After cerebral ischemia, bone marrow mesenchymal stem cells are mobilized and travel from the bone marrow through peripheral circulation to the focal point of ischemia to initiate tissue regeneration. However, the number of bone marrow mesenchymal stem cells mobilized into peripheral circulation is not enough to exert therapeutic effects, and the method by which blood circulation is promoted to remove blood stasis influences stem cell homing. The main ingredient of Xuesaitong capsules is Panax notoginseng saponins, and Xuesaitong is one of the main drugs used for promoting blood circulation and removing blood stasis. We established rat models of cerebral infarction by occlusion of the middle cerebral artery and then intragastrically administered Xuesaitong capsules (20, 40 and 60 mg/kg per day) for 28 successive days. Enzyme-linked immunosorbent assay showed that in rats with cerebral infarction, middle- and high-dose Xuesaitong significantly increased the level of stem cell factors and the number of CD117-positive cells in plasma and bone marrow and significantly decreased the number of CD54- and CD106-positive cells in plasma and bone marrow. The effect of low-dose Xuesaitong on these factors was not obvious. These findings demonstrate that middle- and high-dose Xuesaitong and hence Panax notoginseng saponins promote and increase the level and mobilization of bone marrow mesenchymal stem cells in peripheral blood.

Mesenchymal stromal/stem cells (MSCs) reside in many human tissues and comprise a heterogeneous population of cells with self-renewal and multi-lineage differentiation potential, making them useful in regenerative medicine. It remains inconclusive whether MSCs isolated from different tissue sources exhibit variations in biological features. In this study, we derived MSCs from adipose tissue (AT-MSC) and compact bone (CB-MSC). We found that early passage of MSCs was readily expandable ex vivo, whereas the prolonged culture of MSCs showed alteration of cell morphology to fibroblastoid and reduced proliferation. CB-MSCs and AT-MSCs at passage 3 were CD29⁺, CD44⁺, CD105⁺, CD106⁺, and Sca-1⁺; however, passage 7 MSCs showed a reduction of MSC markers, indicating loss of stem cell population after prolonged culturing. Strikingly, CB-MSC was found more efficient at undergoing osteogenic differentiation, while AT-MSC was more efficient to differentiate into adipocytes. The biased differentiation pattern of MSCs from adipogenic or osteogenic tissue source was accompanied by preferential expression of the corresponding lineage marker genes. Interestingly, CB-MSCs treated with DNA demethylation agent 5-azacytidine showed enhanced osteogenic and adipogenic differentiation, whereas the treated AT-MSCs are less competent to differentiate. Our results suggest that the epigenetic state of MSCs is associated with the biased differentiation plasticity towards its tissue of origin, proposing a mechanism related to the retention of epigenetic memory. These findings facilitate the selection of optimal tissue sources of MSCs and the ex vivo expansion period for therapeutic applications.

The biophysical fine-tuning of cancer cell plasticity is crucial for tumor progression but remains largely enigmatic. Although vascular cell adhesion molecule-1 (VCAM-1/CD106) has been implicated in melanoma progression, here its presentation on endothelial cells was associated with diminished melanoma cell spreading. Using a specific nanoscale modulation of VCAM-1 (tunable from 70 to 670 ligands/μm²) next to integrin ligands (RGD motifs) in a bifunctional system, reciprocal regulation of integrin α4 (ITGA4/VLA-4/CD49d)-dependent adhesion and spreading of melanoma cells was found. As the VCAM-1/VLA-4 receptor pair facilitated adhesion, while at the same time antagonizing RGD-mediated spreading, melanoma cell morphogenesis on these bifunctional matrices was directly regulated by VCAM-1 in a dichotomic and density-dependent fashion. This was accompanied by concordant regulation of F-actin cytoskeleton remodeling, Rac1-expression, and paxillin-related adhesion formation. The novel function of VCAM-1 was corroborated in vivo using two murine models of pulmonary metastasis. The regulation of melanoma cell plasticity by VCAM-1 highlights the complex regulation of tumor-matrix interactions.Implications: Nanotechnology has revealed a novel dichotomic function of the VCAM-1/VLA-4 interaction on melanoma cell plasticity, as nanoscale tuning of this interaction reciprocally determines adhesion and spreading in a ligand density-dependent manner. Mol Cancer Res; 16(3); 528-42. ©2017 AACR.

