Vascular cell adhesion molecule 1 (VCAM-1, CD106) is expressed as a type I transmembrane integrin counter-receptor on activated endothelium and mediates white blood cell attachment. The alternatively spliced 7-domain (7d) form of VCAM-1 contains a potential thrombin cleavage site. Thrombin proteolysis of 7d-VCAM-1 may help regulate adhesive activity ofVCAM-1. We determined whether 7d-VCAM-1 is proteolyzed and rendered inactive by thrombin. Recombinant extracellular domain of 7d-VCAM-1 was cleaved by thrombin to generate 33- and 44-kDa products. Cleavage was in the sequence PGPR/IAAQIG near the N-terminal border of the alternatively spliced fourth immunoglobulin (Ig)-like module. There was no cleavage of 6d-VCAM-1 lacking the fourth module. Expression of full-length 7d-VCAM-1 presented on Chinese hamster ovary (CHO) monolayers, as detected by flow cytometry with an antibody directed to Ig-like modules 1-3, was reduced by thrombin treatment whereas there was no reduction in the expression of full-length 6d-VCAM-1. Adhesion of blood eosinophils to full-length 7d-VCAM-1 was reduced after treatment of CHO cells with thrombin, whereas adhesion to full-length 6d-VCAM-1 was not affected. We conclude that cleavage of 7d-VCAM-1 by thrombin is a potential mechanism for differential regulation of VCAM-1 splice forms in white blood cell adhesion and trafficking.

OBJECTIVE

To investigate the possibilities of human mesenchymal stem cells (hMSCs) migrating toward the oxidative stress injuries of endothelial cells.

METHODS

hMSCs were isolated and cultured from human marrow in vitro and the multipotential differentiation of P3 hMSCs identified by specific medium induced to differentiate into osteoblasts, adipocytes and endothelial cells. And the marker antigen of P3 hMSCs was detected by flow cytometry (FCM) and immunohistochemistry. Then a cellular model of hMSCs migrating toward the oxidative stress injuries of endothelial cells was created, i. e. 1 x 10(5) hMSCs were seeded in Transwell upper chamber, indirectly co-cultured with ECV-304 cells seeded in the Transwell inferior chamber and was injured by adding 3% H2O2 into the medium (final concentration of 0.01 ml/ml) for 1 h, the injured ECV-304 cells + hMSCs group (n = 8), as experimental group, and in the mean time, hMSCs indirectly co-cultured with uninjured ECV-304 cells in Transwell chamber, ECV-304 cells + hMSCs group (n = 8) and hMSCs monoculture group (n = 8) in Transwell chamber as control groups. After a 12-h culture in all groups, the migrating hMSCs in Transwell upper chamber were HE-stained and counted under an inverted phase contrast microscope. To understand the reason why hMSCs migrated to the oxidative stress injured endothelial cells, ELISA was employed to measure the concentration of monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1) of cellular supernatant in ECV-304 cells with H2O2 1-h treating group (H2O2 treatment group) or without H2O2 treating group (control group).

RESULTS

The multipotential differentiation experiment demonstrated that the cultured P3 hMSCs can be induced to differentiate in vitro into osteoblasts, adipocytes and endothelial cells. And the expressions of CD29, CD44, CD90 and CD106 were positive in hMSCs while CD31, CD34, CD45 and CD49b negative by using FCM and immunohistochemistry. And the effects of hMSCs upon in vitro movement toward oxidative stress injuries of ECV-304 cells were averaged (8. 00 +/- 0.22) cells/HP in the injured ECV-304 cells + hMSCs group, significantly higher than those of the ECV-304 cells + hMSCs group [(0.20 +/- 0.05) cells/HP, P < 0.01] and the hMSCs monoculture group [(0.00 +/- 0.00) cells/HP, P < 0.01). The concentrations of MCP-1 and VCAM-1 in cellular supernatant of the H2O2 treatment group were significantly higher than those of the control group [(69.2 +/- 3.5) ng/ml vs (62.5 +/- 3.6) ng/ml, P < 0.05; (114.0 +/- 7.5) ng/ml vs (97.2 +/- 5.0) ng/ml, P < 0.01].

CONCLUSIONS

The oxidative stress injuries of endothelial cells chemoattracted the hMSCs toward the injured site and its mechanism may be correlated with releasing a certain concentration of chemoattractant factor to result in the elevations of MCP-1 and VCAM-1 by oxidative stress injury.

