The study aims to identify the phenotypic marker expressions of different human adult stem cells derived from, namely, bone marrow, subcutaneous fat, and omentum fat, cultured in different media, namely, DMEM-Low Glucose, Alpha-MEM, DMEM-F12 and DMEM-KO and under long term culture conditions >>P20). We characterized immunophenotype by using various hematopoietic, mesenchymal, endothelial markers, and cell adhesion molecules in the long term cultures (Passages-P1, P3, P5, P9, P12, P15, and P20.) Interestingly, data revealed similar marker expression profiles irrespective of source, basal media, and extensive culturing. This demonstrates that all adult stem cell sources mentioned in this study share similar phenotypic marker and all media seem appropriate for culturing these sources. However, a disparity was observed in the markers such as CD49d, CD54, CD117, CD29, and CD106, thereby warranting further research on these markers. Besides the aforesaid objective, it is understood from the study that immunophenotyping acts as a valuable tool to identify inherent property of each cell, thereby leading to a valuable cell based therapy.

Classically, MSC are identified by a CD45-CD106+ phenotype. In this study, we found that mouse MSC achieve this characteristic phenotype only at later passages. With increasing passages, CD45 (hematopoietic marker) expression shifts to negativity, whereas CD106 (vascular cell adhesion molecule-1) expression becomes increasingly positive. These results demonstrate that MSC cells cultured from mouse bone marrow acquire a classical MSC immunophenotype (CD45-CD106+) in later passages.

OBJECTIVE

To investigate the expression of surface antigen of human periodontal ligament cells (PDLCs) and dental pulp cells (DPCs) and the impact of ex vivo expansion to the expression of surface antigen. To provide basis of proper surface antigen for further selection of homogenous stem cell subpopulation from PDLCs and DPCs.

METHODS

PDLCs and DPCs were isolated and cultured by collagenase type I and dispase. The expression of surface antigen was analyzed by immunohistochemistry and flow cytometry.

RESULTS

Positive expression of STRO-1 and CD146 were observed in PDLCs and DPCs by immunocytochemistry. Similar to DPCs, PDLCs expressed mesenchymal stem cell markers STRO-1, CD146, CD29, CD44 and CD106, and displayed negative expression for CD34 at passage 1 by flow cytometry. There were no significant difference of STRO-1, CD29 and CD44 expression level between PDLCs and DPCs (P>> 0.05). PDLCs expressed significantly higher level of CD106 and significantly lower level of CD146 than DPCs (P < 0.001). The proportion of STRO-1 and CD146 positive cells decreased steadily with passages in PDLCs and DPCs.

CONCLUSIONS

PDLCs have some similar surface antigen as DPCs, and the stem cells properties of PDLCs and DPCs decreased steadily with passages.

Life-long regeneration of healthy muscle by cell transplantation is an ideal therapy for patients with degenerative muscle diseases. Yet, obtaining muscle stem cells from patients is very limited due to their exhaustion in disease condition. Thus, development of a method to obtain healthy myogenic stem cells is required. Here, we showed that the four transcription factors, Six1, Eya1, Esrrb, and Pax3, converts fibroblasts into induced myogenic stem cells (iMSCs). The iMSCs showed effective differentiation into multinucleated myotubes and also higher proliferation capacity than muscle derived stem cells both in vitro and in vivo. The iMSCs do not lose their proliferation capacity though the passaging number is increased. We further isolated CD106-negative and α7-integrin-positive iMSCs (sort-iMSCs) showing higher myogenic differentiation capacity than iMSCs. Moreover, genome-wide transcriptomic analysis of iMSCs and sort-iMSCs, followed by network analysis, revealed the genes and signaling pathways associated with enhanced proliferation and differentiation capacity of iMSCs and sort-iMSCs, respectively. The stably expandable iMSCs provide a new source for drug screening and muscle regenerative therapy for muscle wasting disease.