Xenoreactive antibodies (Ab) are important for the development of acute vascular rejection (AVR) of xenografts characterized by monocytes, natural killer (NK) cells and neutrophils infiltrating the graft. The mechanisms by which anti-galactose alpha 1,3galactose (alpha-Gal) IgG influence NK cell migration across porcine aortic endothelium (PAEC) were investigated. NK cell migration across PAEC increased in the presence of anti-alpha-Gal IgG. Anti-alpha-Gal IgG exposure activated PAEC as shown by an increased expression of CD62E and CD106. NK cells adhered, spread and showed motile forms on plastic surfaces coated with human IgG, IgG Fc and on mAb against CD16, but not on mouse IgG or BSA, suggesting that CD16 cross-linking can mediate increased adhesiveness. Increased NK cell motility was observed on Boyden filters coated with human IgG, IgG Fc, and mAb against CD16 and the alpha 4, alpha 5, alpha L, beta 1 and beta 2 integrin chains. No motile response was seen on mouse IgGor CD7, CD56 and alpha 6 integrin mAb. NK cell migration on human IgG and anti-CD16 Ab was blocked by anti-CD16 or anti-beta 2, but not anti-beta 1 Ab, implying that the motile response triggered by CD16 cross-linking is mediated via beta 2 integrins. Preformed or induced anti-alpha-Gal IgG may therefore contribute to AVR by stimulating innate immune cell infiltration of the graft.

OBJECTIVE

To assess the differentiation potential of rat adipose-derived stem cells (ADSCs) into Schwann-like cells in vitro.

METHODS

ADSCs isolated from adult SD rats were cultured in vitro and identified with the cell surface antigens CD44, CD49d and CD106 by immunocytochemistry. The ADSCs of the sixth to eighth passages were inoculated in polylysine-coated culture plate and cultured for 12 days in DMEM/F12 culture medium containing 10% fetal bovine serum, 5 ng/ml platelet-derived growth factor, 10 ng/ml basic fibroblast growth factor, 14 micromol/L Forskolin and 200 ng/ml Heregulin to induce their differentiation in vitro. Immunocytochemistry was performed to identify the expression of the cell surface markers nestin, glial fibrillary acidic protein (GFAP), S-100, and P75.

RESULTS

The isolated and purified ADSCs were positive for CD44 and CD49d expressions but negative for CD106. After 12 days of culture in the conditional culture medium, most of the cells showed positive expressions of GFAP, S-100, and P75, the specific protein markers of Schwann cells.

CONCLUSIONS

Adult rat ADSCs are confirmed to have potentials of neuroglial differentiation and capable of differentiating into Schwann-like cells in vitro.

BACKGROUND

Pyogenic granuloma is a common non-neoplastic connective tissue proliferation. ICAM-1 and VCAM-1 are vascular adhesion molecules and CD34 is a marker for evaluation of angiogenesis. The purpose of this study was to compare the immunohistochemical expression of ICAM-1, VCAM-1 and CD34 in oral pyogenic granuloma and normal gingiva.

METHODS

This study was performed on thirty five formalin-fixed, paraffin embedded samples of gingival pyogenic granuloma. Also we used thirty five paraffined blocks of normal gingiva as control group which were taken from crown lengthening surgery. We employed immunohistochemistry staining for our prepared microscopic slides using monoclonal mouse anti-human antibodies against ICAM-1 (CD54), VCAM-1 (CD106) and CD34. Slides were examined under light microscope and then the mean amount of stained vessels also known as microvascular density (MVD) in highly vascularized areas (hot spots) was measured. Paired t-test and repeated measures ANOVA were used to compare the difference between quantitative variables and Chi-square test for qualitative variables in different groups. Pearson correlation coefficient was used to compare relations between quantitative variables. P<0.05 was considered significant.

RESULTS

The mean of MVD for ICAM-1, VCAM-1 and CD34 was significantly higher in pyogenic granuloma than normal gingiva (p<0.001 and p<0.001 and p<0.001, respectively). Expression of CD34 in pyogenic granuloma was significantly higher than ICAM-1 and VCAM-1 (P<0.001). Besides, expression of ICAM-1 in normal gingiva, was significantly lower than two other markers (p<0.001).

CONCLUSIONS

Regarding the results, it seems that ICAM-1, VCAM-1 and CD34 are useful biomarkers in evaluation of vascular and inflammatory lesions such as gingival pyogenic granuloma and the results indicate the role of these biomarkers in pathogenesis of oral pyogenic granuloma.