Cells expressing CD4, CD8, CD18, CD49d/CD29, CD44, CD54, CD80, CD86, CD106, CD11b/CD18 or DNA breaks were stained in the pancreases of female or male scid/ scid mice after adoptive transfer of lymphocytes from older than 12-week female nonobese diabetic (NOD) or Balb/c mice. After intraperitoneal adoptive transfer of NOD splenocytes to female severe combined immunodeficiency (scid)/scid mice, in situ end labeling (ISEL)+ as well as CD80+ and CD86+ cell infiltrates appeared first in the blood vessel walls and pancreatic interstitial tissue at 2-3 weeks after transfer. CD4+, CD8+, CD18+, CD44+, CD54+, and CD106+ cells then encircled and invaded some islets of the scid/scid mice 2 to 7 weeks after transfer. Cells expressing these surface components, except CD8, were present also in the Balb/c mice, but as individual cells, not as infiltrates. CD8+ cells were observed in the pancreases of all NOD splenocyte-injected scid mice at 2, 3, 4, 6, and 7 weeks after transfer, but in none of the Balb/c splenocyte-injected scid mice. Some scid/scid mice injected with NOD splenocytes also developed severe noninfectious diarrhea and cachexia 4 weeks after transfer. ISEL+ cells were observed in the pancreases of NOD splenocyte-injected female scid mice at all times after transfer, especially in the blood vessel walls and in the islets. Fas ligand was not present in Western blotting. It is proposed that apoptosis commonly occurs in islet-infiltrating lymphocytes and that in the scid/scid adoptive-transfer model, the first islet-infiltrating cells are destroyed by programmed cell death independent of Fas ligand. Further, CD8+ T lymphocytes inevitably play a central role in intraperitoneal adoptive transfer of insulitis.

The aim of the present study was to investigate the morphological characteristics and pluripotent differentiation potential of human bone marrow mesenchymal stem cells (hBMMSCs) in vitro and in vivo. Bone marrow cells were isolated from a rib fragment of an adult surgical patient, hBMMSCs were isolated based on plastic adherence and expanded ex vivo and phenotyping was performed. Pluripotent differentiation assays for adipogenesis, myogenesis and osteogenesis were conducted. Hematopoietic reconstruction of sublethally irradiated nude mice was performed by infusion of hBMMSCs. The gene expression profiles of early and late hBMMSCs were examined. The rate of CD31‑positive cells was 31.1% in passage (P)4 hBMMSCs and 18.6% in P10 hBMMSCs. CD105 and CD106 were expressed in 99 and 95% of P25 hBMMSCs, respectively. Lipid droplets appeared at day 18 post induction. For osteogenesis, palpable masses were grossly observed from day 35 post inoculation of hBMMSCs. Hematoxylin and eosin staining further revealed chondrocytes and bone tissues. For myogenesis, at day six post subcutaneous inoculation, hBMMSCs differentiated into myocytes and were positive for myoglobin and MyoD1. In irradiated nude mice reconstituted by hBMMSCs, the white blood cell count briefly decreased following irradiation; however, it gradually recovered. In the irradiated nude mice reconstituted with hBMMSCs, CD45‑ and CD34‑positive cells were detected 72 h post induction. Gene microarray analysis of P7 and P57 hBMMSCs demonstrated that 20 genes were upregulated >2 fold and 40 genes were downregulated >2 fold in P57 hBMMSCs. In conclusion, the isolated HBMMSCs possessed pluripotent differentiation potential and it was feasible and safe to use hBMMSCs within 30 passages.

This study was aimed to compare partial biological characteristics of bone marrow mesenchymal stem cells (BMMSCs) in AML patients, AML patients with complete remission (CR) and non-leukemia patients. The bone marrow (BM) MSCs were divided into 3 groups: group of MSCs from AML patients, group of MSCs from AML patients with CR, group of MSCs from non-leukemia patients. The morphologic features of MSCs were observed by light microscopy; CFU-F numbers of MSCs were counted after Wright-Giemsa staining in situ; the fusion times of MSCs were determined; the growth curves of MSCs were drawn by counting cell numbers; the immunophenotypes and cell cycle of MSCs were detected by flow cytometry; the DI values of MSCs were calculated. The results showed that the morphologic features of MSCs in 3 groups did not display difference; there was significant difference (p < 0.01) of CFU-F numbers in 3 groups, while CFU-F number of MSCs in AML group was minimal; there was significant difference of MSC fusion time in 3 groups, while fusion time of MSCs in AML group was most long; the growth curves of MSCs in 3 groups were similar; MSCs in 3 groups highly expressed CD105 and CD106, but not expressed CD45; the cell distribution ratios at phase of G(0)/G(1) for MSCs in 3 groups were 89.9 +/- 4.0%, 90.2 +/- 3.0% and 91.0 +/- 3.0% respectively; the DI values of MSCs in 3 groups were between 0.9 and 1.1. It is concluded that no significant difference of biological characteristics of the second generations of MSCs is found between those in leukemia and non-leukemia patients.

