Streptococcus suis serotype 2 is known to be a major pathogen of swine, causing mainly meningitis. It is also a zoonotic agent leading predominantly to meningitis in humans working in close contact with pigs. In this study, we investigated the ability of S. suis to up-regulate the expression of adhesion molecules involved in inflammation, using an enzyme-linked immunosorbent assay. S. suis serotype 2 stimulated the up-regulation of the expression of intercellular adhesion molecule-1 (ICAM-1, CD54), CD11a/CD18 and CD11c/CD18 on human THP-1 monocytes, but did not change that of ICAM-1, vascular cell adhesion molecule-1 (VCAM-1, CD106) and E-selectin (CD62E) on human endothelial cells. The up-regulation of adhesion molecules was time- and bacterial concentration-dependent, and cell wall components were largely responsible for such stimulation. To a lesser extent, purified haemolysin of S. suis also stimulated adhesion molecule expression. Stimulation of monocytes with strains of different origin showed that there was no clear tendency for human strains to induce a higher expression of adhesion molecules than strains from diseased pigs. Finally, monocytes stimulated with S. suis also showed an increase in adherence to endothelial cells. Hence, S. suis is capable of up-regulating important adhesion molecules involved in inflammation, which may result in an increased leucocyte recruitment into sites of infection, thus providing a possible mechanism for some of the inflammatory features of meningitis caused by this pathogen.

Arthritis is one of the most common complications of human active brucellosis, but its pathogenic mechanisms have not been completely elucidated. In this paper, we describe the role of synoviocytes in the pathogenesis of brucellar arthritis. Our results indicate that Brucella abortus infection inhibited synoviocyte apoptosis through the upregulation of antiapoptotic factors (cIAP-2, clusterin, livin, and P21/CIP/CDNK1A). In contrast, infection did not change the expression of proteins that have been involved in apoptosis induction such as Bad, Bax, cleaved procaspase 3, CytC, and TRAIL, among others; or their expression was reduced, as occurs in the case of P-p53(S15). In addition, B. abortus infection induced upregulation of adhesion molecules (CD54 and CD106), and the adhesion of monocytes and neutrophils to infected synoviocytes was significantly higher than to uninfected cells. Despite this increased adhesion, B. abortus-infected synoviocytes were able to inhibit apoptosis induced by supernatants from B. abortus-infected monocytes and neutrophils. Moreover, B. abortus infection increased soluble and membrane RANKL expression in synoviocytes that further induced monocytes to undergo osteoclastogenesis. The results presented here shed light on how the interactions of B. abortus with synovial fibroblasts may have an important role in the pathogenesis of brucellar arthritis.

The high rate of mortality associated with ovarian cancer (OC) is due in part to the development of resistance to chemotherapy, which allows the resistant tumour cells to invade and metastasise. Clarifying the mechanistic basis for drug resistance may reveal novel avenues for treatment. The present study investigated the mechanism of paclitaxel (PTX) resistance in human epithelial OC by evaluating the expression of stem cell-associated cell surface markers endoglin (CD105), CD44 antigen and vascular cell adhesion molecule 1 (CD106), in association with the malignant potential of the human OC OVCAR3 cell line and its PTX-resistant derivative OC3/TAX300. The expression of CD105, CD44 and CD106 was detected by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and flow cytometry, and cell invasion was evaluated using a Transwell invasion assay. CD105, CD44 and CD106 levels were increased in OC3/TAX300 cells compared with the OVCAR3 cells, as determined by flow cytometry (P<0.01) and RT-qPCR (P<0.05). Additionally, the number of invading cells was increased in the OC3/TAX300 group compared with the OVCAR3 group (54.7±6.65 vs. 31.8±6.55; P<0.01). A western blot analysis of cell surface marker expression in 80 clinical epithelial OC tissue samples, differing in terms of sensitivity to drug treatments, disease stage and degree of differentiation, revealed that high CD105, CD44 or CD106 expression was associated with drug resistance, advanced disease stage, poor differentiation and high rate of recurrence. These data indicated that exposure to high doses of PTX enhanced the stem-like properties of OC cells, which are associated with drug resistance and invasion and lead to poor prognosis due to induced chemoresistance and/or metastasis. Therefore, CD105, CD44 and CD106 may serve as potential stem cell-associated cell surface and prognostic markers, and therapeutic targets, in OC.