Infection with Flaviviruses upregulates the cell surface expression of MHC-I, MHC-II, ICAM-1 (CD54), VCAM-1 (CD106) and TAP proteins. Although all these studies have been confirmed using West Nile virus and other Flaviviruses, there are few reports that have examined the effects of Japanese encephalitis virus (JEV) infection directly on nonclassical and classical MHC expression in astrocytes. We show in this report that JEV infection of mouse brain astrocytes results in induction of the nonclassical MHC Class Ib genes, H-2T23, H-2Q4 and H-2T10 in addition to MHC-I, Type I (alpha/beta) IFNs, TAP-1, TAP-2, Tapasin, LMP-2, LMP-7 and LMP-10 but not IFNgamma, CD80, CD86 and MHC-II genes. The increased cell surface expression of these antigens as well as induction of the genes mentioned above as measured by RT-PCR suggests that JEV infection may lead to the induction of classical MHC Class Ia as well as nonclassical MHC Class Ib molecules.

The administration of corticosteroids induced apoptosis of thymocytes in vivo. Among various adhesion molecules examined, vascular cell adhesion molecule-1 (VCAM-1, CD106) was shown to be strongly expressed in these apoptotic cells. Flow cytometric analysis also showed the expression of VCAM-1 in apoptotic thymocytes. An RT-PCR study demonstrated the expression of VCAM-1 mRNA in thymocytes. Splenic lymphocytes and other lymphoid cell lines also expressed VCAM-1 during the process of apoptosis. VCAM-1 mRNA expression was also observed in RT-PCR performed on these cell lines.

Staphylococcal scalded skin syndrome (SSSS) results from the effect of exfoliative-toxins produced by staphylococcal strains. The disease affects predominantly children, and is rare in adults. We report two cases of the adult type of SSSS. Corticotherapy, chronic alcohol abuse and epilepsy-related immune changes might have been predisposing factors in these patients. The immunopathological characteristics of the inflammatory cell infiltrate in adults SSSS have not been thoroughly explored so far in the literature. Biopsies from 2 patients with bullous SSSS skin were studied by means of immunochemistry using a panel of 10 antibodies directed to FXIIIa, CD15, CD31, CD45R0, CD50, CD54, CD62E, CD95, CD106, and L1-protein, respectively. Cutaneous biopsies from related blistering diseases were compared. They included drug-induced toxic epidermal necrolysis (TEN), bullous impetigo and superficial pemphigus. A dense cell infiltrate composed of granulocytes (CD15+), macrophages (L1 protein+) and memory T cells (CD45R0+) and a strong expression of ICAM-3 (CD50) were present in the epidermis. CD95+ keratinocytes were lining the intraepidermal blisters. Type I dermal dendrocytes (Factor XIIIa+) were numerous and plump in the dermis. Bullous impetigo exhibited the same pattern of inflammatory cells, but with a lower density in type I dermal dendrocytes. TEN differed from SSSS by both the absence of CD15+ granulocytes and a stronger expression of the pro-apoptotic CD95 antigen in the epidermis. In superficial pemphigus, CD95 antigen was not expressed, and CD15+ granulocytes, CD45R0+ lymphocytes and L1 protein+ monocytes were much less numerous. It is concluded that the specific binding of SSSS-induced exotoxins to the desmosomes alters the keratinocyte metabolism leading to an inflammatory reaction followed by focal apoptosis. Our findings are in line with the concept that SSSS exotoxins might be superantigens. A common pathomechanism leading to epidermal destruction is likely operative in SSSS and bullous impetigo. The inflammatory cell composition in TEN and superficial pemphigus markedly differs from that in SSSS.

Callithrix jacchus, the common marmoset, is particularly suitable for immunological studies in vivo and in vitro since many antibodies directed against epitopes of human cells do also react with their analogues from this non-human primate. We studied the reactivity of antibodies against human epitopes on primary cultures of thymic epithelial cells from marmosets and humans by flow-cytometry after different culture periods. The antibodies against integrins, including CD61, reacted with thymic epithelial cells from both humans and marmosets, as did anti-CD44 and anti-CD106. Antibodies specific for thymic epithelial cells (TE-3, TE-4, TE-8, TE-15, TE-16, TE-19) also bound to cells from marmosets but expression of all epitopes was not observed in all cultures studied. The expression of CD51, CD54, CD58 and CD106 on human cells declined after 4 weeks of culture. Our findings indicate that marmosets are a valuable model for immunological studies of effects of xenobiotics on the thymic epithelium.

