In rheumatoid arthritis (RA) synovial fibroblasts are activated by growth factors and cytokines to proliferate and to express matrix-degrading proteases and pro-inflammatory cytokines. This contributes to cartilage degradation and joint destruction. To analyse the parameters that lead to activation of synovial fibroblasts, we established a stable human synoviocyte line (K4IM) from a healthy donor by immortalization with SV40 T antigen (TAg). Characterizing the phenotype of the immortalized K4IM cells, we found that they maintained CD44, CD54 (intercellular adhesion molecule; ICAM-1) and CD95 (Fas) expression, but lost the expression of CD106 (vascular cell adhesion molecule 1; VCAM-1) and the receptors for interleukin 1 (IL-1) and platelet-derived growth factor (PDGF). We also monitored normal expression kinetics of transcription factor Egr-1 upon activation with tumor necrosis factor alpha (TNF-alpha) or synovial fluid from RA patients. In addition, we showed that HLA-DR expression could still be upregulated by recombinant interferon gamma (rINF-gamma). The immortalized K4IM cell line therefore represents a valuable and unique tool to study mechanisms that induce or maintain synoviocyte activation.

Metastatic colorectal cancer is usually progressive despite infiltration of the tumours by T lymphocytes, suggesting that these tumour-infiltrating lymphocytes (TILs) are functionally deficient. Recently, TILs from other tumours have been shown to express reduced levels of the T-cell receptor signal-transducing CD3-zeta chain. We were interested to determine whether a similar abnormality existed in TILs from human colorectal hepatic metastasis (CHM) and, if so, whether correcting the abnormality in vitro would restore anti-tumour activity and provide support for the development of immunotherapy for colorectal hepatic metastases. Twelve of 19 TILs from colorectal hepatic metastases were successfully expanded in vitro in high-dose recombinant interleukin 2 (rlL-2) and their specific anti-tumour cytolytic activity was determined. CD3-positive (CD3+) TILs were HLA-Drhigh and CD69high, suggesting that they had been activated by exposure to antigen but expressed low levels of CD25, CD71 and the nuclear proliferation antigen Ki-67. Furthermore, they showed reduced expression of CD3-zeta compared with autologous peripheral blood T cells (PBTs) and failed to proliferate in the absence of high-dose rIL-2. Expansion of TILs in rIL-2 resulted in restoration of CD3-zeta expression and the ability to lyse K562 and Daudi cells but not autologous tumour cells. The absence of autologous tumour-specific cytolytic T-cell (CTL) activity may be due to the poor immunogenicity of colorectal tumour cells, which we found expressed only low levels of MHC I antigens and CD54 and failed to express MHC II antigens or the co-stimulatory molecules CD80, CD86 or CD106. The inability of rIL-2 to generate tumour-specific CTLs despite restoration of CD3-zeta expression and the presence of an intact lytic mechanism suggests that successful immunotherapy may require the development of strategies to increase the immunogenicity of this tumour.

While dental mesenchymal stem cells are well-studied, the origin of these cells is still unclear. Bone marrow-derived cells (BMDCs) have the potential to engraft into several tissues after injury, but whether they can become dental tissue-specific progenitor cells under normal conditions and the relationship of these cells to the tissue-resident cells are unknown. Thus, we transplanted green fluorescent protein (GFP)-labeled BMDCs into irradiated wild-type mice. We found that the engraftment of BMDCs participated in the regeneration and differentiated into periodontal specific cells after injury. Under normal conditions, there were more BMDCs engrafting into the dental mesenchymal tissue than other organs, in which the expression of stromal cell-derived factor-1 (SDF-1) was significantly higher than in other organs, and the engraftment of cells increased with time. A small fraction of GFP+ cells maintained the mesenchymal stem cell phenotype positive for CD105, CD106, and CD90, which were significantly less than the tissue-resident stem cells; meanwhile, GFP+/CD45+ cells were rare. Isolation and characterization of the dental pulp cells showed that the number of GFP+/Ki67+ cells were greater than the GFP-/Ki67+ cells. In addition, some GFP+ cells differentiated into the dental-specific cells and expressed dental-specific proteins, and can be found in the odontoblast layer after implantation of the apical bud. In conclusion, these data suggest that bone marrow progenitor cells communicate with dental tissues and become tissue-specific mesenchymal progenitor cells to maintain tissue homeostasis.