OBJECTIVE

Experimental approaches tested to date for functional restoration of salivary glands (SGs) are tissue engineering, gene transfer, and cell therapy. To further develop these therapies, identifying specific cell surface markers for the isolation of salivary acinar cells is needed. To test a panel of cell surface markers [used in the isolation of mesenchymal stem cells, (MSCs)] for the localization of salivary acinar cells.

METHODS

Human submandibular and parotid glands were immunostained with a panel of MSC markers and co-localized with salivary acinar cell differentiation markers [α-amylase, Na-K-2Cl cotransporter-1, aquaporin-5 (AQP5)]. Additional cell markers were also used, such as α-smooth muscle actin (to identify myoepithelial cells), cytokeratin-5 (basal ductal cells), and c-Kit (progenitor cells).

RESULTS

CD44 identified serous acini, while CD166 identified mucous acini. Cytokeratin-5 identified basal duct cells and 50% of myoepithelial cells. None of the remaining cell surface markers (Stro-1, CD90, CD106, CD105, CD146, CD19, CD45, and c-Kit) were expressed in any human salivary cell.

CONCLUSIONS

CD44 and CD166 localized human salivary serous and mucous acinar cells, respectively. These two cell surface markers will be useful in the isolation of specific populations of salivary acinar cells.

Mesenchymal stem cells (MSCs) have been isolated from many sources, including adults and fetuses. Previous studies have demonstrated that, compared with their adult counterpart, fetal MSCs with several remarkable advantages may be a better resource for clinical applications. In this study, we successfully isolated a rapidly proliferating cell population from limb bud of aborted fetus and termed them "human limb bud-derived mesenchymal stem cells" (hLB-MSCs). Characteristics of their morphology, phenotype, cell cycle, and differentiation properties were analyzed. These adherent cell populations have a typically spindle-shaped morphology. Flow cytometry analysis showed that hLB-MSCs are positive for CD13, CD29, CD90, CD105, and CD106, but negative for CD3, CD4, CD5, CD11b, CD14, CD15, CD34, CD45, CD45RA, and HLA-DR. The detection of cell cycle from different passages indicated that hLB-MSCs have a similar potential for propagation during long culture in vitro. The most novel finding here is that, in addition to their mesodermal differentiation (osteoblasts and adipocytes), hLB-MSCs can also differentiated into extramesenchymal lineages, such as neural (ectoderm) and hepatic (endoderm) progenies. These results indicate that hLB-MSCs have a high level of plasticity and can differentiate into cell lineages from all three embryonic layers in vitro.

This study investigated whether there are marked differences in surface markers between rabbit and human mesenchymal stem cells (MSCs). Murine and rabbit MSCs have been reported to be CD90-negative. Rat MSCs have been reported to be CD71-negative. Our previous study also shows that rabbit MSCs are CD29-negative. However, human MSCs are generally considered to be CD29-, CD71-, and CD90-positive. Therefore, the surface markers of human MSCs might differ from those of other species. Rabbit bone marrow MSCs were obtained that had a multi-differentiation potential. The phenotype of these cells was studied using flow cytometry antibodies for 25 rabbit surface markers, namely, CD13, CD14, CD29, CD31, CD34, CD44, CD45, CD49d, CD49f, CD51, CD54, CD59, CD71, CD73, CD90, CD105, CD106, CD133, CD166, MHC I, MHC II, α-smooth muscle actin (α-SMA), cytokeratin, desmin, and vimentin. The phenotype of commercially available human MSCs was similarly studied using antibodies for human surface markers. CD14, CD31, CD34, CD45, CD49d, CD49f, CD51, CD54, CD71, CD106, CD133, MHC II, and cytokeratin were absent from both rabbit and human MSCs, while CD44, α-SMA, and vimentin were present on both cell lines. CD13, CD29, CD59, CD73, CD90, CD105, CD166, and MHC I were present on human MSCs, but not on rabbit MSCs. However, desmin was present on rabbit MSCs, but not on human MSCs. In total, the surface expression of nine markers differed between human and rabbit MSCs, whereas the surface expression of 16 markers was the same in the two cell lines.

