OBJECTIVE

Histological structures of peritoneum, processus vaginalis, and sacs obtained from girls with inguinal hernia and boys with inguinal hernia, hydrocele, and undescended testis have been compared through immunohistochemical features to evaluate if any clue descriptive for the etiology of inguinal hernia exists.

METHODS

Parietal peritoneums (n = 6), processus vaginalises (n = 4), female hernia sacs (n = 5), male hernia sacs (n 12), and sacs from hydrocele (n = 5) and undescended testis (n = 9) were stained with indirect immunoperoxidase method. Anti-CD9, CD26, CD29, CD31, CD36, CD44, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD54, CD55, CD56, CD62E & P, CD71, CD98, CD102, CD106, CD146, CD151 monoclonals and NFL-NPH, S-100 antiserums were used. The histological structures of each group of samples were identified and compared.

RESULTS

Smooth muscle layers have been encountered within the walls of hernia sacs of both boys and girls. Although the hydrocele sacs have shown smooth muscle bundles distributed as patchy areas, smooth muscle bundles have been observed infrequently among sacs from patients with undescended testis. Peritoneum and processus vaginalis samples have been free of smooth muscle.

CONCLUSIONS

Inguinal hernia during childhood seems to be related to the presence of smooth muscle within the wall of the sac. The smooth muscle bundles may have played a role both in prevention of obliteration and clinical outcome. Because the sacs associated with undescended testis are without smooth muscles, and herniation is not a frequent association, they may not share the same etiologic basis with inguinal hernia.

The cytokine-inducible vascular cell adhesion molecule 1/CD106 is widely distributed in endothelial, epithelial, macrophage, and dentritic cells. We previously have reported a mAb termed sTA that recognizes the CD106 molecule on various TCR gamma delta T cell clones that do not proliferate in response to an anti-CD3 stimulation. In the present report, further biochemical analysis reveals two intracellular precursors (82 and 98 kDa) of the membrane-bound 110-kDa form of CD106. In addition, we detect a 100-kDa soluble form in the culture supernatant of the specific cloned lymphocytes. Phorbol ester raises the amount of the soluble CD106 in the supernatant while simultaneously inducing the disappearance of the membrane-bound form. We show that the membrane-anchored form of CD106 is converted to soluble form by a regulated proteolytic cleavage process involving a metalloprotease. EDTA and 1,10-phenanthroline, two potent inhibitors of metalloproteases, specifically inhibit the conversion of the membrane anchored to the soluble form of the CD106 molecule. In fact, these results implicate a Zn(2+)-activated metalloprotease in the regulation of CD106 expression in a subset of T cells and, therefore, represent a novel pathway of T cell functions.

Ligation of very late antigen (VLA)-4 (alpha 4 beta 1 integrin) with a cross-linked anti-alpha 4 subunit monoclonal antibody (mAb) triggered a biphasic Ca2+ response in Jurkat cell populations and in peripheral human lymphocytes. Cross-linking vascular cell adhesion molecule (VCAM)-1 (the counter-receptor of VLA-4) in ECV 304 endothelial cells generated a biphasic Ca2+ response. Tumor necrosis factor-alpha-primed human umbilical cord vascular endothelial cells also responded to the cross-linked mAb with a biphasic Ca2+ profile. Ligated VLA-4 (Jurkat cells) or VCAM-1 (ECV 304) stimulated the production of myo-inositol 1,4,5-trisphosphate. ECV 304 cells induced a biphasic Ca2+ response in Fura2-loaded Jurkat cells, whereas a transient response was observed when Jurkat cells were added to Fura2-loaded ECV 304 cells. The Ca2+ responses in these experiments involved VLA-4/VCAM-1 interactions since they were significantly reduced (approximately 80%) by prior treatment of the target cells with the relevant noncross-linked mAb. Close contact between the cells triggered mutual Ca2+ signaling as shown by spectrofluorimetric and confocal microscopy time-dependent recordings. Fibronectin and its CS-1 fragment (V25) triggered a sustained Ca2+ response in Jurkat cells (confocal microscopy). Our results suggest that the VLA-4 and VCAM-1 adhesion molecules can transduce a signal that involves activation of the phosphoinositide pathway and the mobilization of Ca2+.