The effect of immobilized hyaluronidase on stem and progenitor cells of the lungs was studied on the model of partially reversible toxic bleomycin-induced pulmonary fibrosis in C57Bl/6 mice. During the inflammation phase, immobilized hyaluronidase reduced infiltration of alveolar interstitium with hemopoietic stem cells Sca-1(+), c-Kit(+), CD34(-), (CD3, CD45R (B220), Ly6C, Ly6G (Gr1), CD11b (Mac1), TER-119)(-). Improvement of histological parameters of bleomycin lungs during the phase of collagen fiber deposition after the treatment was accompanied by accumulation of mesenchymal multipotent stromal cells (CD31(-), CD34(-), CD45(-), CD44(+), CD73(+), CD90(+), CD106(+)decrease in the population of pan-hemopoietic cells (CD45(+)), accelerated restoration of the content of endothelial cells, and inhibition of clonal activity of fibroblast precursors (CD45(-)).

OBJECTIVE

To investigate the differentiation potential of rat adipose tissue-derived cells (ADSCs) into neuron-like cells in vitro using a two-step induction protocol.

METHODS

ADSCs isolated from the epididymal fat pads in male SD rats by means of differential attachment were cultured in vitro and subjected to adipogenic induction. After flow cytometric identification of the cell surface antigens CD106, CD11b, CD45, CD49d, CD90 and CD29, the third-passage ADSCs were induced to transdifferentiate into neural stem cell (NSC)-like cells in DMEM/F12 medium containing 10 ng/ml basic fibroblast growth factor (bFGF), 20 ng/ml epidermal growth factor (EGF) and 2% B27. The resultant NSC-like cells were then induced to differentiate into neuron-like cells in the neurobasal medium containing 10 ng/ml brain-derived neurotrophic factor (BDNF), 10 ng/ml glial cell line-derived neurotrophic factor (GDNF) and 1 µmol/L retinoic acid (RA). Immunocytochemistry was employed to identify the expression of the cell surface markers nestin, MAP2 and NeuN.

RESULTS

The isolated ADSCs were positive for CD90 and CD29, and oil red O staining of the induced adipose-like cells yielded positive results. The third-passage ADSCs induced for 7 days aggregated as floating cell spheres positive for NSC surface antigen nestin. Further induction in neurobasal medium for 4 h resulted in adhesion of the cell spheres and the formation of cell processes extending from some peripheral cells, suggesting a morphological resemblance to neurons. Most of the cells showed positivity for MAP2 and NeuN.

CONCLUSIONS

ADSCs can be induced to differentiate into neuron-like cells in vitro under appropriate conditions.

OBJECTIVE

HOXB4 transcription factor plays an important role in embryonic and adult hematopoiesis. Overexpression of HOXB4 in murine and human embryonic stem cells (ESC) has been used to generate hematopoietic stem cells (HSC) via the embryoid body formation method.

METHODS

We used FuGENE 6-based transfection of YPL2-HOXB4 vector to generate HOXB4-expressing colonies from human ESC line H9 and investigated the potential of these cells for differentiation into primitive CD34(+) hematopoietic cells, via co-culture methodology with OP9 murine bone marrow stromal cells. Expression of HOXB4 in transfected human ESC colonies and their derivatives was verified using immunocytochemistry and reverse-transcription polymerase chain reaction (RT-PCR).

RESULTS

Utilizing OP9 stromal cell co-culture methodology, we generated CD34(+) cells from HOXB4-expressing H9 human ESC at a frequency similar to, and not higher than, non-transfected human ESC. However, we observed that some colonies of HOXB4-expressing human ESC not co-cultured on OP9 cells, differentiated into mesenchymal stromal cells (MSC) while preserving their HOXB4 expression. These HOXB4-expressing MSC expressed CD29, CD73, CD44, CD90, CD105 and HLA-class I, were negative for the expression of CD34, CD45, CD54, CD71, CD106 and HLA-DR, and could be differentiated into adipocytes and osteocytes.

CONCLUSIONS

In our specific experimental system we observed that overexpression of HOXB4 in human ESC did not improve the generation of CD34(+) hematopoietic cells via OP9 co-culture methodology. Furthermore, we could generate MSC from human ESC over-expressing HOXB4.