Human follicular dendritic cell (FDC)-like cells (FLC) have been utilized for the in vitro analysis of germinal center reactions. However, there is no consensus whether FLC represent FDC in vitro. The purpose of the present study has therefore been to determine distinguishing features of FDC and FLC in vitro. The expression of CD40, CD54, CD49d, cytokine (gamma-IFN and IL-4)-dependent MHC-class II, and CD106 was observed to be specific for the determination of FDC in long-term culture. The cytokine-dependent emperipolesis of germinal center B cells was establised as another discriminating property for FDC in vitro. In 2 out of 72 long-term cultures of FDC, we encountered dividing cells among the non-dividing population of FDC. The dividing cells expressed accessory molecules similar to those of FDC but showed emperipolesis only for the initial few days of their growth. FDC did not enhance the CD40-dependent proliferation of germinal center B cells; in contrast, FLC augumented it. Both types of cells produced a significant amount of cytokine-dependent IL-6. Further studies are needed to determine whether FLC originate from FDC in vitro.

Nurse-like stromal cell lines from the synovial tissue of patients with rheumatoid arthritis (RA-SNC) produce, on coculture with lymphocytes, large amounts of proinflammatory cytokines. In the present paper, we analyze the molecular events necessary for the induction of cytokine release from RA-SNC cells, and particularly the roles played by cell adhesion and the transmigration (also known as pseudoemperipolesis) of lymphocytes. For this purpose, the effects of various mAbs on the binding and transmigration of a human B-cell line, MC/car, were examined using a cloned RA-SNC line, RA-SNC77. To analyze the role of lymphocyte binding and transmigration on upregulated cytokine production by the RA-SNC77 cells, we used C3 exoenzyme-treated MC/car cells, which could bind to RA-SNC77 cells but could not transmigrate. Treatment with anti-CD29 or anti-CD49d mAb significantly reduced binding and transmigration of the MC/car cells. In contrast, the neutralizing anti-CD106/vascular cell adhesion molecule 1 mAb did not show any inhibitory effect. Likewise, none of the neutralizing mAbs against CD11a, CD18, CD44, CD49e, or CD54 showed significant effects. Binding of C3-treated or untreated MC/car cells to RA-SNC77 cells induced comparable levels of IL-6 and IL-8 production. In addition, the enhanced cytokine production by RA-SNC77 cells required direct lymphocyte contact via a very late antigen-4 (VLA-4)-independent adhesion pathway. These results indicate that, although both the VLA-4-dependent/vascular cell adhesion molecule 1-independent and the VLA4-independent adhesion pathways are involved in MC/car binding and subsequent transmigration, only the VLA4-independent adhesion pathway is necessary and sufficient for the enhanced proinflammatory cytokine production by RA-SNC77 cells. The transmigration process, which is dependent on Rho-GTPase, is not a prerequisite for this phenomenon.

Inflammation, extracellular matrix proteins (hydroxyproline, connective tissue growth factor, collagen, and fibronectin), stem and progenitor cells (multipotent mesenchymal stromal cells, Clara cells, angiogenesis, precursors, endothelial and epithelial cells) were studied in female C57Bl/6 mice with experimental elastase-induced emphysema. Diffuse emphysema reduced the number of endothelial (CD45(-)CD31(+)CD34(+)) and epithelial (CD45(-)CD117(+)CD49f(+)) cells, induced microcirculation disturbances, and decreased the area occupied by the connective tissue. Emphysematous changes in the lungs were accompanied by infiltration of the alveolar septa with macrophages and lymphocytes, increase in the serum and lung concentrations of transforming growth factor-β, IL-1β, IL-2, IL-5, IL-10, and IL-13, and lung concentration of IL-17. In the lungs, inflammation was associated with marked increase in the number of multipotent mesenchymal stromal cells CD90(+)CD73(+)CD106(+)CD44(+)) and Clara cells (CD45(-)CD34(-)CD31(-)Sca1(+)) and overexpression of extracellular matrix proteins (hydroxyproline, connective tissue growth factor, collagen, fibronectin) and Clara cells protein. On the other hand, elastase reduced the number of angiogenic precursor cells (CD45(-)CD117(+)Flk1(+)).

OBJECTIVE

To assess the capacity to isolate and expand mesenchymal stem cells (MSC) from bone marrow of CBA/Ca, ICR and Balb/c mice.