Prodromal signs of a non-healing wound after revascularisation, which might be strictly linked with impending failure of vascular reconstructions, are associated with an inflammatory response mediated by several circulating adhesion molecules, extracellular endopeptidases, and cytokines. The aim of our study was to investigate the role of selected plasma biomarkers in the prediction of both wound healing and failure of infrapopliteal vein graft or percutaneous trans-luminal angioplasty (PTA) with selective stent positioning of the superficial femoral artery (SFA) in a population affected with critical limb ischaemia. A total of 68 patients who underwent either surgical or endovascular revascularisation of the inferior limb with autologous saphenous vein infrapopliteal bypass or PTA and selective stenting of the SFA were enrolled in our study. Patients were divided into two groups according to treatment: 41 patients were included in Group 1 (open surgery) and 27 in Group 2 (endovascular procedure). Plasma and blood samples were collected on the morning of surgery and every 6 months thereafter for up to 2 years of follow-up or until an occlusion occurred of either the vein bypass graft or the vessel treated endovascularly. Fifteen age-matched healthy male volunteers were considered a reference for biological parameters. Vascular cell adhesion molecule 1 [VCAM-1]/CD106, inter-cellular adhesion molecule-1 [ICAM-1]/CD54), interleukin-1 (IL-1), interleukin-6 (IL-6), tumour necrosis factor alpha (TNF-α), and metalloproteinases (MMP)-2 and -9 plasma levels were measured with enzyme-linked immunosorbent assay (ELISA) kits. The mean observed time to heal of 54 wounds was 13 ± 4 months, with no statistically significant differences among the groups. The healing failure of the remaining wounds was strictly related to an unsuccessful open (n = 12) or endovascular (n = 8) treatment. The 2-year primary patency rate was 65% (SE = .09) in Group 1 and 52% (SE = .1) in Group 2. When compared with mean concentration values of Group 1, VCAM-1 and ICAM-1 were always significantly higher during follow-up in patients of Group 2 (P < .05). Furthermore, in the same group, IL-6 and tumour necrosis factor alpha (TNF-α) were found to be significantly higher at 6- and 12-month (P < .05) when compared with surgically treated patients. Cox regression analysis showed that elevated plasma levels of VCAM-1, ICAM-1, IL-6, and TNF-α during follow up were strongly related to impaired wound healing and/or revascularisation failure (P < .05). Elevated plasma levels of inflammatory markers VCAM-1, ICAM-1, IL-6, and TNF-α may be related to the failure of wound healing and revascularisation procedures. Interestingly, we have observed that endovascular treatments cause a higher level of these inflammation biomarkers when compared with a vein graft, although wound-healing and patency and limb salvage rates are not influenced.

BACKGROUND

Cell adhesion molecule expression by tumour endothelium is involved in the efficacy of several cancer therapies. This study investigated the effect of tumour microenvironmental conditions, i.e. reduced oxygenation and tumour-conditioned medium (TCM), on the expression of adhesion molecules by HUVECs.

METHODS

E-selectin (CD62-E), VCAM-1 (CD106) and PECAM-1 (CD31) were measured using an ELISA assay. Reduced oxygen tension (approximately 0%, 1% vs. 20%) and the presence of TCM from human breast adenocarcinoma MDA-MB-231 was investigated following the treatment of HUVECs with TNFalpha.

RESULTS

E-selectin (peak at 4 hours) and VCAM-1 (peak at 24 hours) expression were significantly increased by TNFalpha but unchanged by reduced oxygenation. TCM significantly attenuated E-selectin (71%), VCAM-1 (74%) and PECAM (62%) response to TNFalpha.

CONCLUSIONS

Tumour-secreted factors have a greater inhibitory action than reduced oxygen tension on HUVECs adhesion molecule expression induced by TNFalpha. Reduced expression of adhesion molecules may limit cancer therapies that mediate their action by host cell infiltration.