Cultures of endothelial (En) cells derived from human brain microvessels were established in order to characterize adhesion molecule expression and to assay the adhesion properties of neoplastic cell lines to monolayers of En cells. Low constitutive expression of beta1 integrin (CD29), and ICAM-2 (CD102) was detected on human brain microvessel En cells. The beta1 chain of the VLA integrin family, ICAM-1, E-selectin (CD62E) and VCAM-1 (CD106) but not ICAM-2 and PECAM-1 (CD31) expression was upregulated by IL1-alpha, and TNF-alpha proinflammatory cytokines. High expression of PECAM-1 was found on non-activated human brain EN cells. In order to study the potential role of adhesion molecules in neoplastic cell adhesion two tumor cell lines were chosen. Adhesion of a cell line (DU145) derived from a cerebral metastasis of prostate carcinoma to human brain microvessel En cell monolayers was less pronounced compared to adhesion of a primary prostate carcinoma cell line (ND1). Adhesion of cerebral metastatic neoplastic cell line (DU145) was not significantly influenced by incubation of endothelial cells with different proinflammatory cytokines. The adhesion capability of primary prostate carcinoma line (NDI) was significantly upregulated by TNF-alpha proinflammatory cytokine. Furthermore, the adhesion of ND1 was partly inhibited using anti-E-selectin and VCAM-1 monoclonal antibodies. There was no significant effect of anti-adhesion antibodies on the adhesion characteristics of the cerebral metastatic (DU145) cell line. Our data demonstrate that different mechanisms are involved in the adhesion of neoplastic cells to cerebral En cells and turn our attention to the importance of adhesion molecule expression in the formation of metastases.

Peripheral lymphoid tissues contain a fibroblastic cell type referred to as stromal cells or reticulum cells which interact with lymphocytes as part of the lymphoid microenvironment. After isolation from human tonsils and expansion in vitro we analyzed the surface phenotype, extracellular matrix components, cytoskeletal products, cytokine production, binding and functional interaction with B lymphocytes of in vitro cultured stromal cells (HTSC) both in resting condition and after activation with tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. Our results show that HTSC do not express specific myeloid, lymphoid, endothelial or epithelial markers. HTSC express CD54 (ICAM-1), CD49a (VLA-1), CD49b (VLA-2), CD49c (VLA-3), CD49e (VLA-5), CD49f (VLA-6), CD29, CD51, CD44 and produce vinculin, beta-tubulin, alpha-actin, vimentin, fibronectin, laminin and collagen types I, III and IV. Activation of HTSC up-regulated CD54 (ICAM-1) and induced HLA-DR and CD106 (VCAM-1). HTSC constitutively produce interleukin (IL)-6 which is enhanced upon activation with TNF-alpha. IL-8 and granulocyte/macrophage colony-stimulating factor are detected only in the supernatants of activated HTSC. Reverse transcriptase polymerase chain reaction analysis revealed that HTSC display mRNA for IL-1 alpha, leukemia inhibitory factor and IL-7. The adhesion of tonsillar B lymphocytes to activated HTSC is mediated by CD11a/CD18 and CD54. Furthermore, HTSC can induce maximal proliferation of IL-2-activated B lymphocytes cocultured in direct cell-cell contact with HTSC. These results clearly distinguish in vitro cultured HTSC from common fibroblasts and other non-lymphoid elements present in the lymphoid parenchyma, such as follicular dendritic cells, and show that HTSC actively participate in the lymphoid microenvironment. In vitro cultures of HTSC could therefore be a useful model system for detailed analysis of the interactions between stromal cells and lymphocytes under physiological and pathological conditions.

MSCs (mesenchymal stem cells) migrate into damaged tissue and then proliferate and differentiate into various cell lineages to regenerate bone, cartilage, fat and muscle. Cell-cell adhesion of MSCs is essential for the MSC-dependent tissue regeneration after their homing into a damaged tissue. However, it remains to be elucidated what kinds of adhesion molecules play important roles in the cell-cell communication between MSCs. In order to identify adhesion molecules that facilitate mutual contact between MSCs, a comprehensive analysis of mRNA expression in adhesion molecules was performed by comparing profiles of expression status of adhesion molecules in MSCs at low- and high-cell density. We found that the expression level of VCAM1 (vascular cell adhesion molecule-1)/CD106 was clearly up-regulated in the human bone marrow-derived MSCs-UE7T-13 cells - under a condition of high cell density. Intriguingly, the migratory ability of the cells was clearly accelerated by a knockdown of VCAM1. Furthermore, the migratory ability of UE7T-13 cells was decreased by the over expression of exogenous VCAM1. In addition, the high cell density-induced expression of VCAM1 was clearly suppressed by NF-κB (nuclear factor-κB) signalling-related protein kinase inhibitors such as an IKK-2 (IκB kinase-2) inhibitor VI. In conclusion, the high cell density-induced VCAM1 expression through the NF-κB pathway inhibits the migratory ability of human bone marrow-derived MSCs.