METHODS

Bone marrow of tibia and femur were flushed, cultured and maintained in supplemented Dulbecco's modified Eagle's medium. MSC immunophenotype of cultures were tracked along increasing passages for positivity to CD106, Sca-1 and CD44 and negativity to CD45, CD11b and MHC class II. Differentiation capacity of MSC towards osteogenic and adipogenic lineages were also assessed.

RESULTS

MSC were successfully cultured from bone marrow of all 3 strains, albeit differences in the temporal expression of certain surface antigens. Their differentiation into osteocytes and adipocytes were also observed. MSC from all 3 mouse strains demonstrated a shift from a haematopoietic phenotype (CD106(-)CD45(+)CD11b(+)Sca-1(low)) to typical MSC phenotype (CD106(+)CD45(-)CD11b(-)Sca-1(high)) with increasing passages.

CONCLUSIONS

Information garnered assists us in the decision of selecting a mouse strain to generate MSC from for downstream experimentation.

Interactions between MSC and B-CLL cells were investigated to better understand the role of adhesion proteins in the biology of B-CLL. The role of β1 and β2 integrins and CD44 in adherence of B-chronic lymphocytic leukemia (CLL) cells to bone marrow stromal cell (MSC) monolayers and the ability of MSC to prevent apoptosis of CLL cells was investigated. Peripheral blood mononuclear cells, from 8 patients with CD5-positive B-CLL, were effectively depleted of CD3-positive cells with an immunomagnetic column. Purity of B-CLL cells, as judged by coexpression of CD19 and CD5 on two-color fluor-ocytometry, was 92±4% (mean±SD) (n = 8). (51)Cr-labelled B-CLL cells, were incubated with isotype murine monoclonal antibodies or blocking MoAb's against the following adhesion proteins: integrins β1, α4, and αL (chain of LFA-1), CD44 and CD106 (VCAM-1). The B-CLL cell adherence to marrow stromal cell (MSC) monolayers at 2hrs was 29±12% (mean±1SD). MoAb's against CD106, α4, and β1 caused a significant inhibition of heterotypic adherence in 2/8, 3/8 and 4/8 experiments. Despite universal expression of αL on B-CLL cells, MoAb against αL did not influence adhesion of B-CLL cells in any of the eight experiments. MoAb's against CD44 caused an increase in B-CLL cell adherence to MSC in 1/8 experiments. No correlation between basal adhesion and intensity of α4 expression was noted. The absence of this correlation can be explained by the highly variable expression of α4 on the B-CLL cells from a limited number of patients. Notably, the intensity of α4 and β1 expression, on the B-CLL cells correlated with the degree of inhibition by anti-α4 and anti-β1 MoAb. A significant positive correlation was noted between baseline adhesion and intensity of β1 expression. Thus, α4β1 and its ligand VCAM-1 are important for adhesion of B-CLL cells to MSC. However, other ligands of α4 and other as yet undescribed adhesion proteins may also play a role in B-CLL cell adhesion to MSC. In addition, when B-CLL cells were cocultured in direct contact with MSC monolayers, the proportion of B-CLL cells undergoing apoptosis decreased significantly.

BACKGROUND

Mesenchymal stem cells (MSCs) promote skin healing. 12/15-Lipoxgenase (LOX) is crucial in producing specific lipid mediators in wounded skin. The consequences of 12/15-LOX deficiency in MSC densities in skin are unknown.

OBJECTIVE

To determine the effect of 12/15-LOX deficiency in MSC densities in wounded and unwounded dermis.

METHODS

Full-thickness skin incisional wounds were made to 12/15-LOX-deficient (12/15-LOX(-/-) ) and wild-type (WT) C57BL/6 mice. Wounded skin was collected at 3, 8, or 14 days postwounding (dpw). MSCs were analysed in skin sections using histology. 12S- or 15S-hydroxy-eicosatetraenoic acid (HETE) was analysed using a reversed-phase Chiral liquid chromatography-ultraviolet-tandem mass spectrometer.