The galactoside-binding protein galectin-3 is increasingly recognized as an important player in cancer development, progression, and metastasis via its interactions with various galactoside-terminated glycans. We have shown previously that circulating galectin-3, which is increased up to 30-fold in cancer patients, promotes blood-borne metastasis in an animal cancer model. This effect is partly attributable to the interaction of galectin-3 with unknown receptor(s) on vascular endothelial cells and causes endothelial secretion of several metastasis-promoting cytokines. Here we sought to identify the galectin-3-binding molecule(s) on the endothelial cell surface responsible for the galectin-3-mediated cytokine secretion. Using two different galectin-3 affinity purification processes, we extracted four cell membrane glycoproteins, CD146/melanoma cell adhesion molecule (MCAM)/MUC18, CD31/platelet endothelial cell adhesion molecule-1 (PECAM-1), CD144/VE-cadherin, and CD106/Endoglin, from vascular endothelial cells. CD146 was the major galectin-3-binding ligand and strongly co-localized with galectin-3 on endothelial cell surfaces treated with exogenous galectin-3. Moreover, galectin-3 bound to N-linked glycans on CD146 and induced CD146 dimerization and subsequent activation of AKT signaling. siRNA-mediated suppression of CD146 expression completely abolished the galectin-3-induced secretion of IL-6 and G-CSF cytokines from the endothelial cells. Thus, CD146/MCAM is the functional galectin-3-binding ligand on endothelial cell surfaces responsible for galectin-3-induced secretion of metastasis-promoting cytokines. We conclude that CD146/MCAM interactions with circulating galectin-3 may have an important influence on cancer progression and metastasis.

Vascular endothelium is an important site for a wide array of immunological processes such as inflammation, atherosclerosis and allograft rejection. Culture methods of mouse vascular endothelium would provide an important in vitro correlate to immunological murine in vivo models. We describe a simple method to culture mouse vascular endothelium from thoracic aorta. Our cultured cells express typical phenotypic (CD105, CD31, CD106), morphological and ultrastructural (intercellular junctions, Weibel-Palade bodies) markers of vascular endothelium. They also possess functional receptors for uptake and processing of acetylated low-density lipoproteins. The mouse vascular endothelium within our system expresses high levels of MHC class I and MHC class II after activation with IFN-gamma. In addition, these cells express the accessory molecules CD80 and CD54, while they lack constitutive expression of CD86 and CD40, providing them the means to function as antigen presenting cells. Alloreactive CD4(+) and CD8(+) T lymphocytes demonstrate evidence of DNA synthesis after co-culture with activated vascular endothelium indicating their commitment to proliferation. In conclusion, we describe a simple culture system to isolate and grow mouse vascular endothelium, which provides a powerful tool to study biological interactions in vitro.

As a rule, T cell large granular lymphocyte (T-LGL) leukemia runs a chronic clinical course without need for therapy. Some cases, however, progress to an aggressive disease after the indolent clinical stage. The transformation mechanism into a high-grade malignancy has not been well studied. We have established 2 leukemia cell lines, MOTN-1 and PLT-2, derived from the same clone of CD56+ T-LGL leukemia in chronic and aggressive phases, respectively. The paired availability of such cell lines is valuable in biologic and genetic investigation of T-LGL leukemia. We used a microarray containing 406 cDNAs to elucidate alterations of gene expression between the 2 cell lines. We found a number of genes that were differentially expressed: 13 genes with increased expression and 3 genes with reduced expression in PLT-2 cells as compared to MOTN-1 cells. Increased expression of the dek, rac, Op18, CD6, CD58, CD106, Id2, ATF4, IRF5, ELL2 and D6 genes, and reduced expression of the GzmA and GzmK genes were confirmed by real-time quantitative reverse transcription-PCR, whose results paralleled the microarray data. These upregulated genes encode oncoproteins, cell surface antigens including molecules related to T cell proliferation, transcription factors, and a chemokine receptor. The two downregulated genes encode granzymes that play an important role for induction of cell death. These findings suggest that there is differential gene expression in different clinical phases of T-LGL leukemia and these differentially expressed genes would be potential targets for further studies to identify the genes involved in the transformation process of T-LGL leukemia.

CD40/CD40 ligand interaction is an important pathway for B and T cell cooperation and function; functional CD40 molecules have recently been found on nonhematopoietic cells. We detected CD40 in vivo on normal human respiratory epithelial cells and showed that its expression is increased on inflamed airway epithelium. Subsequently, we analyzed its expression and function on primary cultures of human airway epithelial cells. Our data show that CD40 is up-regulated by IFN-beta and IFN-gamma, its ligation increases the surface expression of CD54 and CD106 and it may stimulate the release of IL-6 and IL-8. The use of Janus kinase 3 (JAK3) and NF-kappaB inhibitors suggests that both basal and CD40-induced release of the two cytokines is JAK3-dependent. Using colocalization techniques, we revealed the existence of CD40/JAK3 and CD40/TNFR-associated factor 2 interplay. The extent of these interactions may be partial (2-40% of the cells) or massive (80-90% of the cells) in cultured cells. Stimulation via CD40 causes a significant increase in the number of cells expressing colocalization only in the cultures displaying low frequency of initial colocalization. Thus, airway epithelial cells, activated by CD40, may behave as effector cells of the inflammation process and should be considered priority targets for anti-inflammatory therapy. This work identifies CD40 and the correlated JAK3 signaling molecule as potential molecular targets to block the inflammatory functions of epithelial cells.