The germinal centre (GC) is a specialized microenvironment where high-affinity antibodies are produced through hypermutation and isotype switching. Follicular dendritic cells (FDCs) are the stromal cells of the GC. The timely expansion and establishment of an FDC network is essential for a protective GC reaction; however, only a few factors modulating FDC development have been recognized. In this study, we report that interleukin-15 (IL-15) enhances human primary FDC proliferation and regulates cytokine secretion. The FDCs express IL-15 receptor complexes for IL-15 signal transduction as well as for specific binding. Moreover, the secretion of chemokines CCL-2, CCL-5, CXCL-5 and CXCL-8 was reduced by blocking IL-15 signalling while the secretion of other cytokines, and the expression of CD14, CD44, CD54 (ICAM-1) and CD106 (VCAM-1) proteins remained unchanged. These results suggest that IL-15 plays a crucial role in the development of FDC networks during GC reaction, offering a new target for immune modulation.

BACKGROUND

Myeloma plasma cells interact with the bone marrow microenvironment which, in turn, supports their growth and protects them from apoptosis. In vitro studies have demonstrated the antitumor potential of zoledronic acid (ZOL) on myeloma cell lines, but few data are available on its effects on bone marrow stromal cells (BMSCs). The aim of the current study was to evaluate the antiproliferative and apoptotic effect of ZOL on BMSCs, as well as its effect on the expression of adhesion molecules.

METHODS

BMSCs, obtained from bone marrow mononucleated cells of 8 patients with multiple myeloma, were treated with increasing concentrations of ZOL for 3 days. Cytotoxic effect was analyzed by 3-(4-5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide; thiazolyl blue (MTT) assay whereas the induction of apoptosis was evaluated by flow cytometric detection of fluorescein isothiocyanate-labeled annexin V, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, and nuclear changes. Moreover, expression of CD106, CD56, CD50, CD49d, CD44, and CD40 was analyzed by flow cytometry. Data were evaluated by the Friedman test.

RESULTS

After 3 days of exposure at concentrations of 10(-4) to 10(-5) M, ZOL induced a decrease in proliferation (P < 0.0001) and an increase in apoptosis (P < 0.002). Analysis of culture supernatants showed that myeloma BMSCs expressed interleukin (IL)-6, negligible levels of tumor necrosis factor-alpha, and no IL-1beta. In vitro exposure to the lowest concentrations of ZOL decreased IL-6 production by BMSCs. Among the adhesion molecules, CD106, CD54, CD49d, and CD40, which were strongly expressed at baseline, showed a statistically significant reduction compared with controls after exposure to ZOL.

CONCLUSIONS

ZOL interfered with myeloma BMSCs by reducing proliferation, increasing apoptosis, and modifying the pattern of expression of adhesion molecules, especially those involved in plasma cell binding. These effects on BMSCs might explain the antitumor activity of ZOL.

The phenotypes and functions of endothelial cells (EC), a heterogeneous cell population, vary along the vascular tree and even in the same organ between different vessels. The placenta is an organ with abundant vessels. To enhance further knowledge concerning placenta derived EC, we develop a new method for isolation, purification and culture of these EC. Moreover, in order to investigate the peculiarity of placenta derived EC we compare their phenotypic and functional characteristics with human dermal lymphatic endothelial cells (HDLEC) and human umbilical vein endothelial cells (HUVEC). Freshly isolated placenta derived EC displayed an elongated shape with pale cytoplasm and showed the typical cobblestone pattern of EC but also a swirling pattern when confluent. FISH-analyses of the isolated EC from placentae of male fetus revealed an XY genotype strongly indicating their fetal origin. Characterisation of placenta derived fetal EC (fEC) underlined their blood vessel phenotype by the expression of vWF, Ulex europaeus lectin-1, HLA-class I molecules, CD31, CD34, CD36, CD51/61, CD54, CD62E, CD105, CD106, CD133, CD141, CD143, CD144, CD146, VEGFR-1, VEGFR-2, EN-4, PAL-E, BMA120, Tie-1, Tie-2 and α-Tubulin. In contrast to previous reports the expression of lymphatic markers, like VEGFR-3, LYVE-1, Prox-1 and Podoplanin was consistently negative. Haematopoietic surface markers like CD45 and CD14 were also always negative. Various functional tests (Dil-Ac-LDL uptake, Matrigel assay and TNF-α induced upregulation of CD62E and CD54) substantiated the endothelial nature of propagated fEC. At the ultrastructural level, fEC harboured numerous microvilli, micropinocytic vesicles at their basis, were rich in intermediate filaments and possessed typical Weibel - Palade bodies. In conclusion, the placenta is a plentiful source of fetal, microvascular, blood EC with an expression profile (CD34+, CD133+, VEGFR-2+, CD45-) suggestive of an endothelial progenitor phenotype.

