestra por primera vez que la regeneración ósea guiada usando aloinjertos, es más eficiente para la generación de hueso maduro determinado por la expresión de CD44, comparado con el uso de xenoinjertos y mezcla de aloinjerto-xenoinjerto, independientemente del sector anatómico regenerado.

Differential Expression of Stem Cell Markers in Human Adamantinomatous Craniopharyngioma and Pituitary Adenoma

Background/Aims: Although craniopharyngioma (CP) is histologically benign, it is a pituitary tumour that grows rapidly and often recurs. Adamantinomatous CP (ACP) was associated with an activating mutation in ß-catenin, and it has been postulated that pituitary stem cells might play a role in oncogenesis in human ACP. Stem cells have also been identified in pituitary adenoma. Our aim was to characterize the expression pattern of ABCG2, CD44, DLL4, NANOG, NOTCH2, POU5F1/OCT4, SOX2, and SOX9 stem cell markers in human ACP and pituitary adenoma. Methods and Results: We studied 33 patients (9 ACP and 24 adenoma) using real-time quantitative PCR (RT-qPCR) and immunohistochemistry. SOX9 was up-regulated in ACP, exhibiting positive immunostaining in the epithelium and stroma, with the highest expression in patients with recurrence. CD44 was overexpressed in ACP as confirmed by immunohistochemistry. SOX2 did not significantly differ among the tumour types. The RT-qPCR array showed an increased expression of MKI67,OCT4/POU5F1, and DLL4 in all tumours. NANOG was decreased in ACP. ABCG2 was down-regulated in most of the tumours. NOTCH2 was significantly decreased in the adenomas. Conclusion: Our results confirm the presence of stem cell markers in human pituitary tumours as well as the different expression patterns of ACP and adenoma. These findings suggest that ACP may originate from a more undifferentiated cell cluster. Additionally, SOX9 immunodetection in the stroma and the highest expression levels related to the relapse of patients suggest a contribution to the aggressive behaviour and high recurrence of this tumour type.

Impact of extracellular alkalinization on the survival of human CD24-/CD44+ breast cancer stem cells associated with cellular metabolic shifts

Cancer stem cells reside in a distinct region within the tumor microenvironment that it is believed to play a fundamental role in regulating stemness, proliferation, survival, and metabolism of cancer cells. This study aimed to analyze the effect of extracellular alkalinization on metabolism and survival of human CD24-/CD44+ breast cancer stem cells (BCSCs). BCSCs were cultured in alkalinized DMEM-F12 and incubated at 37°C, 5% CO2, and 20% O2 for 30 min, 6, 24, and 48 h. After each incubation period, we analyzed the modulation of various mRNA expressions related to pH and cellular metabolic regulation using the qRT-PCR. Metabolic state was measured using colorimetric and fluorometric assays. To examine cell proliferation and apoptosis, we used trypan blue and annexin V/propidium iodide assay, respectively. This study demonstrated that alkalinization could stimulate extracellular carbonic anhydrase (CAe) activity, as well as CA9 and HIF1α expression. Under alkaline pH and HIF1α regulation, glucose consumption, extracellular lactate production, and LDH activity of BCSCs were upregulated while O2 consumption was downregulated. These metabolic shifts seemed to promote apoptosis and suppress the proliferation of BCSCs. To conclude, modulation of the extracellular environment through alkalinization could change the metabolic states of BCSCs, which in turn affect the cell survival.

Isolation of breast cancer stem cell from MDA-MB231 cell line using vincristine / Aislamiento de células madre de cáncer de mama de la línea celular MDA-MB231 utilizando vincristina

Cancer has been considered as a stem cell disease. Suspension culture combined with anti-cancer drugs has recently been proposed for isolation of cancer stem cells (CSCs). In the current study, Vincristine as an anti-cancer drug combined with suspension culture was used for isolation and purification of CSCs from human breast cancer cell line (MDA-MB231). The cells were treated with different concentrations of vincristine (0, 2, 4, 6 and 8 ng/ml). Stem cells were identified with the expression of OCT4, nanog, SOX2 and nucleostemin genes by RT-PCR. Mammosphere forming unit was measured upon suspension culture containing EGF, bFGF, LIF, B27, insulin and BSA. The isolated mammospheres were investigated for CD44 expression. Results showed that 4 ng/ml of vincristine for 72 hours could be utilized as the best and most reliable dose which eliminates around 80 % of non-cancer stem cells with no destructive effect on CSCs' viability (P> 0.05). RT-PCR demonstrated that drug treated cells expressed OCT4, nanog, SOX2 and nucleostemin. Mammosphere formation unit of cells pretreated with vincristine was significantly higher than unpretreated ones (P>0.05). Immunofluorescence staining for CD44 depicted high expression of CSC marker among the isolated mammospheres. Vincristine combined with suspension culture can be considered as an appropriate method to isolate CSC.