Leukocyte adhesion and transmigration through the endothelial cell (EC) layer plays a crucial role in inflammation. IL-1 alpha and TNF alpha increase EC-adhesiveness for leukocytes by stimulating surface expression of ICAM-1 (intercellular adhesion molecule 1, CD54), VCAM-1 (vascular cell adhesion molecule 1, CD106) and E-selectin (CD62E). In this study, the effects of ibuprofen on IL-1 alpha and TNF alpha-induced expression of ICAM-1, VCAM-1 and E-selectin on cultured human umbilical vein EC (HUVEC) were analyzed. Exposure to IL-1 alpha or TNF alpha resulted in an increased expression of VCAM-1, ICAM-1, and E-selectin. Ibuprofen was identified as a potent inhibitor of IL-1 alpha and TNF alpha-induced surface expression of VCAM-1 and a less potent inhibitor of pyrogen-induced expression of ICAM-1, whereas no effect on E-selectin was found. The effects of ibuprofen on VCAM-1 expression were dose-dependent (IC50 [IL-1 alpha]: 0.5 mM; IC50 [TNF alpha]: 0.5 mM) and time-dependent with maximum responses observed after 18 h. Moreover, ibuprofen abrogated pyrogen-dependent adhesion of leukocytes to HUVEC. Ibuprofen also inhibited VCAM-1 mRNA expression in pyrogen activated EC. VCAM-1-downregulation on EC by ibuprofen may contribute to the anti-inflammatory actions of the drug.

OBJECTIVE

Although gastrointestinal tract dysfunction is a common feature in patients with systemic sclerosis (SSc; scleroderma), few studies have addressed the pathogenetic mechanisms of gastrointestinal tract involvement in SSc. We previously showed that severe fibrosis and increased expression of profibrotic cytokines are important hallmarks in the gastric wall of patients with SSc. The aim of the present study was to investigate whether immune and/or microvascular abnormalities may account for tissue damage in gastric wall specimens obtained from patients with SSc.

METHODS

Gastric biopsy samples from 27 patients with SSc and 15 healthy control subjects were analyzed by immunohistochemistry for CD45/leukocyte common antigen, CD3/T cells, CD4/T helper cells, CD8/cytotoxic T cells, CD20/B cells, CD14/monocytes, CD68/macrophages, cell adhesion molecules CD11a/lymphocyte function-associated antigen 1 (LFA-1), CD49d/very late activation antigen 4 (VLA-4), CD54/intercellular adhesion molecule 1 (ICAM-1), CD106/vascular cell adhesion molecule 1 (VCAM-1), CD31/platelet endothelial cell adhesion molecule 1, and vascular endothelial growth factor (VEGF).

RESULTS

T cell infiltration was a prominent finding in gastric specimens from patients with SSc. The CD4+/CD8+ T cell ratio was significantly increased in SSc specimens compared with controls. T cells were found in both lymphocyte aggregates and diffuse infiltrates and strongly expressed the activation markers VLA-4, LFA-1, and ICAM-1. Endothelial cells showed corresponding surface activation with strong expression of VCAM-1 and ICAM-1. Mature B cells were frequently observed arranged in aggregates and rarely were seen in a diffuse pattern. Most lymphocyte aggregates lacked monocyte/macrophages. No difference in microvascular density was observed between SSc specimens and controls. Both SSc and control specimens showed weak or no expression of VEGF.

CONCLUSIONS

Our findings provide the first evidence that endothelial/lymphocyte activation leading to prominent CD4+ T cell infiltration may play a key pathogenetic role within the gastric wall of patients with SSc and may represent an important therapeutic target.

Regeneration, tissue remodeling, and organ repair after injury, which rely on the regulated activity of tissue-borne stem cells, become increasingly compromised with advancing age. Mesenchymal stroma cells were isolated from bone of differently aged healthy donors. The rare population of mesenchymal stem cells (MSCs) contained in the primary cell isolates barely declined in number, yet the stem cells displayed diminished long-term proliferation potential relative to the donor age and the expression of vascular cell adhesion molecule-1 (VCAM-1; CD106) was elevated on primary MSCs. In CD106(bright) MSCs, the abundance of a panel of stemness transcription factors remained unchanged. Because the CD106 level could be further enhanced by proinflammatory cytokines, we considered the rate of VCAM-1 expression to be a good reflection of an endogenous inflammatory milieu to which the MSCs are exposed. Treatment of MSCs with increasing doses of interferon-γ exerted no immediate influence on their self-renewal capacity. However, it impacted on the differentiation potential toward the adipogenic or osteogenic lineage. Moderately elevated levels of inflammatory stimuli supported osteoblastogenesis whereas the same treatment reduced adipogenic differentiation in MSCs from young and intermediately aged donors. In MSCs from elderly donors, however, osteoblastogenesis was greatly diminished in an inflammatory environment whereas adipogenic differentiation remained unchanged. Conclusively, moderate levels of inflammatory stimuli are being interpreted by MSCs at a young age as instructive signals for osteoblastogenesis, whereas at old age, an inflammatory milieu may effectively suppress bone remodeling and repair by tissue-borne MSCs while uninterrupted adipogenic differentiation may lead to adipose upgrowth.

OBJECTIVE

Human amnion is an easy-to-obtain novel source of human mesenchymal stem cells, which poses little or no ethical dilemmas. We have previously shown that human amnion-derived mesenchymal (HAM) cells exhibit certain mesenchymal stem cell-like characteristics with respect to expression of stem cell markers and differentiation potentials.

METHODS

In this study, we further characterized HAM cells' potential for in vivo therapeutic application.