OBJECTIVE

Bone marrow adherent cells contain conventional bone marrow stromal cells and mesenchymal stem cells and these cells constitute the hematopoietic microenvironment. Mesenchymal stem cells have the capacity to give rise to multiple mesenchymal lineage cells and even ectodermal lineage cells. In the present study, we investigated what types of tumor cells are inducible from BM adherent cells by chemical carcinogens.

METHODS

Bone marrow cells from neonatal C3H/HeN mice were collected within 24 h after birth and then cultured. Four days later, bone marrow adherent cells were obtained and the cells were treated with 3-methylcholanthrene.

RESULTS

By this treatment, some transformed clones consisting of large spindle cells were obtained. The transformed cells were highly positive for CD44 and were positive for Sca-1, CD49d and CD106, whereas the cells were negative for hematolymphoid markers. The cell clones had the ability to support hematopoiesis in vitro. These results indicate that the transformed cell lines have the characteristics of BM stromal cells/mesenchymal stem cells. Moreover, during culture of the transformed cells, spontaneous bone nodule formation was observed. When the transformed cells were inoculated into immunodeficient mice subcutaneously, the neoplasms grew in the subcutaneous tissue of the mice. Microscopically and ultrastructurally, the neoplasms showed the typical morphology of undifferentiated high-grade pleomorphic sarcoma (UHGPS). Bone-related genes have been found to be expressed in both transformed cells and UHGPSs.

CONCLUSIONS

The present study suggests that UHGPSs are derived from BM stromal cells, probably mesenchymal stem cells.

Aging is a critical determinant of regenerative capacity in many organ systems, but it remains unresolved in the lung. This study examines murine lung cell dynamics during age-dependent lung regeneration. Proliferation of lung progenitor cells (EpCAM(neg)/Sca-1(high) lung mesenchymal stromal cells - LMSCs, EpCAM(pos)/Sca-1(low) epithelial progenitor cells, proSP-C(pos) alveolar type II epithelial cells - AECII, and CD31(pos) - endothelial cells) was tracked to day 3 or 7 after pneumonectomy (PNX) or SHAM surgery in 3, 9, and 17 month mice. In 3 month mice, post-PNX LMSC proliferation peaked early (3 days), with 50%-80% more BrdU-positive cells than the other cell types, which peaked later (4-7 days). In older mice (9 and 17 month), abundance and post-PNX proliferation of LMSCs at day 3 were reduced (40%-80%). In both young and old mice, LMSCs were isolated and compared phenotypically with whole lung non-LMSCs. Donor age had no qualitative effect on the phenotype (LMSC vs. non-LMSC), with increased expression of CD90/Thy1, CD105/Eng, CD106/Vcam, CD146/Mcam, and Pdgfrα, and up-regulation of mRNA encoding Fap, Eln, Col1a1, Col3a1, Aldh1a3, Arhgef25, Dner, Fgfr1, and Midkine. However, compared with LMSCs isolated from young mice, LMSCs from older mice exhibited reduced mRNA expression of retinoic acid (Aldh1a3, Rbp4), Fgf/Wnt (Fgfr1, Sfrp1, Wnt2, and Ctnnb1), and elastogenesis (Col1a1, Eln, Fbn1, and Sdc2) pathway genes. Isolated LMSCs from older mice also demonstrated lower colony-forming units (-67%), growth potential (-60% by day 7), ALDH activity (-49%), and telomerase activity (-47%). Therefore, age is associated with declining proliferative potential and regenerative functions of LMSCs in the lung.