El cáncer ha sido considerado como una enfermedad de células madre. Recientemente se ha propuesto cultivo en suspensión en combinación con medicamentos contra el cáncer para aislamiento de las células madre del cáncer (CMC). En este estudio se utilizó la vincristina como fármaco anticanceroso combinado con cultivo en suspensión para el aislamiento y purificación de las células madre cancerosas, de la línea celular de cáncer de mama humano (MDA-MB231). Las células se trataron con diferentes concentraciones de vincristina (0, 2, 4, 6 y 8 ng/ml). Las células madre se identificaron mediante la expresión de los genes OCT4, Nanog, SOX2 y nucleostemin por RT-PCR. La unidad de formación mammosphere se midió a través de cultivo en suspensión que contenía EGF, bFGF, LIF, B27, insulina y BSA. Los mammospheres aislados fueron estudiados para la expresión de CD44. Los resultados mostraron que 4 ng/ml de vincristina durante 72 horas podrían ser utilizados como la mejor y más fiable dosis que permite eliminar alrededor del 80 % de las células madre no cancerosas, sin causar un efecto destructivo sobre la viabilidad de las CMC (P> 0,05). La RT-PCR mostró que en las células tratadas con él fármaco hubo expresión de los genes OCT4, Nanog, SOX2 y nucleostemin. La unidad de formación de las células pretratadas con vincristina fue significativamente más alta que las unidades sin tratamiento previo (P>0,05). La inmunofluorescencia para CD44 muestró una alta expresión del marcador de CMC entre mammospheres aisladas. La vincristina en combinación con el cultivo en suspensión puede ser considerado como un método apropiado para aislar CMC.

Quantitative assessment of expression of cell adhesion molecule (CD44) splice variants: CD44 standard (CD44s) and v5, v6 isoforms in oral leukoplakias: An immunohistochemical study.

Aim: The aim of this study was to semiquantitatively analyze the immunohistochemical expression pattern of CD44 standard (CD44s) and variant (CD44v) isoforms in leukoplakias using a panel of monoclonal antibodies recognizing epitopes of CD44s and of the variant exons v5 and v6. Objective: To evaluate the efficacy of CD44s and CD44 v5, v6 immunoexpression as possible molecular markers in detecting high-risk leukoplakias when screening for this oral precancer. Materials and Methods: Samples of oral leukoplakia (40 cases) and of normal mucosa (10 cases) were evaluated. Oral leukoplakia was graded into: hyperkeratosis without dysplastic change (8 cases), mild dysplasia (13 cases), moderate dysplasia (10 cases), and severe dysplasias (9 cases). Expression of CD44s,v5, v6 was analyzed by immunohistochemistry in a semiquantitative manner. Three areas of epithelium were scored B, S, and C, i.e., stratum basale, stratum corneum, and stratum spinosum, respectively in leukoplakias. Scoring of all specimens followed a two-parameter system, which implemented percentage of positive cells and staining intensities. Statistical analyses for each parameter of all groups and normals, mean, and standard deviation were calculated by using computer software package EPISTAT. Results: In normal epithelium CD44s, CD44v5, and CD44v6 were expressed as membranous proteins localized on the surface of epithelial cells. Both basal and spinous layer of epithelia expressed strong positive staining of CD44s, v5, v6 which then gradually faded into the negative staining of the superficial keratin layer. Profile of CD44s and v5 revealed that the mean levels of stratum B, S, and C in normal cases were comparable to the study cases and by Student 't' test P>0.05 not significant. There was, however, a statistically significant decrease in the expression of v6 with increasing grades of dysplasias when compared with normal mucosa. Conclusion: Among CD44s and its variant isoforms,v5, v6, in this study, variant isoform v6 may serve as a marker in detecting high-risk leukoplakias.