The objective of this study was to investigate whether the superantigen staphylococcal enterotoxin A (SEA), which binds to HLA class II and T-cell receptor Vbeta chains, can direct cytotoxic T cells to lyse cytokine-stimulated endothelial cells (EC). In addition, we wanted to determine whether SEA-primed cytotoxic T cells could be targeted to EC surface molecules as a means of a novel cancer immunotherapy. Human umbilical vein EC (HUVEC), dermal microvascular EC (HMVEC), or the EC line EA.hy926 stimulated with gamma interferon (IFN-gamma) or tumor necrosis factor alpha (TNF-alpha) displayed upregulated HLA class II and adhesion molecule (CD54 and CD106) expression, respectively. SEA-primed T cells induced a strong cytotoxicity against IFN-gamma- and TNF-alpha-activated EA.hy926 which had been preincubated with SEA. Blocking of CD54 completely abrogated the T-cell attack. SEA-D227A, which has a mutated class II binding site, did not promote any cytotoxicity. A strong lysis was observed when a fusion protein consisting of protein A and SEA-D227A was added together with T cells to TNF-alpha-induced EA.hy926 and HUVEC precoated with monoclonal antibodies (MAb) directed against HLA class I, CD54, or CD106 molecules. Finally, an scFv antibody fragment reactive with an unknown EC antigen was fused with SEA-D227A. Both EA.hy926 and HMVEC were efficiently lysed by scFv-SEA-D227A-triggered cytotoxic T cells. Taken together, superantigen-activated T-cell-dependent EC killing was induced when EC expressed an inflammatory phenotype. Moreover, specific MAb targeting of the superantigen to surface antigens induced EC lysis. Our data suggest that directed T-cell-mediated lysis of unwanted proliferating EC, such as those in the tumor microvasculature, can be clinically useful.

Our study aimed to find out the most effective mode for chondrogenic differentiation based on time, dose and culture method. ADSCs were cultured and identified by CD44, CD49d, and CD106 immumohistochemical staining method, and their differentiation potential to chondrocyte were detected by Alizarin red staining. ADSCs induced by different concentrations of GDF-5 for chondrogenic differentiation were detected by blue and toluidine blue staining and collagen type II and X immumohistochemical staining. The expression of collagen I, II, X and aggrecan gene in GDF-induced ADSCs cultured in 2- and 3-dimension was identified by real-time PCR. Cell microstructure and proliferation in three-dimensional scaffolds at day 7, 14, 21 and 28 were analyzed by scanning electron microscopy and MTS assay. The ADSCs were successfully identified by CD44 and CD49d, and their differentiation potential was detected by Alizarin red staining. Real-time PCR showed that collagen and aggrecan were expressed at high levels in 100 or 200 ng/mL GDF-5 treated cells. The collagen types (I, II) and aggrecan genes were higher expressed in GDF-5 induced scaffold group than that in monolayer group. MTS showed that the cell counts were not significantly different among different treated time. Both collagen type II and aggrecan gene were highly expressed at day 14, while collagen types I and X gene expressions peaked at day 21 and 28. The 100 ng/mL GDF-5 is effective and cost-effective for chondrogenic differentiation when cultured at day 14 in vitro under three-dimensional culture conditions.

The in vitro amplification of endothelial progenitor cells (EPCs) is an important method because of its role in gene transferring and regenerative medicine. In this study, we isolated rabbit bone marrow-derived EPCs to further manipulation and overexpression of dimethylarginine dimethylaminohydrolase (DDAH) in EPCs. Isolated EPCs were cultured, expanded in endothelial basal medium. Morphology of EPCs and expression levels of surface markers detected using immunocytochemistry staining and through the use of flow cytometery. Endothelial progenitor cells were transfected with plasmid vectors expressing human DDAH2 (DDAH2-EPCs). Three days after gene transfer, positive transfected-EPCs proliferation and DDAH activity were assayed. We observed colonies conformation and endothelium-like morphology gradually in the third week of culture. Characterization results revealed positive expression of EPC surface markers CD106, Flk-1, vWF, and CD34 using few identification techniques. Overexpression of DDAH2 increased citrulline production after 96 hours of transfection, 235.34 ± 0.69 vs 95.26 ± 5.76 ng/mL; P = .023. These results suggest that cell population with EPC characteristics can be simply isolated from rabbit bone marrow and successfully engineered to overexpress exogenous gene. In this study, we offer a feasible method to isolate and identify EPCs from bone marrow. In addition, an efficient transfection with a plasmid vector (without risk of interference) can be constructed a hybrid structure with EPC and DDAH2 gene to examine their function in vitro.