RESULTS

Flow cytometric analyses of HAM cells show that they express several stem cell-related cell surface markers, including CD90, CD105, CD59, CD49d, CD44 and HLA-ABC, but not CD45, CD34, CD31, CD106 or HLA-DR. HAM cells at the 10th passage showed normal karyotype. More interestingly, the AbdB-like HOXA genes HOXA9, HOXA10 and HOXA11 that are expressed in the mesenchyme of the developing female reproductive tract and pregnant uteri are also expressed in HAM cells, suggesting similarities between these two mesenchymal cell types. Progesterone receptor is also highly expressed in HAM cells and expression of genes or proteins in HAM cells could be manipulated with the aid of lentivirus technology or cell-permeable peptides. To test potentials of HAM cells for in vivo application, we introduced enhanced green fluorescence protein (EGFP)-expressing HAM cells to mice by intrauterine infusion (into uteri) or by intravenous injection (into the circulation). Presence of EGFP-expressing cells within the uterine mesenchyme after intrauterine infusion or in lungs after intravenous injection was noted within 1-4 weeks.

CONCLUSIONS

Collectively, these results suggest that HAM cells are a potential source of mesenchymal stem cells with therapeutic potential.

OBJECTIVE

B lymphocytes accumulate in the inflamed joints of patients with rheumatoid arthritis (RA) and are responsible for production of high amounts of (auto)-antibodies. The aim of this study was to determine the capacity of fibroblast-like synoviocytes (FLS) to contribute to the accumulation of synovial fluid (SF) B cells by extending their life span.

METHODS

Highly purified SF B cells were cultured with FLS in the presence or absence of blocking antibodies directed against cell adhesion molecules, and cell viability was determined after various time intervals by trypan blue, annexin V, propidium iodide, or Hoechst staining. Phenotypic characterization of peripheral blood and SF B cells and FLS was carried out by flow cytometry.

RESULTS

Synovial B cells, which consist predominantly of memory B cells and plasma cells (PC), undergo spontaneous cell death by apoptosis upon removal from their in vivo environment, despite expression of Bcl-2. Coculture with FLS rescued synovial B cells from apoptosis in a cell contact-dependent manner. Blocking studies using monoclonal antibodies demonstrated a role for the molecular interaction of SF B cells with vascular cell adhesion molecule 1 (VCAM-1; CD106) in FLS-induced survival. The ability of FLS to induce SF B cell survival was not related to the rheumatoid origin since FLS from non-RA patients had similar properties.

CONCLUSIONS

These findings indicate a crucial role for FLS in the survival of synovial B cells at the site of inflammation in RA through the interaction with VCAM-1 expressed on FLS. Consequently, memory B cells and PC accumulation arise and persist not only as a result of maturation and recruitment of these cells, but also by active prevention from cell death by the microenvironment.

Endotoxin (lipopolysaccharide (LPS), 100 ng/ml) and muramyl dipeptide (MDP 100 ng/ml), two immunomodulatory bacterial cell wall products, were incubated with human whole blood, and the expression of receptors involved in antigen presentation, costimulation, and cell activation was investigated by use of flow cytometry. On monocytes, LPS and MDP increased surface expression of human leukocyte antigen-DR (HLA-DR), CD18, CD54 (intercellular adhesion molecule-1, ICAM-1), and CD86 (B7-2). On lymphocytes, LPS but not MDP increased HLA-DR expression after 18 h. The expression of CD28, CD49d/CD29, and CD106 (vascular cell adhesion molecule-1, VCAM-1) remained unchanged on both monocytes and lymphocytes. The early increase (1-6 h) of CD18 and ICAM-1 expression led us to hypothesize that CD18-dependent costimulatory signals were involved in the later (6 h) increase of monocyte HLA-DR expression. However, blocking studies using monoclonal antibodies against CD18 (IB4, 15 microg/ml) demonstrated that the LPS- and MDP-induced increase of HLA-DR and ICAM-1 expression on monocytes was not mediated through CD18. LPS induced the expression of the early activation marker CD69 by a CD14-dependent but CD18-independent mechanism, whereas MDP did not induce CD69 expression. Analysis of leukocyte subsets demonstrated that CD4(+) T-cells, CD8(+) T-cell, CD19(+) B-cells, CD56(+) natural killer (NK)-cells, and CD14(+) monocytes increased the expression of CD69 after stimulation with LPS. Collectively, these data demonstrate a stronger immunomodulatory effect of LPS compared with MDP which may, in part, explain the established difference of toxicity between these two bacterial cell wall products.