Adipose were obtained from patients underwent liposuction treatment (total 19 female donors, 31.5 +/- 5.8 years old). Liposuction tissues were digested with type I collagenase, cells were isolated and cultured up to passage 10. To evaluate the proliferation potential of ADCs, growth curve and cumulative population doubling were achieved by cell counting. CD29,CD105, CD106, CD166, CD49d, CD34, CD31, 3G5 were analyzed by flow cytometry and immunocytochemistry to characterize the cell population. The multi-lineage potential of ADCs was testified by differentiating cells with osteogenic,chondrogenic and adipogenic inducer. A total of 5x10(7) nucleared cells could be obtained from 300ml liposuction tissues. After in vitro cultivation,cumulative population doubling number reached 15.53 at passage 10 (average 1.59 +/- 0.224 /passage). Flow cytometry and immunocytochemistry showed that ADCs expressed high level (>60%) of stem cell-related antigen (CD29, CD105, CD106, CD166), while cells expressed hematopoiesis-related antigen CD34 and CD31 around 7.3% and 29.2% respectively. Collagen II (both in mRNA and protein level) was detected in chondrogenic differentiation. The calcified nodules were observed by von Kossa and Alizarn Red staining and the expressions of AKP and Osteonectin were detected by RT-PCR in osteogenic differentiation. PPARr2, GLU-4, and Leptin genes were detected in adipogenic differentiation and intracellular lipid droplets could be observed by Oil Red staining. ADCs can be abundantly harvested and have high proliferative and multi-lineage differentiation potential.

Tumor cell lines are generally killed by lymphokine-activated killer (LAK) cells. In this study, however, we report a LAK-resistant cell line, OKM-2T. The OKM-2T is an adult T-cell leukemia (ATL) cell line, and was compared to other ATL cell lines and non-ATL cell lines. The LAK cells were generated from healthy volunteers. Cell surface markers were determined by a flow cytometry test. The ATL and non-ATL cell lines were killed by the LAK cells, markedly or moderately. However, the OKM-2T was scarcely killed. The adhesion tendency of the OKM-2T to the LAK cells was preserved at the same level as that of the other cell lines, whereas the OKM-2T showed low levels of adhesion molecules CD58 (LFA-3), CD86, and CD106 (VCAM-1). We determined blocking tests using specific antibodies. Anti-CD58 blocked the LAK lysis. Anti-CD54 and anti-CD106 enhanced the blocking effect of the anti-CD58; anti-CD86 did not show such an effect. These results suggest that the low expression of CD58 in the OKM-2T may have an intimate relationship with LAK resistance, and that the low expression of CD106 may also be responsible for it, in part.

BACKGROUND

Exposure to dust from swine confinement buildings, causes an inflammatory response. Monocyte recruitment to the murine lung after instillation of lipopolysaccharide (LPS) has been shown to partially depend on CD106/VCAM1. We wanted to determine whether this can be confirmed in man.

METHODS

Non-naïve persons bearing personal air samples were exposed for 3 hr in a swine confinement building, and were bronchoscopied before and after exposure. Blood samples were taken at the time of bronchoscopy. Expression of adhesion molecules on leukocytes was investigated by flow cytometry in bronchoaveloar lavage (BAL) and blood.

RESULTS

Expression of CD106 on alveolar macrophages, but not on neutrophils or T cells in BAL or blood, was up-regulated in proportion to the amount of LPS that individuals were exposed.

CONCLUSIONS

This argues for requirement of CD106 during inflammatory recruitment of monocytes to the human lung.

BACKGROUND

Cellular rejection infiltration of the interstitium is the basic histological finding in biopsies of transplanted kidneys, and leukostasis in the muscular arteries and glomeruli is an important sign of exacerbating rejection. For better understanding and more accurate interpretation the authors used immunohistochemistry.

RESULTS

The authors examined 282 tissue specimens from 208 grafts using the two- or three-step immunoenzyme method with 28 mono- or polyclonal antibodies specific for a series of differentiation and activation leukocytic antigens, adhesion molecules and selected cytokines. In the compact component of the rejection infiltrate CD4+ lymphocytes with expression of CD 45 RA antigen predominated while in the disperse component there were mostly macrophages (CD68, 14, 11b); their number correlated significantly with the parenchymatous damage, similarly as intraarterial and glomerular accumulation. The disperse infiltrate and adherent cells expressed CD45 RO (rarely CD25) and integrin molecules of the series CD11 and CD49 CD57+ lymphocytes penetrated into the tubules but did not accumulate in the blood vessels. As to adhesive molecules of the "Ig superfamily", CD106 (VCAM-1) was more important than CD54 (ICAM-1) and its arterial and mesangial expression correlated with the rejection damage. Evidence of cytokines (IL1, IL2, TNF alpha, beta) did provide neither unequivocal results nor correlations.

CONCLUSIONS

Immunohistochemistry improves considerably the accuracy of bioptic evaluation of rejection nephropathy and some antigens (e.g., CD68, CD14, CD45 RO., CD57, CD106) are suitable for diagnostic practice. With their aid it is easier to evaluate the activity of rejection, assess the probability of vascular lesions in specimens without affected vessels and detect more sensitively intravascular stasis and adhesion of leukocytes.

BACKGROUND

The heparan sulfate proteoglycan syndecan-1 (CD138) was shown to regulate inflammatory responses by binding chemokines and cytokines and interacting with adhesion molecules, thereby modulating leukocyte trafficking to tissues. The objectives of this study were to examine the expression of syndecan-1 and its role in leukocyte recruitment and chemokine presentation in the microcirculation underlying the parietal peritoneum.