We investigated the role of the 6 domain (6D) and 7 domain (7D) forms of human VCAM-1 as counter-receptors for the alpha 4 beta 1 integrin (VLA-4) in monocyte migration induced by C5a. Across Chinese hamster ovary (CHO) cell monolayers transfected with VCAM-6D or VCAM-7D, monocyte migration was not inhibited by treatment of monocytes with mAb to CD18. Addition of mAb to alpha 4 to the CD18 mAb inhibited monocyte migration by 90% across CHO VCAM-6D and CHO VCAM-7D. mAbs to domain 1 (4B9) or domain 4 (GH12) of VCAM-1 each inhibited migration across CHO VCAM-7D partially, when monocytes were also treated with anti-CD18 mAb. When the VCAM-1 mAbs were combined, migration of these monocytes across CHO VCAM-7D was further inhibited to the same degree as with mAbs to alpha 4 plus CD18. IL-1-treated human umbilical vein endothelium (HUVE) supported CD18-independent, VLA-4-mediated monocyte migration to C5a. A mAb to domain 1 of VCAM-1 almost completely inhibited the CD18-independent migration across HUVE activated with IL-1 for 2 h or 20 h, but was less inhibitory when HUVE was treated with IL-1 for 5 h. However, when mAbs to domain 1 and domain 4 were combined, CD18-independent migration was inhibited completely under all conditions tested. These results suggest that either domain 1 or domain 4 of VCAM-1 can mediate VLA-4-dependent monocyte transendothelial migration, that VLA-4 interaction with these two domains can account for all of the VLA-4-mediated migration, and that the expression of VCAM-1 variants on HUVE depends partly on the duration of IL-1 activation.

BACKGROUND

Decidual stromal cells (DSCs) have classically been considered fibroblastic cells, although their function, cell lineage and origin are not fully understood. We previously demonstrated that human DSCs showed similarities with follicular dendritic cells (FDCs): DSCs expressed FDC-associated antigens, both types of cells are contractile and both are related to mesenchymal stem cells (MSCs). To further characterize DSCs, we investigated whether DSCs and FDCs share any distinctive phenotypical and functional characteristics.

METHODS

Human FDC lines were obtained from tonsillectomy samples, human DSC lines from elective termination of pregnancy samples and human MSC lines from bone marrow aspirates. We isolated DSC, FDC and MSC lines and compared their characteristics with flow cytometry and enzyme-linked immunosorbent assay. Cell lines were cultured with tumour necrosis factor (TNF) and lymphotoxin (LT)α(1)β(2), cytokines involved in FDC differentiation. Cell lines were also differentiated in culture after exposure to progesterone and cAMP, factors involved in the differentiation (decidualization) of DSC.

RESULTS

Like MSCs, DSCs and FDCs expressed MSC-associated antigens (CD10, CD29, CD54, CD73, CD106, α-smooth muscle actin and STRO-1) and lacked CD45 expression, and all three types of cell line showed increased expression of CD54 (ICAM-1) and CD106 (VCAM-1) when cultured TNF and LTα(1)β(2). DSCs and FDCs, however, exhibited characteristics not observed in MSCs: DSCs expressed FDC-associated antigens CD14, CD21 and CD23, B cell-activating factor and secreted C-X-C motif chemokine 13. Moreover, DSC lines but not MSC lines inhibited the spontaneous apoptosis of B lymphocytes, a typical functional attribute of FDC. During culture with progesterone and cAMP, FDCs, like DSCs but in contrast to MSCs, changed their morphology from a fibroblastic to a rounder shape, and cells secreted prolactin.

CONCLUSIONS

Our results suggest that DSCs and FDCs share a common precursor in MSCs but this precursor acquires new capacities when it homes to peripheral tissues. We discuss these shared properties in the context of immune-endocrine regulation during pregnancy.