Uncultured mesenchymal stromal cells (MSCs) are increasingly used in therapies; however, the effects of donor age on their biological characteristics and gene expression remain unclear. The aim of this study was to investigate age-related changes in bone marrow (BM) MSCs following minimal or no culture manipulation. Iliac crest BM was aspirated from 67 healthy donors (19-89 years old) and directly used for the colony-forming unit-fibroblast (CFU-F) assay or CD45^lowCD271⁺ cell enumeration. The colonies were analysed for colony area and integrated density (ID) when grown in standard MSC media or media supplemented with human serum from young (YS) or old (OS) donors. There was a notable age-related decline in the number of MSCs per millilitre of BM aspirate revealed by the CFU-F assay (r = -0.527, p < 0.0001) or flow cytometry (r = -0.307, p = 0.0116). Compared to young donors (19-40 years old), colony IDs were significantly lower in older donors (61-89 years old), particularly for smaller-sized colonies (42% lower, p < 0.01). When cultured in media supplemented with OS, young and old donor MSCs formed colonies with lower IDs, by 21%, p < 0.0001, and 27%, p < 0.05, respectively, indicating the formation of smaller sparser colonies. No significant differences in the expression of selected adipogenic, osteogenic, stromal, and bone remodelling genes as well as CD295, CD146, CD106, and connexin 43 surface molecules were found in sorted CD45^lowCD271⁺ MSCs from young and old donors (n = 8 donors each). Altogether, these results show similar trends for age-related decline in BM MSC numbers measured by the CFU-F assay and flow cytometry and reveal age-related effects of human serum on MSC colony formation. No significant differences in selected gene expression in uncultured CD45^lowCD271⁺ MSCs suggest that old donor MSCs may not be inferior in regard to their multipotential functions. Due to large donor-to-donor variation in all donor groups, our data indicate that an individual's chronological age is not a reliable predictor of their MSC number or potency.

Mesenchymal stem cells (MSCs) are being tested in several biological systems and clinical settings with the aim of exploring their therapeutic potentials for a variety of diseases. MSCs are also known to be heterogeneous populations with variable functions. In the context of this multidimensional complexity, a recurrent question is what source or population of MSCs is suitable for specific clinical indications. Here, we reported that the biological features of MSCs varied with the individual donor, the tissue source, the culture condition and the subpopulations. Placental chorionic villi (CV) derived MSCs exhibited superior activities of immunomodulation and pro-angiogenesis compared to MSCs derived from bone marrow (BM), adipose and umbilical cord (UC). We identified a subpopulation of CD106(VCAM-1)+MSCs, which are present richly in placental CV, moderately in BM, and lowly in adipose and UC. The CD106+MSCs possess significantly increased immunomodutory and pro-angiogenic activities compared to CD106-MSCs. Analysis of gene expression and cytokine secretion revealed that CD106+MSCs highly expressed several immnumodulatory and pro-angiogenic cytokines. Our data offer new insights on the identification and selection of suitable source or population of MSCs for clinical applications. Further efforts should be concentrated on standardizing methods which will ultimately allow the validation of MSC products with defined biomarkers as predictive of potency in suitable pre-clinical models and clinical settings.

Pulmonary fibrosis is a grave diagnosis with insidious progression, generally considered as a consequence of aberrant epithelial wound healing and excessive scarring. This process is commonly modeled in animals by local bleomycin administration, resulting in peribronchial inflammation and subsequent fibrosis. We have previously described initiation and early development of distal pulmonary fibrosis following repeated subcutaneous bleomycin injections (systemic administration). The aim of this study was to identify mechanisms for the development of pulmonary fibrosis, which we hypothesize is related to endothelial stress and activation. Bleomycin was administered subcutaneously 3 times/week during 0.33-4w, and parenchymal alterations were studied. In addition, we used microvascular endothelial cells to investigate effects of bleomycin in vitro. Our results confirmed that systemic administration of bleomycin exerts oxidative stress indicated by an increase in Sod1 at 0.33, 1, and 4w (P<0.05). Endothelial cells were activated (increased CD106 expression) from 1w and onwards (P<0.05), and p21 expression was increased 2-3 times throughout the study (P<0.05) as were the number of β-catenin-positive nuclei (P<0.001). Wnt3a was increased at 0.33, 1, and 4w (P<0.01) and Wnt5a from 1w and onwards (P<0.001). The present study suggests that bleomycin-induced reactive oxygen species (ROS) causes DNA stress affecting the endothelial niche, initiating repair processes including Wnt signaling. The repeated systemic administrations disrupt a normally fine-tuned balance in the Wnt signaling. In addition, pericyte differentiation was affected, which may have significant effects on fibrosis due to their ability to differentiate into myofibroblasts. We conclude that the endothelial niche may have an important role in the development of pulmonary fibrosis and warrants further investigations.