METHODS

Wild-type BALB/c and syndecan-1 null mice were stimulated with an intraperitoneal injection of Staphylococcus aureus LTA, Escherichia coli LPS or TNFα and the microcirculation of the parietal peritoneum was examined by intravital microscopy after 4 hours. Fluorescence confocal microscopy was used to examine syndecan-1 expression in the peritoneal microcirculation using fluorescent antibodies. Blocking antibodies to adhesion molecules were used to examine the role of these molecules in leukocyte-endothelial cell interactions in response to LTA. To determine whether syndecan-1 co-localizes with chemokines in vivo, fluorescent antibodies to syndecan-1 were co-injected intravenously with anti-MIP-2 (CXCL2), anti-KC (CXCL1) or anti-MCP-1 (CCL2).

CONCLUSIONS

Syndecan-1 was localized to the subendothelial region of peritoneal venules and the mesothelial layer. Leukocyte rolling was significantly decreased with LPS treatment while LTA and TNFα significantly increased leukocyte adhesion compared with saline control. Leukocyte-endothelial cell interactions were not different in syndecan-1 null mice. Antibody blockade of β2 integrin (CD18), ICAM-1 (CD54) and VCAM-1 (CD106) did not decrease leukocyte adhesion in response to LTA challenge while blockade of P-selectin (CD62P) abrogated leukocyte rolling. Lastly, MIP-2 expression in the peritoneal venules was not dependent on syndecan-1 in vivo. Our data suggest that syndecan-1 is expressed in the parietal peritoneum microvasculature but does not regulate leukocyte recruitment and is not necessary for the presentation of the chemokine MIP-2 in this tissue.

BACKGROUND

The pathogenesis of nasal polyps (NPs) is incompletely understood. The aim of this study was to investigate the distribution of inflammatory cells, adhesion molecules, intermediate filaments, and chemokine receptors in subgroups of NP patients.

METHODS

In total, 35 patients were enrolled (group 1, 10 patients with Samter syndrome; group 2, 10 patients with diffuse polyposis without signs of Samter syndrome; group 3: 5 patients with solitary nasal polyps; group 4, 10 controls). Immunohistochemical staining was performed for CD105, CD106, CD62E, CD4, CD8, CXCR4, CD147, CD90, CD104, BF45, vimentin, pancytokeratin, and muscle-specific actin (MSA) in all patients' specimens.

RESULTS

Expression of CD4, CD8, and CD106 were similar between the groups. Number of patients expressing CD4 in groups 1, 2, and 3 were higher than the controls. Number of patients expressing CD8 antigen were significantly higher in all three groups than in the control group. Expression of CD147 in groups 3 and 4 was significantly higher than in groups 1 and 2. CD98 expression was higher in groups 1, 2, and 3 than in group 4. The number of patients expressing vimentin in groups 1, 2, and 3 was significantly higher than in group 4. Immunostaining for pancytokeratin was positive in all patients.

CONCLUSIONS

In conclusion, inflammatory cell, adhesion molecule, intermediate filament, and chemokine receptor profiles in nasal polyps differ among different patient groups and control subjects. Additional specific immunohistochemical studies are necessary for development of more specific immunotherapies.

The aim of the present study was to investigate the tropism of mesenchymal stem cells (MSCs) to the tumor microenvironment, and to evaluate the feasibility of bone marrow mesenchymal stem cells differentiating into myofibroblasts in vitro. A total of 1 ml bone marrow was extracted from the greater trochanter of one male New Zealand rabbit, and MSCs were obtained by density gradient centrifugation and cultured routinely. The surface markers were analyzed by flow cytometry. A VX2 tumor was aseptically excised from another male New Zealand rabbit and primary cultured. The tropism of MSCs for 30% and 50% VX2 conditioned medium was determined by using Transwell migration assays. MSCs were incubated in 30% VX2 conditioned medium for 7 or 14 days. The messenger (m)RNA levels and protein expression of α-smooth muscle actin (α-SMA) and vimentin were measured by reverse transcription-polymerase chain reaction and western blotting. MSCs were observed to have a spindle shape. The cultured MSCs were cluster of differentiation (CD)44+, CD105+, CD106+ and CD34-. VX2 cells demonstrated a spindle or polygon shape. In the Transwell assay, it was observed that the migrated cells appeared more frequently in the 30% VX2 conditioned medium group compared with the other groups when microscopically examined, which was additionally confirmed by the results of a colorimetric assay. The mRNA levels and protein expression of α-SMA and vimentin significantly increased in the test group compared with the control group at 7 days (P<0.01), and further increased in the test group at 14 days (P<0.01). The results of the present study demonstrated that MSCs have tropism for the tumor microenvironment and furthermore, may differentiate into myofibroblasts in the tumor microenvironment in vitro. The present study suggested that MSCs may migrate to the tumor and subsequently differentiate into myofibroblasts due to the tumor microenvironment, which may lead to promotion of the growth of the tumor. The present study additionally suggested that MSCs may be the precursors of tumor/carcinoma-associated myofibroblasts.