Intravascular large cell lymphoma (IVLL) is a rare neoplasm characterized by a proliferation of lymphoma cells within the blood vessels. Its cell origin and clinicopathological characteristics have not been well understood. The study uses 5 male and 4 female patients who were diagnosed as having IVLL from 1978 to 1996. We examined cell lineage and adhesion molecules using immunohistochemical staining and performed a molecular analysis by using polymerase chain reaction (PCR) on the IgH gene, on T-cell receptor chain genes, and the Epstein-Barr virus (EBV) and in situ hybridization on EBV. The immunohistochemical and PCR results disclosed 8 cases of B- cell and one of T-cell lymphoma. Three of four cases whose frozen specimens were available expressed CD5. PCR showed EBV in 7 of 9 cases, although EBV was found by in situ hybridization in only 3 cases. Lymphoma cells express CD11a and CD49d (VLA-4), while endothelial cells expressed CD54 (CD11a ligand) and CD106 (CD49d ligand). Such interaction of these adhesion molecules might contribute to the intravascular proliferation of lymphoma cells. Furthermore, the CD5 expression of lymphoma cells suggests that IVLL most likely originates from a unique subtype of B cells, although their normal counterpart remains uncertain.

This study characterized peripheral blood mononuclear cells (PBMC) in terms of their potential in cartilage repair and investigated their ability to improve the healing in a pre-clinical large animal model. Human PBMCs were isolated with gradient centrifugation and adherent PBMC's were evaluated for their ability to differentiate into adipogenic, chondrogenic and osteogenic lineages and also for their expression of musculoskeletal genes. The phenotype of the PBMCs was evaluated using Stro-1, CD34, CD44, CD45, CD90, CD106, CD105, CD146 and CD166 cell surface markers. Osteochondral defects were created in the medial femoral condyle (MFC) of 24 Welsh mountain sheep and evaluated at a six month time point. Four cell treatment groups were evaluated in combination with collagen-GAG-scaffold: (1) MSC alone; (2) MSCs and PBMCs at a ratio of 20:1; (3) MSCs and PBMC at a ratio of 2:1 and (4) PBMCs alone. Samples from the surgical site were evaluated for mechanical properties, ICRS score and histological repair. Fresh PBMC samples were 90% positive for hematopoietic cell surface markers and negative for the MSC antibody panel (<1%, p = 0.006). However, the adherent PBMC population expressed mesenchymal stem cell markers in hypoxic culture and lacked CD34/45 positive cells (<0.2%). This finding demonstrated that the adherent cells had acquired an MSC-like phenotype and transformed in hypoxia from their original hematopoietic lineage. Four key genes in muskuloskeletal biology were significantly upregulated in adherent PBMCs by hypoxia: BMP2 4.2-fold (p = 0.0007), BMP6 10.7-fold (p = 0.0004), GDF5 2.0-fold (p = 0.002) and COL1 5.0-fold (p = 0.046). The monolayer multilineage analysis confirmed the trilineage mesenchymal potential of the adherent PBMCs. PBMC cell therapy was equally good as bone marrow MSC therapy for defects in the ovine large animal model. Our results show that PBMCs support cartilage healing and oxygen tension of the environment was found to have a key effect on the derivation of a novel adherent cell population with an MSC-like phenotype. This study presents a novel and easily attainable point-of-care cell therapy with PBMCs to treat osteochondral defects in the knee avoiding any cell manipulations outside the surgical room.

Monocytes are key players in atherosclerotic. Human monocytes display a considerable heterogeneity and at least three subsets can be distinguished. While the role of monocyte subset heterogeneity has already been well investigated in coronary artery disease (CAD), the knowledge about monocytes and their heterogeneity in peripheral artery occlusive disease (PAOD) still is limited. Therefore, we aimed to investigate monocyte subset heterogeneity in patients with PAOD. Peripheral blood was obtained from 143 patients suffering from PAOD (Rutherford stage I to VI) and three monocyte subsets were identified by flow cytometry: CD14++CD16- classical monocytes, CD14+CD16++ non-classical monocytes and CD14++CD16+ intermediate monocytes. Additionally the expression of distinct surface markers (CD106, CD162 and myeloperoxidase MPO) was analyzed. Proportions of CD14++CD16+ intermediate monocyte levels were significantly increased in advanced stages of PAOD, while classical and non-classical monocytes displayed no such trend. Moreover, CD162 and MPO expression increased significantly in intermediate monocyte subsets in advanced disease stages. Likewise, increased CD162 and MPO expression was noted in CD14++CD16- classical monocytes. These data suggest substantial dynamics in monocyte subset distributions and phenotypes in different stages of PAOD, which can either serve as biomarkers or as potential therapeutic targets to decrease the inflammatory burden in advanced stages of atherosclerosis.