RESULTS

There were more stem cell antigen (Sca)1(+) CD29(+) MSCs (cells/field) at 3, 8, and 14 dpw, more Sca1(+) CD106(+) MSCs at 3 and 14 dpw in the wounded dermis, more MSCs in unwounded dermis of WT mice compared with 12/15-LOX(-/-) mice, and more MSCs in the wounded dermis than in the unwounded dermis. For 12/15-LOX(-/-) dermis, Sca1(+) CD106(+) MSCs peaked and Sca1(+) CD29(+) MSCs reached a flat level at 8 dpw. However, for the WT dermis, MSCs increased from 8 to 14 dpw. There were more Sca1(+) CD106(+) MSCs than Sca1(+) CD29(+) MSCs in the 12/15-LOX(-/-) wounded dermis at 8 dpw. However, there were more Sca1(+) CD29(+) MSCs in the 12/15-LOX(-/-) than Sca1(+) CD106(+) MSCs in the WT wounded dermis at 3 dpw, and Sca1(+) CD106(+) MSCs and Sca1(+) CD29(+) MSCs were at comparable levels in other conditions. 12/15-LOX deficiency suppressed levels of 12/15-LOX protein and their products, 12S-HETE and 15S-HETE, in wounds.

CONCLUSIONS

12/15-LOX deficiency reduces MSC densities in the dermis, which correlates with the suppressed 12/15-LOX pathways in wounded and unwounded skin.

Endothelial cell lines have been established from cells that were isolated from porcine yolk sacs from day 18 and day 22 embryos and propagated in vitro under various growth conditions. After expansion in vitro, the general properties of the cells proved similar for the different media used. The endothelial cells expressed cell surface receptors for acetylated low-density lipoprotein and also expressed cell surface-associated angiotensin-converting enzyme. The cells showed a characteristically high level of binding for Bandeiraea simplicifolia lectin I and Dolichos biflorus agglutinin but did not bind significant amounts of Ulex europaeus lectin I. The cells expressed low but serologically detectable levels of Class I major histocompatibility complex (MHC) antigens but failed to bind antibodies directed against Class II MHC antigens. Alpha5beta1 integrins were weakly expressed, whereas vascular cell adhesion molecule-1 (CD106) and alphavbeta3 integrins were not detected. Three-dimensional tube formation was readily observed in cultures grown on Matrigel and occurred even in uncoated plastic dishes in the absence of Matrigel. In contrast to most of the adult porcine endothelial cells, yolk sac-derived endothelial cells did not possess serologically detectable receptors for porcine growth hormone (GH), an observation consistent with the finding that GH did not increase the proliferative rate of these cells. Electron microscopic examination demonstrated the presence of Weibel-Palade bodies, tight endothelial cell junctions, and typical rough endoplasmic reticulum. Exposure of the cells to either concanavalin-A-stimulated porcine splenocyte culture supernatants or to human tumor necrosis factor alpha did not cause upregulation of VCAM-1 or Class II MHC antigens. Addition of porcine interferon-gamma led to an increase in the level of expression of Class I MHC. Yolk sac endothelial cells from day 22 embryos showed a low but detectable level of expression of Class II MHC antigens, whereas the endothelial cells from day 18 embryos showed no expression of Class II antigens after interferon-gamma stimulation. The cells maintained competence to develop vascular structures in vitro and could do so after coinjection with murine tumor cells into adult, immunocompromised mice.

BACKGROUND

Extracellular vesicles are particles ranged from 30 nm to 5μm and subcategorized into three groups; exosomes, microvesicles and apoptotic bodies, each of which have different biological impact. Lack of a standard method for the detection and isolation of MVs has led to a challenging issue that is a worth considering. In this study, we isolated MVs from the conditioned medium of UC-MSCs by four different schemes of ultracentrifugation.

METHODS

We examined the efficacy of differential centrifugation ranging from 10,000×g to 60,000×g on UCMSCs- derived microvesicles yield and purity. The fractions were evaluated by Dynamic Light Scattering (DLS) method, total protein quantification and flow cytometry.

RESULTS

UC-MSCs were spindle cells that adhered to plastic culture flasks. These cells expressed MSC markers such as CD44 and CD73, whereas were negative for hematopoietic markers CD45 and CD34. UC-MSCparticles were successfully isolated. Particles were heterogeneous vesicles of approximately 50 to 1250 nm in diameter that bear the surface-expressed molecules UC-MSCs such as; CD90, CD106, CD166 and CD44, and negative for CD34, CD63, and CD9. According to the results of DLS method, centrifugation at 10,000, 20,000, 40,000 and 60,000 ×g, all gave MVs of less than 1000 nm. It is of notion that only at the centrifugation rates of 40,000 and 60,000×g, particles of less than 100 nm in diameter were also obtained.