Transplantation of allogeneic embryonic neural tissue is a potential treatment for patients with Parkinson's and Huntington's diseases. The supply of human donor tissue is limited, and alternatives such as the use of animal (e.g., porcine) donor tissue are currently being evaluated. Before porcine grafts can be used clinically, strategies to prevent neural xenograft rejection must be developed. Knowledge on how human T lymphocytes recognize porcine embryonic neural tissue would facilitate the development of such strategies. To investigate the ability of porcine embryonic brain microvascular endothelial cells (PBMEC) to stimulate human T-cell proliferation, PBMEC were immuno-magnetically isolated and cocultured with purified human CD4 or CD8 single-positive T cells. PBMEC had a cobblestone-like growth pattern and expressed the endothelial cell markers CD31 and CD106. PBMEC stimulated with the supernatant of phytohemagglutinin-activated porcine peripheral blood mononuclear cells or porcine IFN-gamma, but not nonstimulated PBMEC, induced proliferation of both CD8 and CD4 T cells as assessed by [3H]thymidine incorporation. Flow cytometric analyses showed that the degree of CD8 and CD4 T cell proliferation correlated with the expression levels of class I and II major histocompatibility complex (MHC) antigens, respectively. PBMEC expressed a CTLA-4/Fc-reactive molecule, most likely CD86, suggesting that these cells are able to deliver a costimulatory signal to the T cells. Human TNF-alpha, but not human IFN-gamma, induced class I, but not class II, MHC expression on PBMEC. Within a neural graft or the regional lymph nodes, PBMEC might stimulate human T cells via the direct pathway, and should therefore be removed from the donor tissue prior to transplantation.

OBJECTIVE

The last decade has been profoundly marked by persistent attempts to use ex vivo expanded and manipulated mesenchymal stem cells (MSCs), as a tool in different types of regenerative therapy. In the present study we described immunophenotype and the proliferative and differentiation potential of cells isolated from pulp remnants of exfoliated deciduous teeth in the final phase of root resorption.

METHODS

The initial adherent cell population from five donors was obtained by the outgrowth method. Colony forming unit-fibroblast (CFU-F) assay was performed in passage one. Cell expansion was performed until passage three and all tests were done until passage eight. Cells were labeled for early mesenchymal stem cells markers and analysis have been done using flow cytometry. The proliferative potential was assessed by cell counting in defined time points and population doubling time was calculated. Commercial media were used to induce osteoblastic, chondrogenic and adipogenic differentiation. Cytology and histology methods were used for analysis of differentiated cell morphology and extracellular matrix characteristics.

RESULTS

According to immunophenotype analyses all undifferentiated cells were positive for the mesenchymal stem cell markers: CD29 and CD73. Some cells expressed CD146 and CD106. The hematopoietic cell marker, CD34, was not detected. In passage one, incidence of CFU-F was 4.7 +/- 0.5/100. Population doubling time did not change significantly during cell subcultivation and was in average 25 h. After induction of differentiation, the multicolony derived cell population had a tri-lineage differentiation potential, since mineralized matrix, cartilage-like tissue and adipocytes were successfully formed after three weeks of incubation.

CONCLUSIONS

Altogether, these data suggest that remnants of deciduous teeth dental pulp contained cell populations with mesenchymal stem cell-like features, with a high proliferation and tri-lineage differentiation potential and that these cultures are suitable for further in vitro evaluation of cell based therapies.

Interactions between stromal cells or extracellular matrix and hematopoietic cells are important factors for the very complex processes associated with differentiation and maturation in the bone marrow. To elucidate these multifold processes, the expression pattern of various adhesion molecules was studied on enriched fibroblastic populations derived from healthy volunteers. CD44 (homing cell adhesion molecule) and very late activation antigen beta 1 (VLA beta 1; CD29) could be demonstrated on almost all fibroblasts without an alteration following cytokine stimulation. On the other hand, VLA-2 (CDw49b), VLA-3 (CDw49c), VLA-4 (CDw49d), VLA-5 (CDw49e), intercellular adhesion molecule-1 (ICAM-1; CD54) and vascular cell adhesion molecule-1 (VCAM-1; CD106) were only represented by certain cell fractions. In our studies recombinant human granulocyte macrophage-colony stimulating factor failed to alter the expression pattern of these adhesion molecules, whereas recombinant human interleukin-3 (rhIL-3 showed a tendency for downregulation of the VLA antigens except for VLA beta 1. However, recombinant human transforming growth factor-beta 1 (rhTGF beta 1) exerted a reducing effect on the expression of VLA-3 and induced an increase in the VLA-5-positive fraction. In the immunoglobin class VCAM-1 revealed a decrease staining capacity after stimulation with rhTGF beta 1 and rhIL-3. Contrary to this finding, the presentation of ICAM-1 increased after administration of these mediators.