Aims-To analyse the topographical distribution of adhesion molecules involved in lymphocyte recirculation in human lymph nodes and tonsils. The study focused on the expression of LECAM-1 (CD62L), VLA-alpha4 (CD49d), VLA-beta1 (CD29), LFA-1 alphaL (CD11a), LFA-beta2 (CD18), VCAM-1 (CD106), ICAM-1 (CD54), and H-CAM (CD44).Methods-Reactive lymph nodes and palatine tonsils were studied using immunofluorescence methods with fluorescein isothiocyanate (FITC) labelled monoclonal antibodies directed against cell adhesion molecules. To investigate the expression patterns of these molecules in the T and B cell populations, double labelling experiments were performed using Texas Red labelled antibodies against CD2 or CD19, respectively. The images from each fluorochrome were then simultaneously analysed using a laser scanning confocal microscope.Results-LECAM-1, VLA-alpha4 and H-CAM were predominantly expressed by mantle zone B cells, VCAM-1 and ICAM-1 by germinal centre cells, most of which exhibited a reticular staining pattern suggestive of follicular dendritic cells, whereas LFA-1 alphaL and LFA-beta2 were mainly found in extrafollicular and germinal centre T cells. All high endothelial venules expressed VLA-beta1 and ICAM-1, whereas VCAM-1 was present in only a few, with variable intensity.Conclusions-The data show that all of these adhesion molecules are differentially distributed within the distinct functional microenvironments of both organs. The differences observed in the expression patterns among the B and T cells belonging to different compartments probably depend on the momentum of cell traffic, the stage of maturation/activation, as well as on their functional role in the immune response.

OBJECTIVE

To construct the eukaryotic expression vector of human bone morphogenetic protein 7 (hBMP-7) gene so as to observe its expression in rabbit adipose-derived stem cells (ADSCs) and its effects on osteogenic phenotype.

METHODS

Several healthy 3-month-old Japanese rabbits of clean grade were chosen, female or male and weighing 3-4 kg. ADSCs were isolated and cultured with collagenase digestion, then were detected and identified by CD44, CD49d, and CD106 immunofluorescence staining. The eukaryotic expression vector of hBMP-7 gene (pcDNA3.1-hBMP-7) was constructed, which was transfected into rabbit ADSCs (3rd passage) by Lipofectamine 2000 after identified, then the expression of hBMP-7 in transfected ADSCs was detected. The alkaline phosphatase (ALP) level and the collagen type I expression were detected by intracellular ALP spectrophotometry and immunofluorescence, respectively to assess the effect of hBMP-7 gene on the osteoblastic differentiation of ADSCs.

RESULTS

ADSCs mostly presented fusiform and polygon shape with positive expressions of CD44 and CD49d and negative expression of CD106. The eukaryotic expression vector of pcDNA3.1-hBMP-7 gene was successfully constructed and the expression of hBMP-7 was confirmed in ADSCs by immunohistochemical staining. The intracellular ALP quantitative detection showed that the activity of ALP was significantly higher in pcNDA3.1-hBMP-7 transfected group (experimental group) than in pcDNA3.1 transfected group (control group) at 7, 10, and 14 days after transfection (P < 0.05). The expression of collagen type I was higher in experimental group than in control group at 7 and 14 days after transfection (P < 0.05).

CONCLUSIONS

The eukaryotic expression vector of pcDNA3.1-hBMP-7 gene is successfully constructed, which can express in ADSCs. The expressions of collagen type I and ALP in experimental group are higher than those in control group, which lays a basis for the local gene therapy of skeletal regeneration.

OBJECTIVE

To investigate the effect of salidroside on rat bone marrow mesenchymal stem cells (BMSCs) differentiation into the cholinergic nerve cells, so as to provide the theory basis of the combination of salidroside and stem cells for clinical therapy of nervous system diseases.

METHODS

BMSCs were isolated from 2 Wistar rats (aged 4-6 weeks,weighing 120 g), which were identified by CD34, CD45, CD90, and CD106 with flow cytometry. According to inducing method, BMSCs at passage 2 were divided into 3 groups: In groups A and B, BMSCs were induced by salidroside (20 microg/mL) and retinoic acid (5 micromol/mL) respectively for 1, 3, 6, and 9 days, in group C, BMSCs were cultured with serum-free DMEM/F12 medium as control. MTT assay was used to detect the cellular proliferation activity. The immunofluorescence chemical technology was used to detect the expressions of nerve growth factor (NGF) and relevant marker molecule of nerve cells, including neuron-specific enolase (NSE), microtubule-associated protein 2 (MAP2), beta-Tubulin III, glial fibrillary acidic protein (GFAP), and the marker of cholinergic neuron, such as Acetylcholine (Ach) and NGF. RT-PCR was used to detect mRNA expressions of NSE, beta-Tubulin III, GFAP,brain derived neurotrophic factor (BDNF),and gamma-aminobutyric acid (GABA). ELISA was used to detect the levels of BDNF and NGF, and the expression level of NGF protein was analyzed by Western blot.