Mesenchymal stromal cells (MSCs) from gestational tissues represent promising cell populations with stem cell-like properties for use in regenerative medicine. Previously, we reported that MSCs in the chorionic villi of the human placenta reside in a vascular niche. However, the niche(s) in which MSCs reside in the fetal membranes, another rich source of MSCs, remains to be determined. The cell surface markers STRO-1 and 3G5 were previously employed to identify niches in a variety of tissues and here we use these markers to report the location of the MSC niche in the human decidua parietalis. The cultured decidua parietalis MSCs (DPMSCs) isolated from the choriodecidua component of the fetal membranes possessed stem cell-like properties such as adherence to plastic, colony forming ability, and multipotent differentiation potential. Fluorescence in situ hybridization analysis showed cultured DPMSCs were of maternal origin. Immunocytochemistry demonstrated that cultured DPMSCs stained positively with stem cell surface markers 3G5, CD105, CD106, STRO-1, CD146, CD49a, and α-SMA but were negative for hematopoietic markers (CD117, CD34) and vascular markers (CD34, von Willebrand factor [vWF]). Immunohistochemistry with antibodies to stem cell surface markers and the endothelial markers on term fetal membranes revealed a vascular niche for DPMSCs, which was confirmed by immunofluorescence analysis. Both STRO-1 and vWF fluorescence signals showed substantial overlap, while CD146 and vWF signals showed partial overlap. These observations were consistent with a vascular niche.

Bone marrow (BM) environment is thought to support the growth of myeloma cells and thus to play an important role in the pathogenesis of multiple myeloma (MM). Because interleukin-6 (IL-6) is an essential growth factor in MM, we have examined the effects of two myeloma cell lines (U266 and ARH-77) on the IL-6 production by BM stromal cells in a co-culture system. These cell lines strongly stimulate the IL-6 production and IL-6 triggering was partially dependent on physical contact between lines and stroma. The percentages of cell adhesion to stromal layers were 39% and 25% respectively for ARH77 and U266 cell lines. Inhibition studies with blocking monoclonal antibodies showed the importance of CD49d/CD106 and CD11a/CD54 interactions in the stimulation of IL-6 production by stromal cells. However, cell-to-cell contact was not an absolute requirement for IL-6 production. Cytokines, of which TNF-alpha and IL-1beta produced by MM or accessory cells, were also able to stimulate IL-6 production by fibroblasts and show additive effects. In adhesion assays, TNF-alpha and IL-1beta were able to increase the adhesion of MM cells to stromal cells. CD54 was upregulated by IL-1beta, TNF-alpha or a contact with MM cells while CD106 expression was not, suggesting only a functional change of this molecule. However, the role of monoclonal antibodies, directed against these factors, confirmed the role of TNF-alpha in the IL-6 production by stromal cells, while any IL-1beta intervention was not shown in our co-culture system. IL-6 favoured and maintained adhesion of MM cells to stromal cells spontaneously since its reintroduction in the favoured co-culture system restored their decreased adhesion observed on a glutaraldehyde fixed stromal layer. Overall our data suggest a functional overlap between cytokines and adhesion molecules for the paracrine IL-6 production.

Although mesenchymal stromal cells (MSCs) have demonstrated great therapeutic potential, the heterogeneity of MSCs may be responsible for the incongruent data obtained in MSC-based preclinical studies and clinical trials. Here, four mouse clonal MSC lines, termed MSC1, MSC2, MSC3, and MSC4, were isolated and extensively characterized. MSC4 cells grew most rapidly and formed colonies of the largest size, whereas MSC3 cells exhibited the slowest growth and formed only a few tiny clusters. MSC4 cells could differentiate into adipocytes, osteoblasts, and chondrocytes in vitro, and more importantly, establish hematopoietic microenvironment in vivo; whereas the other lines displayed uni-adipogenic, osteo-chondrogenic, or non-differentiation potential. All lines were positive for Sca-1, CD106, and CD44; MSC4 was also positive for CD90.2. In terms of immunosuppressive capacity, MSC2, MSC3, and MSC4 cells exerted clear inhibitory effects on lymphocyte proliferation, whereas MSC1 did not. Further investigation revealed that the NO and not the PGE2 pathway may play a role in the different immunomodulatory effects of the cell lines. To clarify the molecular basis of this heterogeneity, we employed RNA sequencing to compare the gene expression profiles of the four subtypes, revealing a relationship between gene expression and variability in subtype function. This study provides novel information about the heterogeneity of MSCs and insight into the selection of optimal cell sources for therapeutic applications.