Side population (SP) cells were isolated by FACS from a human amnion mesenchymal cell (AMC) layer soon after enzyme treatment. The yield of SP cells from AMC layer (AMC-SP cells) was about 0.1-0.2%. AMC-SP cells grew well with cell doublings of 40-80 days of culture. FACS profiles and immunocytostaining showed that AMC-SP cells were composed of two different cells immunologically: HLA I(-)/II(-) and HLA P/II(-). Oct-3/4 was detected in the nucleus of AMC-SP cells, when the culture was examined at the third, sixth, and 10th passages. RT-PCR showed that AMC-SP cells expressed the Oct-4, Sox-2, and Rex-1 genes. Immunocytochemistry revealed that all AMC-SP cells were vimentin+, CK19+, and nestin+. In addition, flow cytometry analysis showed that SP cells had high expression of CD13, CD29, CD44, CD46, CD49b, CD49c, CD49e, CD59, CD140a, and CD166 but low expression of CD 49d, and CD51. No evidence of expression was obtained for CD34, CD45, CD49a, CD56, CD90, CD105, CD106, CD117, CD133, CD271, or Flk-1. Upon appropriate differentiation protocols, AMC-SP cells differentiated to several cell lineages such as neuroectodermal, osteogenic, chondrogenic, and adipogenic cells. These results indicate that AMC-SP cells have multilineage potential to several cell lineages with unique immunological characteristics such as HLA I(-)/II(-) or HLA I+/II(-). AMC-SP cells should be of considerable value for regenerative medicine because they do not induce acute rejection after allotransplantation, they do not cause ethical issues, and there is no limit of supply.

Endothelial activation is a pivotal event in the development and progression of inflammation. Central to endothelial activation is the up-regulation of cellular adhesion molecules (CAMs) including E-selectin (CD62E), ICAM-1 (CD54), VCAM-1 (CD106) and PECAM-1 (CD31). These CAMs are also found in soluble forms (sCAMs). In this in vitro study of endothelial activation, we examined whether the levels of sCAMs correlate with the endothelial surface expression of CAMs in a dose-dependent and time-dependent manner. Such a correlation would support the use of sCAMs as surrogate markers for endothelial activation in inflammatory conditions. Human umbilical vein endothelial cells (HUVEC) were cultured with various concentrations of TNF-α for 8 hr and at a fixed concentration of TNF-α for various durations. The levels of soluble and surface-bound E-selectin, ICAM-1, VCAM-1 and PECAM-1 were quantified by flow cytometry. TNF-α stimulation increased CAM and sCAM expression in a dose-dependent and time-dependent manner. There was a significant positive correlation between the levels of ICAM-1 and sICAM-1 and between the levels of VCAM and sVCAM-1 in both the dose-response and time-response experiments. A positive correlation between the levels of E-selectin and sE-selectin was observed in the time-response experiment. This study supports the use of sCAMs as potential biomarkers of endothelial activation. In particular, the use of sICAM-1, sVCAM-1 and sE-selectin seems promising.

Adherence of leukocytes to cells undergoing apoptosis has been reported to be dependent on a variety of recognition pathways. These include alpha V beta 3 (CD51/CD61, vitronectin receptor), CD36 (thrombospondin receptor), macrophage class A scavenger receptor, phosphatidylserine translocated to the outer leaflet of apoptotic cell membranes, and CD14 (LPS-binding protein). We investigated the mechanism by which leukocytes adhere to apoptotic endothelial cells (EC). Peripheral blood mononuclear leukocytes and U937 monocytic cells adhered to human or bovine aortic EC induced to undergo apoptosis by withdrawal of growth factors, treatment with the promiscuous protein kinase inhibitor staurosporine, with the protein synthesis inhibitor and protein kinase activator anisomycin, or with the combination of cycloheximide and TNF-alpha. Expression of endothelial adherence molecules such as CD62E (E-selectin), CD54 (ICAM-1), and CD106 (VCAM-1) was not induced or increased by these treatments. A mAb to alpha V beta 3, exogenous thrombospondin, or blockade of phosphatidylserine by annexin V did not inhibit leukocyte adherence. Further, leukocyte binding to apoptotic EC was completely blocked by treatment of leukocytes but not EC with mAb to beta 1 integrin. These results define a novel pathway for the recognition of apoptotic cells.