RESULTS

The results of the flow cytometry showed that the cultured cells were CD90 and CD106 positive, and CD34 and CD45 negative,which indicated that the cells were BMSCs. The cellular proliferation activity in groups A and B were significantly higher than that in group C at 6 days and 9 days (P < 0.05). RT-PCR results showed that the expression level of NSE,BDNF, beta-Tubulin III,GFAPmRNA were increased in group A at 6 days; In group B, that expression level of NSE mRNA was up-regulated at 6 days, that expression level of BDNF mRNA increased at 1 days and reached the peak at 6 days, and that expression level of beta-Tubulin III mRNA was up-regulated at 3 days, which was significantly higher than that at the other time points, and than that in group C (P < 0.01). But no GABA mRNA expression was detected in each group. Immunofluorescence chemical technology staining showed that the positive rates of NSE, MAP2, beta-Tubulin III, and GFAP were significantly higher in group A than those in group C at 3 days; the positive rates of Ach were significantly higher at 3, 6, and 9 days than those at 1 day in groups A and B, and in groups A and B than in group C (P < 0.01); the positive rates of NGF in groups A and B were significantly higher than those in group C (P < 0.01). The levels of BDNF and NGF in groups A and B were significantly higher than those in group C at 1, 3, 6, and 9 days (P < 0.01), but no significant difference of BDNF was found between groups A and B (P>> 0.05). The expression level of NGF protein in groups A and B were significantly higher than that in group C (P < 0.01). The NGF expression reached the peak at 6 days in group A and at 3 days in group B.

CONCLUSIONS

Salidroside could induce rat BMSCs differentiate into cholinergic nerve cells in vitro.

Clinical presentations of dengue fever (DF) are diverse and non-specific, causing unpredictable progression and outcomes. Its progression and severity have been associated with cytokine levels alteration. In this study, dengue patients were classified into groups following the 2009 WHO dengue classification scheme to investigate the cytokine signature at different severity of the disease: dengue without warning sign symptoms (A); dengue with warning signs (B); severe dengue (C); other fever (OF) and healthy (Healthy). We analyzed 23 different cytokines simultaneously, namely IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-17A, IL-33, CD14, CD54, CD62E, CD62L, CD62p, CD106, CD121b, CD154, CD178, GM-CSF, IFN-g, MIF, ST2 and TNF from patients admitted to National Cheng Kung University Hospital during the 2015 Taiwan dengue outbreak. Cytokines TNF, CD54, CD62E, CD62L, CD62P, GM-CSF, IL-1b, IL-2, IL-6, IL-8, IL-10, IL-12p70, IL-17A, INF-g and MIF were elevated while CD106, CD154, IL-4 and L-33 were decreased when compared to the control. IL-10 demonstrated to be a potential diagnostic marker for DF (H and A group; AUC = 0.944, H and OF group; AUC = 0.969). CD121b demonstrated to be predictive of the SD (A and B group; AUC = 0.744, B and C group; AUC = 0.775). Our results demonstrate the cytokine profile changes during the progression of dengue and highlight possible biomarkers for optimizing effective intervention strategies.

Keywords: biomarker prediction; cytokines; dengue; flaviviruses; severe dengue.

Patients with end-stage renal failure undergoing chronic hemodialysis manifest alterations in cell-mediated immune response despite adequate urea clearance. We conducted a clinical trial in 8 patients (6 male, 2 female; 57.6 +/- 19.0 years) to investigate cell subsets and adhesion molecules on mononuclear blood cells during hemodialysis in patients on either cuprophane (n = 60 or polysulfone (n = 2). Cells were analyzed from the arterial line before, at 5, 10, 15, 30, 90 and 240 min with a panel of 33 surface markers including antibodies towards CD3, CD4, TCR-alpha/beta, CD7, CD8, CD11a-c, CD14, CD18, CD19, CD25, CD28, CD29, CD38, CD44, CD49a-e, CD51/61, CD54, CD56, CD58, CD62, CD106, and HLA class II using single fluorescence analysis. In vivo results were compared with in vitro tests. Our results suggest a substantial difference in the expression of various surface markers and cell adhesion molecules (lymphocytes: CD4, CD25, CD28, CD51, CD54; monocytes: CD49, CD54, CD58, CD62E, CD62P) compared to healthy persons, and a distinct modulation during hemodialysis (lymphocytes: CD11, CD18, CD49, CD51, CD61; monocytes: CD29, CD49, CD51, CD54, CD61) which might affect the immune response beyond the single dialysis session.