Trafficking of antigen-specific T cells into the central nervous system (CNS) is an important initiating step in inflammation in the brain. In spite of the extensive knowledge about the role of adhesion molecules in T cell migration across peripheral vessels, the mechanism of the entry of antigen-specific T cells into the CNS is not known. This work was designed to study the regulatory roles of adhesion molecules in antigen-specific T cell migration into the CNS. Antigen-specific T cells were tracked in an in vivo migration assay using T cell receptor (TCR) transgenic mice having 95% of T cells specific for a defined antigen. pigeon cytochrome c (PCC). TCR transgenic mice were cannulated intraventricularly (IVT) for PCC antigen infusion and cerebrospinal fluid (CSF) sampling. Upon PCC infusion into the CNS, the number of alpha/beta TCR+ Vbeta3+ Mac1- cells in the CSF was characterized in the presence or absence of anti-adhesion molecule reagents. We found that antibodies against VCAM-1 (CD106), VLA-4 (CD49d/CD29), ICAM-1 (CD54), and LFA-1 (CD11a/CD18) did not influence the increased number of antigen-specific T cells in the CSF However, upon intravenous (i.v.) injection, anti-PECAM-1 (CD31) antibody or PECAM-Ig chimeric molecule inhibited the trafficking of alpha/beta TCR+ Vbeta3+ Mac1- cells into the CNS. The expression of PECAM-1 (CD31) was also up-regulated on antigen-specific T cells in a time-dependent manner in vitro upon antigenic stimulation. The antigen-induced activation of T cells in vivo was measured by CD44 and LFA-1 expression and found to be comparable between mPECAMIg-treated mice and wild-type serum control-treated groups. This indicates that CD31 inhibition of antigen-specific T cell accumulation in the CNS is probably not due to a functional inhibition of these cells. Finally, adoptive transfer of CFSE-labeled AND transgenic cells into naïve animals resulted in the accumulation of these cells in the CNS upon PCC IVT immunization that was also inhibited by mPECAMIg treatment. Hence, PECAM-1 (CD31) might play an important role in regulating antigen-specific T cells trafficking in CNS inflammatory diseases.

Adipose tissue is a source of adult multipotent stem cells that can differentiate along mesenchymal lineage. When mature fat cells obtained from human subcutaneous adipose tissue were maintained with attachment to the ceiling surface of culture flasks filled with medium, two fibroblastic cell populations appeared at the ceiling and the bottom surface. Both populations were positive to CD13, CD90, and CD105, moderately positive to CD9, CD166, and CD54, negative to CD31. CD34, CD66b, CD106, and CD117, exhibited potential of unlimited proliferation, and differentiated along mesenchymal lineage to produce adipocytes, osteoblasts, and chondrocytes. The population that appeared at the ceiling surface showed higher potential of adipogenic differentiation. These observations showed that the cells tightly attached to mature fat cells can generate two fibroblastic cell populations with multiple but distinct potential of differentiation. Since enough number of both populations for clinical transplantation can be easily obtained by maintaining fat cells from a small amount of subcutaneous adipose tissue, this method has an advantage in preparing autologous cells for patients needing repair of damaged tissues by reconstructive therapy.

Recent studies have highlighted the importance of paracrine growth factors as mediators of pro-angiogenic effects by endothelial progenitor cells (EPCs), but little is known about the release of lipid-based factors like endocannabinoids by EPCs. In the current study, the release of the endocannabinoids anandamide and 2-arachidonoylglycerol by distinct human EPC sub-types was measured using HPLC/tandem mass-spectrometry. Anandamide release was highest by adult blood colony-forming EPCs at baseline and they also demonstrated increased 2-arachidonoylglycerol release with TNF-alpha stimulation. Treatment of mature endothelial cells with endocannabinoids significantly reduced the induction of the pro-inflammatory adhesion molecule CD106 (VCAM-1) by TNF-alpha.

BACKGROUND

The present study was aimed at determining whether routine prenatal measurements of circulating soluble intercellular adhesion molecule (sICAM-1; CD54) and soluble vascular cell adhesion molecule (sVCAM-1; CD106) in midgestation have predictive value for the identification of pregnant women destined to develop preeclampsia or other complications of pregnancy during late gestation.

METHODS

Plasma sICAM-1 and sVCAM-1 were analyzed between weeks 22 and 29 of gestation in 1543 pregnant women and related to the outcome of pregnancy in a prospective longitudinal study.

RESULTS

Plasma sICAM-1 and sVCAM-1 in uncomplicated pregnancies were normally distributed and varied over a small range (sICAM-1, SD = 22.5%; sVCAM-1, SD = 25.5%). Of all analyzed uncomplicated pregnancies, 54 (3.95%) were identified with concentrations of sICAM-1 or sVCAM-1 above the mean + 2 SD. In contrast, of 177 pregnancies with complications (prevalence, 11.5%), 97 (55%) had sICAM-1 or sVCAM-1 concentrations above the same cutoffs weeks before the onset of disease. The sensitivities of sICAM-1 and sVCAM-1 measurements were 66% for preeclampsia and hemolysis, elevated liver enzymes, and low platelets (HELLP syndrome), 42% for gestational hypertension, 50% for fetal retardation, 46% for preterm labor, 50% for gestational diabetes mellitus, 67% for gestational proteinuria, and 70% for infections during pregnancy. Taken together, routine prenatal sICAM-1 and sVCAM-1 measurements had an overall predictive value of 64%.

CONCLUSIONS

Midgestation measurements of circulating sICAM-1 and sVCAM-1 have a high predictive value (area under the curve of combined sICAM-1 and sVCAM-1 measurements determined by ROC analysis, 0.85) and may identify up to 55% of pregnant women who will later develop a severe pregnancy-related complication.

OBJECTIVE

To investigate the expression of adhesion molecules belonging to the immunoglobulin superfamily on human primary articular chondrocytes and to determine their response pattern to cytokines with respect to the adhesion of lymphocytes.