Adipose tissue represents an abundant and accessible source of adult stem cells that can differentiate into cells and tissues of mesodermal origin, including osteogenic cells.

This paper describes the procedure to obtain a 5-cm3 saline sample, containing the adipose-derived stem cells (ASCs) pellet, starting from lipoaspirate obtained from a conventional abdominal liposuction.

A mean of 2.5×106 cells is isolated for each procedure; 35% (875000) of these are CD34+/CD45- cells, which express a subset of both positive (CD10, CD13, CD44, CD59, CD73, CD90, HLAABC) and negative (CD33, CD39, CD102, CD106, CD146, HLADR) cell-associated surface antigens, characterizing them as ASCs.

This procedure is easy, effective, economic and safe. It allows the harvesting of a significant number of ASCs that are ready for one-step bony regenerative surgical procedures.

BACKGROUND

The advent of monoclonal antibody stem cell marker technology has made it possible to identify a variety of human stem cells and their progeny. Specific markers exist for cells related to bone healing and bone regeneration. These include but are not limited to hematopoietic, mesenchymal, endothelial, angiogenic, and vasculargenic precursor cells.

OBJECTIVE

The purpose of this investigation was to (1) to identify, by means of cell markers, the presence of stem cells needed for bone formation within peripheral blood and bone-marrow aspirate, and (2) to ascertain whether more of those stem cells were present in the bone-marrow aspirate than the peripheral blood.

METHODS

Samples of autogenous bone-marrow aspirate and peripheral blood from 6 patients ranging in age from 23 to 73 were analyzed with 6-column flow cytometry using cell markers for stem cells relating to bone growth and bone healing. Six monoclonal antibody cell markers were utilized: CD14, CD34, CD36, CD105, CD106, and CD309 (also known as vascular endothelial growth factor receptor or KDR). Subgroups reacting to these markers or combinations of markers were then further tested with other marker combinations.

RESULTS

: The expression of specific monoclonal antibody cell markers revealed that bone marrow contained more osteogenic stem cells than peripheral blood. Bone marrow contained a higher percentage of cells that reacted with the CD34 and CD14 markers. This suggests that bone marrow contains more hematopoietic stem cells that proliferate to become myeloid progenitor cells, megakaryocytes, monocyte/macrophages, and osteoclast progenitors. When the fractions of bone marrow and peripheral blood samples that reacted with both CD34 and CD14 were further tested for CD105, more of the fraction from bone marrow reacted to CD105 than that from peripheral blood, suggesting more osteogenic potential in the bone marrow than the peripheral blood. When the fraction of bone marrow and peripheral blood samples that reacted with CD34 and CD14 were tested for the combination of CD105, CD106, and CD36, a smaller percentage of cells from the bone marrow reacted with CD36 than those from peripheral blood, suggesting that CD36 does not express for mesenchymal stem cells (MSCs).

CONCLUSIONS

Bone-marrow aspirate seems to contain a significantly greater percentage of hematopoietic, endothelial, and MSCs than peripheral blood. Of particular significance is the higher percentage of bone-marrow cells reacting to CD105, an indication of the presence of MSCs. The ability of multipotent MSCs to form osteoblasts for bone regeneration makes transplanted bone-marrow aspirate a promising tool for enhancing bone regeneration.

Semliki Forest virus A7 (SFV-A7) is a neurotropic alphavirus that leads to an asymptomatic encephalitis in adult immunocompetent mice. We studied the expression of leukocyte and endothelial cell adhesion molecules in the spleen and in the central nervous system (CNS) during SFV-A7 infection. Kinetics of the expression of LFA-1 alpha/CD11a, LFA-1 beta/CD18, Mac-1/CD11b, VLA-4/CD49d, ICAM-1/CD54 and L-selectin/CD62L was determined on splenic CD4+ and CD8+ T-cells and macrophages by flow cytometry. Time course of the expression of these antigens and VCAM-1/CD106 as well as viral antigens in the CNS was studied by immunoperoxidase staining. In the spleen, a sustained increase in LFA-1-expression and a temporary increase at day 7 in the expression of VLA-4, Mac-1 and ICAM-1 were detected on CD8+ T-cells. L-selection was down-regulated on CD4+ cells. Adhesion molecules on macrophages remained unchanged. In the CNS, expression of Mac-1+, VLA-4+ and LFA-1+ cells increased in parallel with the kinetics of the expression of their ligands ICAM-1 and VCAM-1 on brain vessels. Upregulation of adhesion of molecules peaked between days 5-8 and was most prominent in the cerebellar and brain stem white matter where viral antigens were most abundant. We conclude that the adhesion molecules profile of splenic T cells is altered during SFV-A7 infection which may influence their homing into the CNS. Macrophages are probably recruited non-specifically as a consequence of activation of the brain vascular endothelium in the inflamed areas of the brain.