BACKGROUND

The inappropriate elevation of parathormone (PTH), which regulates the process of angiogenesis in parathyroid tissue, causes the changes of activity of enzymes responsible for the removal of free radicals. Parathyroidectomy (PTX) in patients with primary hyperparathyroidism (PHPT) lowers the level of PTH and leads to the reduction of risk of cardiovascular and all-cause mortality by normalization of the antioxidant status. Therefore, the aims of the study were to assess the activity of antioxidant enzymes and free radical reaction products in patients after parathyroidectomy, and to evaluate the correlation between the systemic oxidative stress and angiogenic parameters.

METHODS

Patients with PHPT treated surgically were enrolled into the study. Total antioxidant capacity (TAC), total oxidative status (TOS), oxidative stress index (OSI), superoxide dismutase (SOD), ceruloplasmin (CER), lipid hydroperoxides (LHP) and malondialdehyde (MDA) were measured before and after parathyroidectomy. The immunohistological expression of angiogenic factors in parathyroid specimens was assessed by the BrightVision method from ImmunoLogic using murine monoclonal anti-human: anti-VEGF, anti-CD31 and anti-CD106 antibodies.

RESULTS

The significant increase of TAC, CER, reduction of TOS, MDA, SOD, especially for cytoplasmic form, and significant decrease of OSI, LHP were observed after PTX. There was no significant correlation between changes of oxidative stress markers and angiogenic parameters: VEGF, CD-31, CD-106 in parathyroid tissue. The correlation level was low and medium.

CONCLUSIONS

Parathyroidectomy causes down-regulation of lipid peroxidation processes and leads to reduction of oxidative stress in patients with PHPT. The decrease in the OSI is the results of down-regulation of oxidative stress in the postoperative period. The change of the antioxidant status has no impact on angiogenesis processes in parathyroid tissue.

OBJECTIVE

To study the ability of bone marrow mesenchymal stem cells (BMSCs) to repair the internal environment of the testis in male azoospermia rats.

METHODS

We established azoospermia models in 22 six-week-old male SD rats by intraperitoneal injection of busulfan at 20 mg per kg body weight. We transplanted allogeneic rat BMSCs (rBMSCs) into the testicular seminiferous tubules of the model rats and, 30 days after transplantation, observed the composition and structure of the seminiferous tubular cells by HE staining and detected the expressions of CD44, CD106, and c-kit in the rBMSCs by immunohistochemistry.

RESULTS

The number of epididymal sperm was significantly reduced in the model rats as compared with the normal controls (P < 0.01). CD44 and CD106, but not c-kit, were expressed in the isolated rBMSCs. At 30 days after transplantation of rBMSCs, lots of new cells were observed in the seminiferous tubules, some expressing CD106 and some expressing the germ cell surface marker c-kit.

CONCLUSIONS

BMSCs can transdifferentiate into germ cells and repair the damaged seminiferous tubules of sterile rats.

Objective To observe the effect of panax notoginsenosides (PNS) on cardiac function of rats after acute myocardial infarction (AMI) and investigate the influence of PNS on the mobilization of bone marrow-derived mesenchymal stem cells (BM-MSCs). Methods A total of 48 rats were randomly assigned into the sham group, AMI group, low-dose PNS group [100 mg/(kg.d)] and high-dose PNS group [500 mg/(kg.d)]. The rat model of AMI was established by coronary ligation, and 6 rats were sacrificed in each group after 7 and 21 days of treatment with high and low doses of PNS. The heart function of rats was detected by echocardiography before execution, and peripheral blood and heart tissue were collected. Flow cytometry was used to test the proportions of CD90, CD105, CD54 or CD106 positive cells in the peripheral blood. ELISA was performed to measure the levels of stem cell factor (SCF) in the peripheral blood. TTC staining was applied to evaluate the infarct size of myocardial tissues. TUNEL assay was carried out to determine the apoptosis of myocardial tissues and immunohistochemistry to determine the expression levels of CD105 in the infarction area. Results Compared with the sham group, the percentage of apoptotic cells in the AMI group significantly increased. Seven days or 21 days after the intervention with PNS, the infarction area and the apoptotic rates in the PNS treated groups were observably alleviated when compared with the AMI group. In addition, at 7 days and 21 days after operation, the LVEF and LVFS decreased, whereas the LVIDs, LVIDd, LVEDV and LVESV significantly decreased in the AMI groups when compared with the sham group. Treatment with PNS could effectively improve the above alterations. When compared with the AMI group, PNS treatment significantly increased the proportions of CD90 or CD105 positive cells and the concentration of SCF, whereas decrease the proportions of CD54 or CD106 positive cells in the peripheral blood in a dose-dependent manner. Moreover, the level of CD105 in the marginal zone of AMI was significant higher in the PNS treated groups when compared with that in the AMI group. Conclusion PNS treatment improves left ventricular function after AMI, which may be related to PNS inhibiting the apoptosis of myocardocytes and promoting the mobilization of BM-MSCs.