METHODS

The expression of adhesion molecules was studied by flow cytometry (cultured cells), immunohistochemistry (cartilage), reverse transcription-polymerase chain reaction, and Northern blotting. Adhesion of T cells to chondrocytes was measured using the Jurkat T cell line.

RESULTS

Vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) were found to be constitutively expressed on large percentages of unstimulated chondrocytes in culture and in cartilage ex vivo. ICAM-2, ICAM-3, and very late activation antigen 4 (VLA-4; alpha4beta1 integrin), the ligand for VCAM-1, were not detected. Interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) further induced VCAM-1 and ICAM-1 messenger RNA and protein expression. Transforming growth factor beta (TGFbeta) had no effect on ICAM-1 and decreased the expression of VCAM-1. Another adhesion molecule, VLA-2 alpha2beta1 integrin) that was also expressed on unstimulated chondrocytes, was differentially regulated by cytokines. While neither IL-1beta nor TNFalpha had any effect on expression of VLA-2, TGFbeta markedly increased the alpha2 subunit of VLA-2. Adhesion of Jurkat T cells to chondrocytes was further induced by IL-1beta and TNFalpha. Pretreatment of chondrocytes with monoclonal antibodies to VCAM-1 and ICAM-1 inhibited adhesion of T cells to chondrocytes.

CONCLUSIONS

VCAM-1, ICAM-1, and VLA-2 are constitutively expressed by human articular chondrocytes. Expression is regulated by cytokines. As shown for other chondrocyte genes, IL-1beta/TNFalpha and TGFbeta antagonistically modulate the expression of adhesion molecules. VCAM-1 and ICAM-1 contribute to adhesion of T lymphocytes to chondrocytes, and may thus participate in host defense mechanisms during inflammatory joint conditions such as rheumatoid arthritis and after cartilage transplantation.

OBJECTIVE

To evaluate the inflammatory status and the cartilage regenerative potential of pathological synovial fibroblasts from patients with osteoarthritis (OA) compared with non-inflamed synovium (NS)-derived cells from patients with chondropathy.

METHODS

The inflammatory cell phenotype was investigated based on the constitutive and inducible surface expression and secretion of various effector molecules using flow cytometry or ELISA assays. The capacity of cells to produce cartilage-like extracellular matrix was assessed using acid Alcian blue staining and type II collagen immunostaining after treatment with transforming growth factor beta1 (TGF-beta1).

RESULTS

OA and NS fibroblasts consistently expressed CD29, CD44, CD49e, CD54, CD90 and CD106. Expression of high-affinity receptors for IL-4, IL-15, CXCL8 and CXCL12 was also detected but only intracellularly. All types of fibroblasts spontaneously released abundant amounts of CXCL12, CCL2, IL-6 and tissue inhibitor of metalloproteinase 1, while the production of IL-11, TGF-beta1, matrix metalloproteinase 1 (MMP-1) and MMP-9 was detected at moderate levels. Several other secreted factors remained undetectable. No statistically significant differences were noted between the two groups of fibroblasts. Treatment with the proinflammatory cytokine tumour necrosis factor alpha (TNF-alpha) up-regulated the same set of surface and secreted molecules, including CD54, CD106, membrane IL-15, CCL2 and CCL5. Under TGF-beta1 treatment and adipogenic culture conditions, both OA and NS fibroblasts displayed chondrogenic and adipocytic activities that were reduced in OA compared with NS cells.

CONCLUSIONS

OA synovial fibroblasts did not display a distinct activated inflammatory phenotype compared with NS cells. However, they did differ in their reduced ability to produce cartilage-like matrix. This difference may be an additional important factor contributing to OA pathogenesis.