The product generated by skeletal muscle and bone marrow mesenchymal stem cell cocultures has been demonstrated to improve the functional outcomes after cell therapy in postinfarction or Chagas myocardiopathy. This coculture method allows cell interactions in vitro, diminishing the operational costs of the culture/expansion as well as leading to angiogenesis and myogenesis for regeneration of the injured heart. Flow cytometric analysis may better characterize the cellular types in this model. Our objective was to use flow cytometry to analyze the immunophenotype expressed in this coculture model. The coculture was performed in accordance with Carvalho for 21 days. Flow cytometry was performed before and after coculture to characterize the immunophenotypic profile of cellular subsets, namely, the surface markers CD31, CD34, CD44H, CD45, CD49d, CD54, CD73, CD90, CD105, CD106, Myo-D, M-cadherin, and Connexin-43. Statistics were performed by the nonparametric Friedman test (P < .05) with post-hoc analysis by the nonparametric Wilcoxon test (P < or = .017, Bonferroni correction). The results demonstrated statistical significance for CD45(+) in 89.49% of mononuclear cells, 3.58% in skeletal muscle cells, and 4.74% among cocultured cells (P = .0094); and CD90(+) in 36.18% of mononuclear cells, 6.01% in skeletal muscle cells, and 48.94% among cocultured cells (P = .0420). The cocultured cells expressed the markers CD73(++), CD90(+++), CD45(-), CD34(+), CD105(-/+), CD106(-/+), M-cadherin(-/+), and Connexin-43(-/+). In conclusion, flow cytometric analysis showed a heterogeneous adherent cell population in this coculture model.

Tissue engineering using biomaterials is a promising solution for cartilage replacement. The purpose of this study was to investigate whether the fibrin sealant Tissucol(R) provides a suitable scaffold for re-implanting stem cells during chondrogenic replacement therapy. Pluripotent stem cells were isolated from adult human bone marrow (hMSCs), cultured and characterized by FACS (CD105+/CD106+, CD45-/CD14-/CD34-). A large-holed porous hMSC-containing fibrin matrix was built that allowed hMSCs to survive throughout the period of culture (42 days) in either proliferation or chondrogenic differentiation medium under normoxic (21% O2) or hypoxic (3% O2) conditions. Morphology (as determined by electron microscopy) and proliferation (Ki67 staining) of the embedded hMSCs did not markedly vary under normoxic and hypoxic culture even after 42 days in culture. The stem cell marker Oct-4 was expressed during the whole culture period. Under chondrogenic differentiation conditions, especially under hypoxic conditions, we observed rounded chondrocyte-like cell types and a chondral phenotype assessed by mRNA expression of collagen II and Alcian blue staining. hMSCs seeded into large-holed porous preparations of Tissucol survive, proliferate and keep their stem cell character. Furthermore, culturing the cells in a corresponding medium induces chondrogenic differentiation, which could be remarkably and significantly enhanced under hypoxic conditions.

Interactions of leukocytes with vascular endothelium are important components of inflammation tissue reactions and have been implicated in cardiac transplant rejection and demonstrated to be mediated by cell adhesion molecules (CAM's). The expression of ICAM-1, VCAM-1 and E-selectin in human myocardium is variable and little is known about the expression of LFA-1 and Mac-1 during allograft rejection. This study investigated these CAM's in myocardial biopsies of transplanted hearts (HTX) and of coronary artery disease eligible for coronary artery bypass grafting (CABG) as non-inflammatory controls and explicitly examines vascular endothelium, cardiomyocytes and infiltrating cells. Immunohistochemistry was performed using the APAAP-method and directing specific mouse anti-human monoclonal antibodies against ICAM-1 (CD54), VCAM-1 (CD106), E-selectin (CD62E), alpha-LFA-1 (CD11a), alpha-Mac-1 (CD11b), alpha-p150/95 (CD11c) and the beta2-integrin chain (CD18). CD18, LFA-1 (CD11a), Mac-1 (CD11b) and p150/95 (CD11c) were markedly expressed on infiltrating immunocytes in HTX compared to CABG where no expression of beta2-integrins was observed. Cardiac allografts demonstrated a strong expression of ICAM-1 on vascular endothelium and on infiltrating cells. ICAM-1 was not detected on cardiomyocytes. In CABG a weak expression of ICAM-1 was observed on endothelial cells but not on myocytes. VCAM-1 was expressed on vascular endothelium and perivascular infiltrating cells in HTX but not in CABG. VCAM-1 was not found to be expressed on myocytes. There was no evidence for the presence of E-selectin in any of our biopsy specimens. Our study shows that the study of cell adhesion molecules adds to the pathophysiological understanding of inflammation after transplantation in cardiac disease. This offers a potential for the development of diagnostic tools and new therapeutic strategies.