The wound healing is a complex process wherein inflammation, proliferation and regeneration evolve according to a spatio-temporal pattern from the activation of coagulation cascade to the formation of a plug clot including fibrin matrix, blood-borne cells and cytokines/growth factors. Creating environments conducive to tissue repair, the haemoderivatives are commonly proposed for the treatment of hard-to-heal wounds. Here, we explored in vitro the intrinsic regenerative potentialities of a leucocyte- and platelet-rich fibrin product, known as CPL-MB, defining the stemness grade of cells sprouting from the haemoderivative. Using highly concentrated serum-based medium to simulate wound conditions, we isolated fibroblast-like cells (CPL-CMCs) adhering to plastic and showing stable in vitro propagation, heterogeneous stem cell expression pattern, endothelial adhesive properties and immunomodulatory profile. Due to their blood derivation and expression of CXCR4, CPL-CMCs have been suggested to be immature cells circulating in peripheral blood at quiescent state until activation by both coagulation event and inflammatory stimuli such as stromal-derived factor 1/SDF1. Expressing integrins (CD49f, CD103), vascular adhesion molecules (CD106, CD166), endoglin (CD105) and remodelling matrix enzymes (MMP2, MMP9, MMP13), they showed a transendothelial migratory potential besides multipotency. Taken together, our data suggested that a standardized, reliable and economically feasible blood product such as CPL-MB functions as an artificial stem cell niche that, under permissive conditions, originate ex vivo immature cells that could be useful for autologous stem cell-based therapies.

Increased thrombocyte activation leads to a higher likelihood of coagulation in sickle-cell disease. On the other hand, chronic inflammation and endothelial cell activation promote vaso-occlusion. The effect of circulating microparticles derived from erythrocytes, monocytes, thrombocytes, and endothelial cells on the vaso-occlusive process is unclear. This study aims to analyze the relationship between sickle-cell disease and miscellaneous organ complications by defining the circulating microparticles during the steady-state and painful crisis periods in 45 patients with sickle-cell disease. Microparticle analysis was conducted using an eight-parameter flow cytometric method, using CD61 PERCP, CD142PE, CD106 FITC, CD14 APC-H7, CD235a FITC, and Annexin-V APC monoclonal antibodies. Microparticle levels of sickle-cell patients were found to be significantly higher during both painful crisis and steady-state situations compared with the control group (for all, p < 0.001). Among these microparticles, levels of erythrocyte microparticles (eMPs) were significantly higher during crisis than in the steady-state period (eMP steady state vs. painful crisis: 7.59 ± 12.24 vs. 7.59 ± 12.24, respectively; p < 0.01). Microparticles, including eMPs, were not affected by hydroxyurea treatment. Their level did not reflect the high frequency of crisis (>3 times/year). Thrombocyte microparticle levels were found to be higher in patients with nephropathia than in those without (48.05 ± 40.23 vs. 7.67 ± 6.75, respectively; p < 0.049). Circulating microparticles seem to be involved in the pathogenesis of sickle-cell disease. eMPs may help with the management of crisis. Thrombocyte microparticles might predict renal damage induced by vaso-occlusion.

BACKGROUND

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.

Endothelial pathology is considered to play a key role in septic shock. Since endothelial-derived microvesicles (MV) are elevated in various diseases associated with endothelial pathology, they are considered surrogate markers of the endothelial state. By analyzing the signature of circulating MV with high-sensitivity flow cytometry (hsFC), we wanted to test the hypothesis whether endothelial-derived MV are increased in septic shock.

MV in blood from healthy volunteers and patients with septic shock treated in a medical intensive care unit were quantified by hsFC, which has an improved detection limit of approximately 0.3 μm.

Patients with septic shock (n = 30) showed 3-fold higher levels of CD31+/CD41- MV (58.5 (26.4-101.2) [median (25th-75th percentile)] vs. 19.5 (12.8-25.4) MV/μL; P <0.001) compared with healthy volunteers (n = 18). Absolute counts of CD144+, CD62E+, and CD106+ MV, specific for endothelial-derived MV, were low in all groups. The number of CD31+/CD41- MV correlated significantly with leukocyte count (rs = 0.64; P <0.001). Platelet-derived CD41+ MV were significantly elevated in the group dying within 48 h after inclusion (639.1 (321.3-969.7) vs. 221.5 (119.5-456.9) MV/μL; P = 0.037). Patients dying within 48 h had also significantly higher levels of CD31+/CD41-/AnnexinV- MV (51.9 (24.9-259.8) vs. 18.9 (9.7-31) MV/μL; P = 0.028).

Despite an improved detection limit for MV by using hsFC, counts of endothelial-specific MV are unexpectedly low in patients with septic shock. Increased amounts of CD41+ and CD31+/CD41-/AnnexinV- MV indicate release by activated platelets and possibly leukocytes correlating with unfavorable outcome.