Rapid Isolation of Adipose Tissue-Derived Stem Cells by the Storage of Lipoaspirates

PURPOSE: This study examined a rapid isolation method decreasing the time and cost of the clinical application of adipose tissue-derived stem cells (ASCs). MATERIALS AND METHODS: Aliquots (10 g) of the lipoaspirates were stored at 4degrees C without supplying oxygen or nutrients. At the indicated time points, the yield of mononuclear cells was evaluated and the stem cell population was counted by colony forming unit-fibroblast assays. Cell surface markers, stem cell-related transcription factors, and differentiation potentials of ASCs were analyzed. RESULTS: When the lipoaspirates were stored at 4degrees C, the total yield of mononuclear cells decreased, but the stem cell population was enriched. These ASCs expressed CD44, CD73, CD90, CD105, and HLA-ABC but not CD14, CD31, CD34, CD45, CD117, CD133, and HLA-DR. The number of ASCs increased 1x1014 fold for 120 days. ASCs differentiated into osteoblasts, adipocytes, muscle cells, or neuronal cells. CONCLUSION: ASCs isolated from lipoaspirates and stored for 24 hours at 4degrees C have similar properties to ASCs isolated from fresh lipoaspirates. Our results suggest that ASCs can be isolated with high frequency by optimal storage at 4degrees C for 24 hours, and those ASCs are highly proliferative and multipotent, similar to ASCs isolated from fresh lipoaspirates. These ASCs can be useful for clinical application because they are time- and cost-efficient, and these cells maintain their stemness for a long time, like ASCs isolated from fresh lipoaspirates.

The association of co-expression of CD44v4/MMP-9 with different nodal status in high-grade breast carcinoma patients.

BACKGROUND: Breast carcinoma is one of the most common tumors in female patients, and its metastasis is a major cause of death. An experimental model has recently found the association of CD44 with MMP-9 that facilitates tumor cell invasion and metastasis. MATERIAL AND METHOD: The CD44v4 and MMP-9 were performed on tissue in paraffin blocks of 50 cases of high-grade breast carcinoma with node positive and 50 cases with node negative. RESULTS: Increased expression of MMP-9(60%) significantly observed in high-grade breast carcinoma patients with node positive (p = 0. 004), whereas CD44v4 displays no significant difference between the two groups (p-value = 0.81). Significant co-expression of CD44v4+ / MMP-9+ (46%) was observed and correlated with node-positive patients whereas the CD44v4+ / MMP-9- (54%) express in node-negative patient (p-value = 0.01). CONCLUSION: The solely expression of CD44v4 does not associate with node status. MMP-9 plays an important role to enhance breast carcinoma cell invasion and associates with lymph node metastasis. The combined expression of CD44v4 (overexpression) and derangement of MMP-9 expression was significantly associated with nodal status.

Increased expression of osteopontin in the spinal cords of Lewis rats with experimental autoimmune neuritis

To investigate the pattern of expression of osteopontin (OPN) in tissues of the central nervous system (CNS) responding to peripheral immunological stimulation, the expression of OPN was studied in the spinal cord of rats with experimental autoimmune neuritis (EAN). In this model system, the sciatic nerves and spinal nerve roots are the target organs of EAN and the spinal cord is a remote organ that may be indirectly affected. OPN was constitutively expressed in some astrocytes adjacent to the pia mater and neurons in normal rats. In rats with EAN, OPN was increased in the same cells and in some inflammatory cells, including macrophages in the subarachnoid space. Expression of CD44, a receptor of OPN, was weak in normal spinal cord tissue and increased in the entire spinal cord parenchyma in rats with EAN, as well as in inflammatory cells. These findings suggest that inflammatory cells as well as reactive astrocytes are major sources of OPN and CD44 in the spinal cord of rats with EAN. Further study is needed to elucidate the functional role of OPN in the spinal cord affected by EAN.

Detection of Epstein-Barr Virus by PCR and Expression of LMP1, p53, CD44 in Gastric Cancer

BACKGROUND: Epstein-Barr virus (EBV) is associated with various lymphoproliferative disorders and nasopharyngeal carcinoma. Recently, some gastric cancer cells were observed to contain the EBV sequence. We detected EBV in gastric cancer by using PCR to determine the frequency of EBV-associated gastric cancer, and performed immunohistochemical staining for the latent membrane protein (LMP1), p53 and CD44 to investigate the possible mechanism in EBV-associated gastric cancer. METHODS: Eighty-seven formalin-fixed and paraffin-embedded blocks (40 gastric adenocarcinomas, 34 adjacent normal tissues, 13 metastatic lymph nodes) from 40 surgically resected gastric specimens were studied. All patients were diagnosed with gastric cancer at the Kang-Nam St. Mary's Hospital between April 1995 and April 1997. After DNA was extracted from each paraffin block, we performed PCR and immunohistochemical staining for the LMP1, p53 and CD44. RESULTS: EBV was detected in 4 of 40 cases (10%). In 1 of 4 EBV-positive cases, EBV was also detected in a metastatic lymph node. The immunohistochemical staining for the LMP1, p53 and CD44 were negative in all the EBV-positive cancer patients. Of the patients having these cancers, 2 had a poorly differentiated adenocarcinoma with a lymphoepithelioma-like morphology. DISCUSSION: The frequency of EBV-associated gastric cancer is about 10% in Korea. Considering the negative result of the immunohistochemical staining for the LMP1, p53 and CD44, EBV-associated gastric cancer seems to have a different mechanism of tumorigenesis from ordinary gastric cancer or other EBV-associated cancers. This specific mechanism must be determined by further large scale studies.