Inflammation plays an important role in both the initiation of atherosclerosis and development of atherothrombotic events. The adherence of leukocytes/monocytes to the endothelium is an early event in atherogenesis. Phytotherapeutica as garlic and garlic extracts were shown to have beneficial modulating effects in patients with atherosclerotic disease. The aim of this study was to evaluate in vitro the influence of water-soluble garlic (Allium sativum) extract on the cytokine-induced expression of endothelial leukocyte adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1, CD54) and vascular cell adhesion molecule-1 (VCAM-1, CD106). Cytokine-induced expression of cellular adhesion molecules was measured on primary human coronary artery endothelial cell (HCAEC) cultures. HCAEC were cultured in microvascular endothelial cell growth medium and preincubated with garlic extract at various concentrations (0.25-4.0 mg/ml), after which human interleukin-1alpha (IL-1alpha, 10 ng/ml) was added for 1 day. Fluorescein isothiocyanate (FITC)-labeled anti-ICAM-1 and FITC-labeled anti-VCAM-1 were used to analyze the IL-1alpha-induced expression of ICAM-1 and VCAM-1 by flow cytometry. Incubation of HCAEC with garlic extract significantly decreased ICAM-1 and VCAM-1 expression induced by IL-1alpha. In addition, we examined the effects of garlic extract on the adhesion of monocytes to endothelial cells, using the monocytic U937 cell line. The presence of garlic extract significantly inhibited the adhesion of monocytes to IL-1alpha-stimulated endothelial cells. These results indicate that garlic extract modulates the expression of ICAM-1 and VCAM-1, thus potentially contributing to the beneficial effects traditionally attributed to garlic.

OBJECTIVE

To compare the adipose-derived mesenchymal stem cells (ADMSCs) isolated from the greater omentums and subcutaneous adipose tissues of rats for their characteristics in cell morphology, growth kinetics and immunophenotypes.

METHODS

ADMSCs were isolated from the greater omentums and inguinal fat pads of 6 SD rats and cultured in vitro. The morphologies of the ADMSCs were observed using phase-contrast microscopy, and their growth curves were generated and the doubling times determined. The phenotypic marker profiles including CD11b, CD29, CD45, CD49d, CD90 and CD106 of the ADMSCs in the fourth passage were determined using flow cytometry.

RESULTS

The ADMSCs harvested from the greater omentums and inguinal fat pads showed almost identical morphologies. The growth curves and the mean doubling time of the ADMSCs from the two different sources showed no obvious difference. With similar positivity rates for CD11b, CD29, CD106 and CD90, the two ADMSCs exhibited different expression rates of CD45 and CD49d.

CONCLUSIONS

The immunophenotypic characteristics of the ADMSCs from the greater omentums and subcutaneous adipose tissues are not totally identical.

Amniotic fluid is a rich source of multipotent mesenchymal stem cells (MSCs). Amniotic fluid stem cells (AFSCs) have become a new source of stem cells; they have low immunogenicity and are easily harvested. For this reason, they may be useful in clinical tissue engineering. Moreover, AFSCs have anti-inflammatory properties and can repair tissues. This study evaluated the utility of AFSC injection to treat bilateral ovarian dystrophy in Holstein-Friesian cows. Bovine AFSCs (BAFSCs) were collected at slaughter from Holstein-Friesian cows during the third or fourth month of pregnancy and cultured in vitro. The BAFSCs began to show a fibroblast-like morphology. They were positive for β-integrin, CD44, CD73, CD106 and Oct4 and negative for CD34 and CD45. After induction, the cells differentiated into mesodermal lineages. Bilateral ovarian dystrophy was confirmed by ultrasonography in 16 lactating cows. The subsequent experiment lasted 15 weeks. Serum was collected weekly to analyse progesterone concentrations, and weekly ultrasonography recorded ovarian changes. Each cow was equipped with an automatic heat detection system to facilitate oestrus observation and breeding records. The progesterone concentration of two cows in the treatment group (25%) significantly increased during weeks 10-15. On ultrasonography, the treatment group demonstrated mature follicles after BAFSCs injection, and foetuses were visualized approximately 40 days after artificial insemination (AI). Oestrus rates in the control and treatment groups were 0% (0/8) and 50% (4/8), respectively; pregnancy rates were 0% (0/8) and 25% (2/8), respectively. Calves were successfully delivered in both cases of pregnancy. These results show that BAFSCs can alleviate bovine ovarian dystrophy and restore fertility.