Stem cells are undifferentiated cells located in different parts of the body. The major role of stem cells is to restore of injured tissues. Since the discover of stem cells, they gained a big attention due to their differentiation and regeneration capacity. The main source of stem cells was known as bone marrow. However, different sources for obtaining stem cells were discovered. Dental tissues, a new source for stem cells, provide cells having mesenchymal stem cell characteristics such as fibroblast-like structure, expression of surface antigens specific for mesenchymal stem cells, regeneration ability, multilineage differentiation capacity and immunomodulatory features. Dental pulp stem cells (DPSCs), dental follicle progenitor cells (DFPCs), stem cells from apical papilla (SCAP), tooth germ stem cells (TGSCs) and periodontal ligament stem cells (PDLSCs) are stem cells derived from dental tissues as well as stem cells from exfoliated deciduous teeth (SHED). Dental stem cells express mesenchymal stem cell markers like Stro-1, CD146, CD106, CD90, CD73 CD29 and CD13. However, they do not express hematopoietic stem cell markers such as CD11b, CD45 and CD34. Dental stem cells are able to undergo myogenic, chondrogenic, adipogenic, neurogenic, osteogenic and odontogenic differentiation. Thanks to these differentiation ability of dental stem cells, they can easily be manipulated in regenerative medicine. Dental stem cells, that can effortlessly be transfected, can also be used in cell therapy application. Immunomodulatory features of dental stem cells make them suitable candidates for the therapy of immune-related disorders. Dental stem cells with high potentials such as ability of self-renewal, mesenchymal stem cell characteristics, multilineage differentiation and immunomodulation are promising tool for in vitro and in vivo differentiation studies as well as the therapy of immune-related diseases.

Despite complete matching of siblings for the HLA loci, after bone marrow transplantation (BMT), approximately 20% develop graft-versus-host disease (GVHD). This is presumably due to incompatibility of minor histocompatibility antigens (mHa). We investigated the polymorphisms of 14 adhesion molecules (CD2, CD28, CD31, CD34, CD36, CD42, CD44, CD48, CD49b, CD54, CD62L, CD86, CD102, and CD106) in Japanese subjects and their association with the occurrence of GVHD after allogeneic HLA identical BMT. Six molecules (CD2, CD31, CD42, CD49b, CD54, and CD62L), which were found to be polymorphic, were then examined in 118 HLA identical sibling donors and recipients who had undergone BMT. Association of the incompatibility of the polymorphic molecules with the presence or absence of GVHD was examined. In these six, we observed a significant correlation between acute GVHD and the compatibility of CD31 (codons 563/670) (Pcorrected = .018), and CD31 (codons 563/670) + CD62L (Pcorrected = .018) in patients with the HLA-B44-like superfamily. In patients with the HLA-A3-like superfamily, the compatibility of CD62L (Pcorrected = .03) and CD62L + CD49b (P = . 004, Pcorrected = .078) was associated with acute GVHD. Therefore, CD31, CD49b, and CD62L might be candidates for immunodominant mHa.

The isolation of primary endothelial cells from murine tissues has long been a challenge and remains a difficult task. Using GFP transgenic C57/BL6 mice as donors, we describe a reliable method to isolate pulmonary endothelial cells by flow cytometry after staining with DiI-Ac-low density lipoprotein (LDL). After mechanical dissociation of murine lung tissue and enzymatic digestion, adherent cells can be quickly stained and sorted by flow cytometry. The isolated cells express endothelial cell markers such as CD31, MECA32 and CD106 and stained positive for Isolectin B4. After 50-fold expansion using standard endothelial growth media, cells could be transplanted into lethally irradiated allogeneic hosts and were detectable using fluorescence microscopy up to 24-h post-transplantation in pulmonary tissue.

Many researchers world over are currently investigating the suitability of stromal cells harvested from foetal tissues for allogeneic cell transplantation therapies or for tissue engineering purposes. In this study, we have investigated the chondrogenic potential of mesenchymal stromal cells (MSCs) isolated from whole sections of human umbilical cord or mixed cord (UCSCs-MC), and compared them with cells isolated from synovial membrane (SMSCs), Hoffa's fat pad (HFPSCs) and cartilage. All MSCs were positive for surface markers including CD73, CD90, CD105, CD44, CD146 and CD166, but negative for CD11b, CD19, CD34, CD45 and HLA-DR in addition to CD106 and CD271. Chondrogenic potential of all cell sources was studied using 3D pellet cultures incubated in the presence of different combinations of anabolic substances such as dexamethasone, IGF-1, TGF-β1, TGF-β3, BMP-2 and BMP-7. BMP-2 and dexamethasone in combination with TGF-β1 or TGF-β3 excelled at inducing chondrogenesis on SMSCs, HFPSCs and chondrocytes, as measured by glycosaminoglycans and collagen type II staining of pellets, quantitative glycosaminoglycan expression, quantitative PCR of cartilage signature genes and electron microscopy. In contrast, none of the tested growth factor combinations was sufficient to induce chondrogenesis on UCSCs-MC. Moreover, incubation of UCSCs-MC spheroids in the presence of cartilage pieces or synovial cells in co-cultures did not aid chondrogenic induction. In summary, we show that in comparison with MSCs harvested from adult joint tissues, UCSCs-MC display poor chondrogenic abilities. This observation should alert researchers at the time of considering UCSCs-MC as cartilage forming cells in tissue engineering or repair strategies.