Homing-Associated Cell Adhesion Molecules and Cell Cycle Status on the Nucleated Cells in the Bone Marrow, Mobilized Peripheral Blood and Cord Blood

Homing-associated cell adhesion molecules (H-CAM) on the CD34+ cells play an important role for the engraftment process following hematopoietic stem cell transplantation (HSCT). However, it seems that not only CD34+ cells but also other nucleated cells (NCs) with H-CAM could be implicated in the engraftment process and the proliferation of hematopoietic stem cells. We investigated the differences of HCAM and cell cycle status on the NCs in cord blood (CB), bone marrow (BM), and mobilized peripheral blood (PB). The proportions of CXCR4+ cells within the NC populations were greater in CB than in PB or BM (p=0.0493), although the proportions of CXCR4+, CD44+, and CD49d+ cells within the CB CD34+ cell populations were same within BM or PB. A lower proportion of CD34+CD49d+ cells within the CD34+ cell populations was more noted in CB than in PB or BM (p=0.0085). There were no differences in cell cycle status between CB and BM or PB. Our results suggest that the migrating potential of CB would be enhanced with increased CXCR4 expression on the NCs, but the adhesion potential of CB CD34+ cells would be less than that of PB and BM. These findings may help explain why the lower cell dose is required and engraftment is delayed in cord blood stem cell transplantation.

Evidence for clustered mannose as a new ligand for hyaluronan- binding protein (HABP1) from human fibroblasts.

We have earlier reported that overexpression of the gene encoding human hyaluronan-binding protein (HABP1) is functionally active, as it binds specifically with hyaluronan (HA). In this communication, we confirm the collapse of the filamentous and branched structure of HA by interaction with increasing concentrations of recombinant-HABP1 (rHABP1). HA is the reported ligand of rHABP1. Here, we show the affinity of rHABP1 towards D-mannosylated albumin (DMA) by overlay assay and purification using a DMA affinity column. Our data suggests that DMA is another ligand for HABP1. Furthermore, we have observed that DMA inhibits the binding of HA in a concentration-dependent manner, suggesting its multiligand affinity amongst carbohydrates. rHABP1 shows differential affinity towards HA and DMA which depends on pH and ionic strength. These data suggest that affinity of rHABP1 towards different ligands is regulated by the microenvironment.

Immunohistochemical Analysis of CD44s and CD44v6 in Endometriosis and Adenomyosis: Comparison with normal, hyperplastic, and malignant endometrium

The expression patterns of CD44s and CD44v6 were immunohistochemically compared with those of normal, hyperplastic and malignant endometrium. In normal endometria (n=37), endometrioses (n=46) and adenomyoses (n=20), the surface and glandular epithelial cells were negative for CD44s and CD44v6 in a proliferative pattern and positive in a secretory pattern, whereas the stroma was only positive for CD44s in both proliferative and secretory patterns. The endometrial hyperplasia (4 simple and 9 complex) had the identical patterns with normal proliferative phase of endometrium. Only one case showing complex hyperplasia with atypia was focally positive for CD44s and CD44v6 in glandular epithelia. CD44s and CD44v6 were positive in all endometrial adenocarcinomas (13), except one CD44s-negative case. In summary, the expressions of CD44s and CD44v6 in endometriosis and adenomyosis recapitulated those of normal cyclic endometrium. The expression patterns in endometrial hyperplasia were similar to those in normal proliferative endometrium, whereas the endometrial adenocarcinoma showed abnormal expressions for CD44s and CD44v6. Thus it was considered that the ectopic endometrium in endometriosis and adenomyosis was not aberrant as in endometrial carcinoma on the aspects of immunohistochemical expressions of CD44s and CD44v6.