CONCLUSIONS

The choice of exact speed greatly influences the purity of MVs and their yield. Our findings indicate that centrifugation at 20,000×g is appropriate for the purification of UC-MSC-MVs.

BACKGROUND

Cell adhesion plays a pivotal role in most ocular surface inflammatory diseases. Adhesion molecules mediate cell-to-cell and cell-to-matrix adhesion. Their expression is up-regulated by pro-inflammatory stimuli such as cytokines, histamine or complement-derived anaphylatoxins. The dipeptide N acetyl-aspartyl glutamic acid (NAAGA) is used as unpreserved topical eyedrops in the treatment of allergic conjunctivitis. NAAGA is known to inhibit leukotriene synthesis, histamine release by mast cells, and complement-derived anaphylatoxin production.

OBJECTIVE

To investigate the potential capability of NAAGA to interfere with leukocyte adhesion to endothelial cells and modulate cytokine-induced expression of adhesion molecules.

METHODS

Human blood-derived leukocytes were co-cultured with human umbilical vein endothelial cells (HUVECs) in the absence or the presence of 1000 UI/mL human recombinant TNFalpha, 10(-4) M histamine di-hydrochloride or 5x10(-6) M human recombinant C5a, and in the absence or presence of NAAGA (final concentration 2.45%). Adhesion of leukocytes to HUVECs was calculated by subtracting the number of nonadherent leukocytes from the total number of leukocytes. Expression of adhesion molecules was assessed by flow cytometry using anti-CD11b, anti-CD49d, anti-ICAM-1 (CD54), anti-ICAM-2 (CD102), anti-VCAM-1 (CD106) and anti-ELAM-1 (CD62E) monoclonal antibodies.

RESULTS

NAAGA was found to totally inhibit adhesion of unstimulated leukocytes, or leukocytes activated with C5a, TNFalpha, or histamine, to TNFalpha-stimulated HUVECs (P=0.0001). Adhesion of leukocytes to unstimulated HUVECs was not modified by NAAGA. Similar results were obtained with endothelial cells stimulated by histamine or C5a. Taken together, these data indicate that NAAGA totally abrogates cell adhesion under inflammatory conditions, without interfering with the physiological adhesion of leukocytes to normal endothelium. At the molecular level, NAAGA inhibited histamine-induced expression of CD11b (P=0.0004) and CD49d (P=0.0045) on granulocytes. On TNFalpha-activated HUVECs, NAAGA induced a significant decrease in the VCAM-1 expression level (P<0.0001) and totally reversed TNFalpha-induced overexpression of ICAM-1 (P=0.0069), ICAM-2 and ELAM-1 (P<0.0001), without interfering with baseline expression of these molecules.

CONCLUSIONS

These results show that the antiallergic compound NAAGA directly inhibits leukocyte adhesion to endothelial cells induced by pro-inflammatory stimuli, and abrogates TNFalpha-induced expression of adhesion molecules on granulocytes and endothelial cells. This capacity to block overexpression of selectins and integrins induced by pro-inflammatory stimuli confers to NAAGA a potential as an anti-inflammatory drug, since interfering with adhesion molecule expression is probably one of the most efficient ways to curb leukocyte recruitment to inflammatory sites.

Human cartilage is reported to contain multipotent stromal cells. We evaluated the effect of human cartilage-derived stromal cells (CDSCs) on heart function when transplanted into the infarcted myocardium of rats. CDSCs were isolated and cultured from human articular cartilage and subjected to fluorescence-activated cell sorting (FACS) analysis. The CDSCs were consistently negative for CD14, CD34, CD38, CD45, CD49f, CD104, CD105, CD106, CD117, HLA-DR, and ABCG-2, and positive for CD10, CD44, CD71, CD73, CD90, CD147, and HLA-A, -B, and -C by FACS analysis. Myocardial infarction (MI) was created in rats by ligation of the left anterior descending artery. Three weeks after MI, the CDSCs labeled with Hoechst stain were injected into the infarct and border zone. Echocardiography, histological examination, and reverse transcription-polymerase chain reaction (RT-PCR) were performed 4 weeks after cell transplantation. Echocardiography indicated that CDSC transplantation could improve heart function. The number of capillaries increased in the injection regions in the transplantation group. Histological examination showed that Hoechst-labeled CDSCs in islands within the infarcted region were stained positively for desmin and smooth muscle actin but negatively for alpha-sarcomeric actin and troponin-I. RT-PCR results indicated the expression level of collagen I, collagen III, tissue inhibitor of metalloproteinase-1, transforming growth factor-beta1, and vascular endothelia growth factor were much higher in the scar tissue in the transplantation group than in the medium and control groups. Our findings suggested that CDSCs might promote angiogenesis, prevent left ventricular remodeling, and improve the heart function when transplanted into injured heart in the rat model of myocardial infarction.