The role of major histocompatibility complex (MHC) and adhesion molecule expression by alveolar epithelium on the modulation of immune responses in the lung is not understood. We have developed efficient methods to isolate, purify and culture human alveolar epithelial cells (type II pneumocytes) in vitro. The expression of MHC and adhesion molecules by isolated, cultured and cytokine-stimulated alveolar epithelial cells was quantified by flow cytometry, and demonstrated the presence of T-cell ligands including class I MHC, HLA-DR and HLA-DP, intracellular adhesion molecule-1 (ICAM-1; CD54) and lymphocyte function-associated antigen (LFA-3; CD58), but not vascular cell adhesion molecule-1 (VCAM-1) (CD106) or B7 (CD80). The proinflammatory cytokine interferon-gamma (IFN-gamma) caused an up-regulation of class I MHC and ICAM-1. In contrast, tumour necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) had little effect on the expression of these surface antigens by human alveolar epithelial cells. The functional activity of alveolar epithelial adhesion molecules was then studied by determining their ability to bind allogeneic lymphocytes. An increase in lymphocyte adherence to monolayers of alveolar epithelial cells was observed following in vitro activation. However, up-regulation of alveolar epithelial counter receptors with the proinflammatory cytokine gamma-IFN did not enhance adhesion. The adhesive interaction between CD18 on lymphocytes and ICAM-1 on alveolar epithelial cells was demonstrated by the use of blocking antibodies specific for both ligands. Blockade of LFA-3 on alveolar monolayers also suppressed lymphocyte adherence. In conclusion, alveolar epithelial cells expressed MHC HLA-A, B, C, HLA-DR and -DP, and functional adhesion molecules including ICAM-1 and LFA-3.

Adult mesenchymal stem cells (MSCs) hold great promise for the repair of heart defects. Both bone marrow-derived mesenchymal stem cells (BMSCs) and adipose tissue-derived stem cells (ASCs) are multipotent and may be induced by 5-azacytidine to differentiate into cardiomyocytes. However, the differentiation potential of human MSCs into sinus node-like cells has not been studied extensively. The aim of this study was to analyze the differences in proliferation and phenotype of ASCs and BMSCs from the same donors and to evaluate their capacity to differentiate into sinus node-like cells in vitro. Five passaged cells from bone marrow and adipose tissue were treated with 10 µM 5-azacytidine for 48 h and further cultured in complete medium for 4 weeks. A comparative study of cultured ASCs and BMSCs was carried out, and the morphological parameters, proliferative capacity, expression of surface markers and differentiation potential to sinus node-like cells were characterized. No morphologic differences were observed between ASCs and BMSCs. Flow cytometric analysis revealed that ASCs and BMSCs both expressed CD29, CD44, CD90 and CD105 and did not express CD34 and CD14, while CD49d, CD106 and CD34 were differentially expressed. Growth curves and doubling time determined with the Cell Counting Kit-8 (CCK-8) demonstrated that ASCs had a stronger proliferative ability than BMSCs. Histological immuofluorescence staining suggested that ASCs and BMSCs were capable of differentiating into sinus node-like cells and that the positive expression ratios of cTNI were higher in ASCs compared to BMSCs at 4 weeks. Expression of the HCN2 and HCN4 genes was detected by reverse transcriptase polymerase chain reaction, and the results revealed that the expression of the HCN genes appeared earlier in ASC-derived sinus node-like cells. ASCs expressed HCN2 and HCN4 shortly after induction with 5-azacytidine for 2 weeks, although BMSCs expressed these genes after 4 weeks. The expression levels of HCN2 and HCN4 mRNA in ASC-derived cells were higher compared to those of BMSCs at 4 weeks. In conclusion, ASCs may be a better candidate as a novel source of cell therapy in sinus bradycardia disorders than BMSCs.