The replacement of endothelium by endothelial progenitor cells (EPCs) for therapeutic use in order to ameliorate the vascular status of ischemic organs is now in the focus of vascular research. The aim of our studies was to investigate whether EPCs derived from peripheral blood mononuclear cells (PBMNCs-derived EPCs) or EPCs propagated from CD34(+) hematopoietic stem cells (HSCs-derived EPCs), both isolated from human cord blood, are able to differentiate into early mature endothelial cells (ECs) under certain in vitro conditions. We characterized both cell populations by flow cytometry, phase contrast microscopy, fluorescence microscopy and confocal laser scanning microscopy as well as ultrastructurally using transmission and scanning electron microscopy. While PBMNCs gave rise to clusters of spindle-like EPCs after few days but did not further mature under in vitro conditions, mature ECs could only be successfully propagated from a starting population of isolated HSCs. Both, PBMNCs- and HSCs-derived EPCs, took up Dil-labeled acetylated low density lipoprotein (Dil-Ac-LDL) and could be positively stained for CD31, CD105, the vascular endothelial growth factor receptor 2 (VEGFR-2, KDR) and ulex europaeus agglutinin 1 (UEA-1) at the cell surface. EPC showed surface expression of CD54 and CD106. However, only a small portion of HSCs-derived EPCs was positive for CD54 but negative for CD106. Intracellular staining for von Willebrand factor (vWF) provided a homogenous stain in PBMNC-derived EPCs while in HSCs-derived EPCs, during cultivation for 2-3 weeks, more and more a typical punctuated staining pattern related to Weibel-Palade bodies (WPBs) was visible. By phase contrast and scanning electron microscopy, an arrangement of PBMNCs-derived EPCs in cord-like structures could be demonstrated. In these formations, cells showed parallel alignment but exhibited only few cell contacts. Well-developed WPBs could never be found in PBMNCs-derived EPCs. In contrast, differentiating HSCs-derived EPCs developed adherence junctions, interdigitating junctions as well as syndesmos. During maturation, spindle-like cell types appeared with abundant WPBs as well as cobblestone-like cell types with a fewer content of these organelles. WPBs, in the spindle-like cell types displayed conspicuous shapes and were concentrated in close proximity to mitochondria-rich areas. HSCs-derived EPCs exhibited signs of high synthetic activity such as a well-developed rough endoplasmic reticulum (RER) and multiple Golgi complexes. In the trans-Golgi network (TGN), close to the Golgi complex, a new formation of WPBs could be observed. These morphological features correlated well with a high growing capacity. Although it was not possible to demonstrate the complete differentiation line from HSCs to early matured ECs by immunologic markers because of the limited number of cells available for such investigations, distinct morphologic maturation stages could be shown at light and electron microscopical levels. In conclusion, the study presented here characterizes not only the different cell populations involved in the differentiation of early EPCs into mature ECs but also the transition stage where the maturation step takes place by demonstration of the new formation of WPBs. In this respect, these investigations provide new insights into the in vitro differentiation which could have some in vivo correlation.

OBJECTIVE

To localise and characterise follicular dendritic cells (FDC) present in autoimmune lesions of primary Sjögren syndrome.

METHODS

Cryostat sections of labial salivary glands from 15 patients with primary Sjögren syndrome were examined by an indirect immunoperoxidase technique and monoclonal antibodies to a panel of dendritic cell markers. Tonsils from two controls were also examined for the same markers.

RESULTS

FDC were localised in the centre of 75% of lymphoid focal structures in labial salivary glands biopsies. FDC in labial salivary glands of patients with primary Sjögren syndrome expressed CD35, CD11c, and CD106 (VCAM-1) in a pattern similar to FDC in tonsils, but they did not express either CD14 or CD11b. This indicates that they may not be of myeloid origin, while FDC in tonsillar lymphoid follicles strongly expressed both CD14 and CD11b. FDC in labial salivary glands of patients also lacked VLA-2 alpha and VLA-3 alpha, which were expressed by FDC in tonsils.

CONCLUSIONS

The characteristic phenotype and origin of these cells may be of importance in the immune responses involved in Sjögren syndrome and the retention of infiltrating lymphocytes in the glands.

BACKGROUND

Multipotent stromal cells (MSCs) are among the key candidates in regenerative medicine. However variety of MSC sources and general heterogeneity lead to controversial data in functional characterization. Furthermore, despite intensive usage as preclinical animal model, little is known about MSCs of the common marmoset monkey.

METHODS

MSCs derived from placental amnion and bone marrow samples from human and common marmoset were characterized in parallel over 12 passages to monitor similarities and significant differences (p ≤ 0.05, Student's t-test) in MSC markers and major histocompatibility complex (MHC) class I expression by immunohistochemistry, flow cytometry, real-time PCR, metabolic activity test, with special focus on pluripotency associated genes.

RESULTS

Human and non-human primate MSCs were characterized for expression of MSC markers and capability of differentiation into mesenchymal lineages. MSCs could be cultured more than 100 days (26 passages), but metabolic activity was significantly enhanced in amnion vs. bone marrow MSCs. Interestingly, MHC class I expression is significantly reduced in amnion MSCs until passage 6 in human and marmoset, but not in bone marrow cells. For MSC markers, CD73 and CD105 levels remain unchanged in amnion MSCs and slightly decline in bone marrow at late passages; CD166 is significantly higher expressed in human MSCs, CD106 significantly lower vs. marmoset. All cultured MSCs showed pluripotency marker expression like Oct-4A at passage 3 significantly decreasing over time (passages 6-12) while Nanog expression was highest in human bone marrow MSCs. Furthermore, human MSCs demonstrated the highest Sox2 levels vs. marmoset, whereas the marmoset exhibited significantly higher Lin28A values. Bisulfite sequencing of the Oct-4 promoter region displayed fewer methylations of CpG islands in the marmoset vs. human.

CONCLUSIONS

Little is known about MSC characteristics from the preclinical animal model common marmoset vs. human during long term culture. Studied human and common marmoset samples share many similar features such as most MSC markers and reduced MHC class I expression in amnion cells vs. bone marrow. Furthermore, pluripotency markers indicate in both species a subpopulation of MSCs with true 'stemness', which could explain their high proliferation capacity, though possessing differences between human and marmoset in Lin28A and Sox2 expression.