Normal human immunoglobulin G (IgG) has anti-inflammatory and immuno-regulatory properties, which are exploited in the therapy of selected diseases. A putative mechanisms of action is the direct regulation of endothelial cell function by natural antiendothelial cell antibodies. Endothelium activation is a critical event in atherosclerosis. We have verified the ability of normal human IgG to modulate endothelial responses to the atherogenic stimuli tumour necrosis factor-alpha (TNFalpha) and oxidized low-density lipoproteins (oxLDL) in vitro. Confocal microscopy was used to visualize vascular cell adhesion molecule-1 (CD106) expression on endothelial cells, cytoplasmic free calcium ([Ca++]i) modifications and fluorescein-coupled oxLDL internalization. Cytokine secretion was measured by ELISA on cell supernatants. IgG prevented TNFalpha induced CD106 membrane expression and an increase in [Ca++]i, and inhibited the secretion of interleukin-6 (IL-6) and macrophage-colony-stimulating factor (M-CSF). IgG also inhibited CD106 expression induced by oxLDL and one pathway of their internalization, but were ineffective on oxLDL induced [Ca++]i rise and apoptosis. F(ab)'2 fragments from IgG, but not monoclonal IgG, reproduce IgG effects. These findings point to a regulatory role for specific antibodies included in circulating normal IgG towards proinflammatory responses of endothelial cells in atherogenesis and suggest possible development of new therapeutic strategies.

BACKGROUND

Washing of red blood cells (RBC) can reduce unwanted biological response modifiers (BRMs) that can mediate transfusion complications in infants. The aim of this study was to examine the in vitro quality and the changes in BRMs following washing in paediatric RBC units.

METHODS

A pool and split design was used to prepare RBC (either 1 or 4 days old; n = 26 pairs). One unit was washed with 0·9% saline by centrifugation and then resuspended in SAG-M, while the other remained unwashed. Each RBC unit was divided to produce four units of paediatric-sized components. Samples were taken after 3 h and subsequently on days 1, 2, 7 and 14 post-wash.

RESULTS

Washing of RBC resulted in some red cell loss, with a minor increase in haemolysis. Washing effectively reduced supernatant potassium and IgA, as well as cytokines and complement proteins. RBC microparticles were significantly reduced in RBC washed at 1, but not 4 days post-collection. Incubation with supernatant from unwashed but not washed RBC led to endothelial cell activation, with increased cell surface expression of CD62E (E-selectin) and CD106 (VCAM).

CONCLUSIONS

Although washing affected some aspects of the in vitro quality of RBC, it effectively reduced the concentration and activity of BRMs in the supernatant of RBC. Such a reduction may be clinically beneficial in selected patient groups.

Porphyromonas gingivalis (P. gingivalis) has been implicated as an important pathogen in periodontal diseases, and its protease (Arg-gingipain) is one of the most important disease-causing factors. We have examined the effect of Arg-gingipain on the cell adhesion molecules of human periodontal ligament fibroblasts (HPLF). The Arg-gingipain was purified from the culture supernatant of P. gingivalis. Confluent monolayer cultures of HPLF were incubated for up to 24 hr with Arg-gingipain. As for the morphological changes, HPLF detached from the dish at 16 hr. The growth rate as evaluated by the MTT assay increased at 12 hr, and then the rate decreased. The cell adhesion molecules (ICAM-1, intercellular adhesion molecule-1; CD54, VCAM-1, vascular cell adhesion molecule-1; CD106, and VLA-4, very late antigen-4; CD49d/cd29) were constitutively expressed on HPLF by flow cytometric analysis. The amounts of fibronectin were increased at 12 hr and then, its production was decreased. The levels of ICAM-1, VCAM-1 and VLA-4 expressions were increased; however, after 16 hr, the levels of expression of all cell adhesion molecules were decreased. Our experiments showed that adhesion decreases as a result of destruction of the extracellular matrix when bacterial protease brings about rapid cell destruction. This may suggest that the increase of expression of cell adhesion molecules is due to a mechanism that compensates for the decreased cellular-extracellular matrix. Alternatively, it may suggest that after the expression is activated, cell adhesion molecules help to increase bacterial adhesion to cells.