CVID is immunologically characterized by defective antibody production. Various additional immunological abnormalities have been reported, but little is known of the role of adhesion molecules in CVID. In 31 CVID patients serum levels of L-selectin (CD62L), vascular cell adhesion molecule-1 (VCAM-1) (CD106) and intercellular adhesion molecule-1 (ICAM-1) (CD54) were significantly elevated compared with controls. In 15 CVID patients investigated, the number of L-selectin-positive cells was significantly reduced in both CD4+ and CD8+ lymphocytes compared with controls, and these changes were observed in both CD45RA+ and CD45RO+ subsets. In CD19+ lymphocytes the percentage of ICAM-1+ cells was significantly increased compared with controls. Fifty percent of the patients had splenomegaly. These patients demonstrated even higher serum levels of adhesion molecules, a lower percentage of L-selectin-positive and a higher percentage of CD38+ cells in many T lymphocyte subsets compared with both other CVID patients and controls. Finally, in this patient group the percentage of L-selectin-positive CD19+ lymphocytes was significantly reduced compared with both other patients and controls. These findings indicate a state of ongoing T lymphocyte activation in CVID, especially in the subgroup of patients with splenomegaly, which may contribute to the impaired antimicrobial defence observed in these patients.

The mechanism by which osteoblasts (OB) interact and modulate the phenotype and proliferation of T lymphocytes during inflammation is not well known. The effects of two regulatory cytokines, TNFalpha and IFNgamma, on the expression of CD54 (ICAM-1) and CD106 (VCAM-1) adhesion molecules and the CXCR3 ligands (CXCL9, CXCL10, CXCL11), were assessed in a primary culture of human OB by real-time PCR, flow cytometry, and immunohistochemistry. In addition, we functionally evaluated the recruitment and proliferation of T lymphocytes grown with resting or stimulated OB. According to the present data IFNgamma, either alone or in combination with TNFalpha, significantly up-regulates the expression of CD54 and CD106 and induces the expression and release of CXCL9, CXCL10, CXCL11 in OB. The supernatant of TNFalpha- and IFNgamma-activated OB induces the recruitment of T lymphocytes more significantly than stimulation by CXCR3 ligands. T lymphocyte proliferation is significantly enhanced by direct contact with TNFalpha- and IFNgamma-activated OB or by incubation with the supernatant of TNFalpha- and IFNgamma-activated OB. Blocking experiments with anti-CD11a, anti-CD49d, anti-CXCR3, and Bordetella pertussis toxin demonstrate that adhesion molecules and the CXCR3 chemokine receptor play a key role in the proliferation of T lymphocytes. The present study demonstrates the involvement of adhesion molecules (CD11a and CD49d) and chemokine receptor (CXCR3) in the mechanism by which OB recruit, interact, and modulate T lymphocyte proliferation under inflammatory conditions.

Interleukin-15 (IL-15) is a neutrophil agonist that plays a role in inflammatory disorders, including a variety of pulmonary diseases. Adhesion of neutrophils onto pulmonary cells is a major event leading to development of inflammation. Recently, elevated levels of IL-15 have been associated with different pulmonary diseases. There is no clear evidence that IL-15 modulates cell surface expression of adhesion molecules in neutrophils, or that IL-15 is involved in neutrophil adhesion onto pulmonary cells. Also, it is not clear if IL-15 induces a neutrophilic inflammation in vivo. This study was aimed at elucidation of these issues. Neutrophils were treated with IL-15 and cell surface expression of CD11a, CD11b, CD11c and CD18 was monitored by flow cytometry. The human respiratory epithelial A549 cell line was used as a substrate for the neutrophil adhesion assay and cell surface expression of CD50, CD54 and CD106 was monitored in IL-15-induced A549 cells. The murine air pouch model was used for investigating potential neutrophilic inflammation induced by IL-15 in vivo. IL-15 significantly increased neutrophil cell surface expression of CD11b and CD18 and up-regulated A549 cell surface expression of CD54. Moreover, A549 cells were found to express IL-15R components and adhesion of neutrophils onto A549 cells was increased when neutrophils or A549 cells were treated with IL-15. Finally, IL-15 induced neutrophilic inflammation in vivo and concentrations of IL-6 and CXCL2/MIP-2 were increased in IL-15-induced pouches. IL-15 might participate in inflammatory pulmonary diseases by attracting neutrophils, modulating cell surface expression molecules and increasing neutrophil adhesion onto pulmonary cells.