The isolation of undifferentiated adult stem/progenitor cells remains a challenging task primarily due to the rare quantity of these cells in biological samples and the lack of unique markers. Herein, we report a relatively straightforward method for isolation of human mesenchymal stem cells (MSCs) based on their unusual resistance to osmotic lysis, which we term "osmotic selection" (OS). MSCs can remarkably withstand significant exposure to hypotonic conditions >> 30 min) with only a reversible impairment in cell proliferation and with no loss of stem cell potential after exposure. Comparison of MSCs to other circulating nonhematopoietic cells revealed a time regime, by which purification of these cells would be attainable without considerable cell loss. OS showed a 50-fold enrichment of fibroblast colony-forming units from umbilical cord blood samples when compared to commonly employed techniques. After upstream processing, isolated cells using OS were immunophenotyped to be CD14-, CD34-, CD45-, CD44+, CD105+, and CD106+, and displayed multipotent differentiation. Preliminary investigations to determine mechanisms responsible for osmolytic resistance revealed MSCs to have an ineffective volume of 59%, with the ability to double cell volume at infinite dilution. Disruption of filamentous actin polymerization by cytochalasin D sensitized MSCs to osmotic lysis, which suggests a cytoskeletal element involved in osmolytic resistance.

We undertook this study to characterize changes in the proliferative capacities, chondrogenic phenotypes, and gene expression profiles of human synovium-derived progenitor cells from osteoarthritic patients during in vitro expansion. Cells isolated from osteoarthritic synovia were cultured, and growth rates during serial passages were evaluated. Surface molecule expressions were determined by flow cytometry and cytogenetic analyses were performed. After chondrogenic differentiation in cell pellets, we evaluated type II collagen and glycosaminoglycan (GAG) synthesis. To assess whether the in vitro expansion of synovium-derived cells affects gene expression, we performed microarray analyses on cells at passage 0, 1, 2, 4, 6, and 8. Synovium-derived cells were rapidly expanded in vitro through passage 8 (about 130 days), and after passage 6, the proliferation rates decreased slightly with a wide range of individual variations. The expressions of CD166, CD49a, and CD106 decreased, whereas those of CD10, CD29, CD44, CD73, CD90, and CD105 showed no significant change. Karyotype analysis revealed no evidence of chromosome abnormalities. The staining of type II collagen and GAG in differentiated cell pellets showed rapid weakening. Genome-wide microarray analysis showed that synovium-derived cells from late passages over-expressed genes associated with cell cycle prolongation and cell aging, and less-expressed genes associated with cell growth stimulation. The in vitro expansion of synovium-derived cells was accompanied with decreased proliferative capacity and the chondrogenic phenotype, which might be modulated by change in gene expression patterns.

Lymphatic filariasis, a mosquito-transmitted disease commonly known as Bancroftian filariasis, is characterized by debilitating pathology linked to the progression of lymphoedema to a chronic state of elephantiasis. We performed longitudinal measurements of endothelial adhesion and angiogenic molecules in 63 Polynesian patients living in an hyperendemic focus of Wuchereria bancrofti. Decreased serum concentrations of soluble (s-) L selectin (CD62L) were noticed in sera of of patients with chronic conditions (hydrocele and elephantiasis). Chyluria was associated with increased vascular endothelial growth factor (VEGF) levels, whereas elephantiasis presented a high endothelin-1 (ET-1) profile. By contrast, increased serum concentrations of soluble intercellular (sICAM-1, CD54), but not of vascular cell (sVCAM-1, CD106), adhesion molecules were observed in sera of patients with bacterial lymphangitis used as controls. These trends are consistent with the increased permeability of vascular structures, a major clinical feature observed in acute lymphatic pathology (of bacterial or filarial origin), and of fundamental differences in the pathogenesis of hydrocele and elephantiasis. Using markers correlated with the clinical status (high ET-1 and VEGF levels for elephantiasis and chyluria, respectively; low CD62L levels for hydrocoele and elephantiasis) it should be possible to monitor disease progression in lymphatic filariasis.