OBJECTIVE

To develop a sensitive and reproducible technique for measuring the adherence of blood lymphocytes to vessel walls exposed in sections of human retina and for examining the role of lymphocyte and vascular adhesion molecules in these events.

METHODS

Cryostat sections of human retina were overlaid with blood lymphocytes from healthy subjects, and experimental conditions were sought by which preferential attachment of the cells occurred to blood vessel walls in the retinal sections. Adherent lymphocytes were identified by staining with methyl green-thionine, and transected blood vessels were identified by their structure and by staining of basement membranes with periodic acid-Schiff. The adherence of enriched preparations of CD4+ (T-helper) and CD8+ (T-cytotoxic) lymphocytes, of interleukin-2 (IL-2)-activated cells, and of lymphocytes from patients with ocular Behçet's disease was examined. The distribution of adhesion molecules on retinal vessel walls was determined by immunohistochemistry, and the contribution of leukocyte integrins to lymphocyte binding was studied by blocking experiments with monoclonal antibodies.

RESULTS

The optimal selectivity of blood lymphocyte attachment to retinal vessel walls occurred when purified lymphocytes were suspended in culture medium with 10% fetal calf serum and overlaid onto retinal sections for 30 minutes at 23 degrees C with gentle agitation. Under these conditions, 92% of the lymphocytes that adhered to the section were confined to the retinal microvasculature, and CD4+ T cells were more adherent than CD8+ T cells (P < 0.01). Prior exposure of normal lymphocytes to IL-2 enhanced their binding to retinal blood vessels, and lymphocytes from patients with Behçet's disease showed supranormal vascular adherence (P < 0.005). Many transected vessels stained positively for CD31; PECAM (mean 62%), CD54; ICAM-1 (mean 73%), CD62E; E-selectin (mean 35%), CD62P; P-selectin (mean 61%), and CD106; VCAM-1 (mean 42%). However, these vascular adhesion molecules occupied < 20% of the area of the blood vessel walls. Lymphocyte adhesion to the retinal vessels was more dependent on CD29 (the common chain of the beta 1 integrins) expression than either CD11a/CD18 or CD49d.

CONCLUSIONS

This technique allows measurements to be made of lymphocyte adherence to vascular and nonvascular structures of retina ex vivo. Extension of this approach to the study of leukocyte adherence to sections of pathologic retina may be of clinical and experimental applicability in understanding mechanisms of retinal inflammation.

Forty-five human stromal cell lines were established from long-term bone marrow cultures transformed with a new vector, pNu MTSVts, which contains the Zn-inducible metallothionein promoter and the temperature-dependent SV40 T antigen from SV40 A58 mutant. Six of these cell lines were studied because of their growth capacity. All cell lines differed with respect to growth potential, expression of cell surface markers, and cytokine transcripts. Major histocompatibility complex (MHC) class I, CD29, CD49d, and CD51 were present on all stromal cell lines, MHC class II and CD34 were consistently absent, and CD11a (LFA-1), CD18 (ICAM-1R), CD54 (ICAM-1), CD58 (LFA-3) CD56 (N-CAM), CD106 (V-CAM), laminin, and collagen IV were diversely expressed. All cell lines contained interleukin (IL)-1alpha, IL-1beta, IL-2, IL-5, and macrophage colony-stimulating factor (M-CSF) transcripts, whereas granulocyte M-CSF, TNFalpha, IL-3, IL-4, and IL-7 were diversely expressed. The most characteristic feature of these cells was their varying capacity to expand cord blood CD34+ cells. One of these stromal cell lines ensured more than twofold expansion of the initial CD34+CD10-CD19- population in the first 2 weeks. Differentiation toward the B cell lineage was limited, producing only very small numbers of CD19+ cells after 6 weeks of culture.