BACKGROUND

The study aims to characterise human corneal endothelial cell (HCEnC) cultures generated by the peel-and-digest method based on their surface protein/carbohydrate expression pattern.

METHODS

Quantitative polymerase chain reaction was used to compare expression of vimentin, CD90, Cytokeratin-19, ZO-1 and Claudin 14 in cultured HCEnC and cell line B4G12 versus stromal cells. Fluorescence-activated cell sorting was used to assess surface protein distribution of cultured and uncultured HCEnC. Distribution of surface proteins/carbohydrates was visualised by immunofluorescent and lectin staining.

RESULTS

Human corneal endothelial cell and B4G12 showed lower expression level for vimentin, CD90, Cytokeratin-19 compared with stromal cells; while ZO-1 was expressed in endothelial cells, Claudin 14 was detected in B4G12 only. Fluorescence-activated cell sorting analyses revealed CD166, CD47, CD44, CD54, CD73, CD90, CD105, CD106, CD112, CD146 and CD325 to be present, with CD34 to be absent from cultured HCEnC. Freshly isolated, non-cultivated HCEnCs were CD90, CD73, CD146 and CD325 positive. Carbohydrates were detected by lectins LCA, PHA E, PHA L, PSA, sWGA, Con A, RCA 120 and WGA, but cultured HCEnC showed negative for GSL I, SBA, DBA, PNA and UEA I.

CONCLUSIONS

Cultures established by the peel-and-digest method are probably not prone to stromal contamination, but the cells are likely to undergo endothelial-to mesenchymal transition as suggested by apparent morphological changes.

BACKGROUND

Paragangliomas (glomus tumors) of the head and neck are rare tumors, arising from the paragangliomatous tissue either of the carotid region or the jugular or the tympanic region. This study was conducted to investigate the possible differences in the tumor biology of these lesions depending on their site of origin.

METHODS

Nineteen specimens (10 jugulotympanic and 9 carotid glomus tumors) were investigated by quantitative DNA measurements and immunohistochemical assessment of proliferation markers (PCNA, Ki67), oncogenes (p53, nm23), different cell surface antigenes (CD44 4/5 and 6, CD54, CD106), and bcl 2 as a marker for apoptosis.

RESULTS

Depending on the location of the tumors these measurements revealed significant differences in tumor biology with higher proliferation scores and a higher number of aneuploid tumors in those of the carotid region, suggesting a more aggressive behavior compared to those of the jugulotympanic region.

CONCLUSIONS

The results also indicate a difference between the two groups in the risk of developing metastases or recurrent disease. They generally help to enhance our understanding of the biology of paragangliomas of the head and neck.

Objective To analyze the differences in biological functions between bone marrow(BM)-derived CD106 ⁺mesenchymal stem cells(MSCs)and the CD106 ^- subgroup. Methods The MSCs from normal BM were isolated and expanded.The subgroups of CD106 ⁺ and CD106 ^-MSCs were sorted.The cell proliferation and adhesion functions,chemotactic activities,adipogenic and osteogenic potentials,senescence,and senescence protein 21(p21)were detected.The capacity of translocation into nucleus of nuclear factor-kappa B(NF-κB)when stimulated by tumor necrosis factor(TNF-α)was measured. Results The proliferative ability was higher in CD106 ⁺MSCs than that in CD106 ^-MSCs.In 48 hours,the value of optical density(OD)was significantly higher in CD106 ⁺MSCs than that in CD106 ^- subgroup(1.004±0.028 vs. 0.659±0.023,t=3.946,P=0.0225).In 72 hours,this phenomenon was even more pronounced(2.574±0.089 vs. 1.590±0.074,t=11.240,P=0.0000).The adhesive capacity of CD106 ⁺MSCs was significantly stronger than that of CD106 ^- subgroup(0.648±0.018 vs. 0.418±0.023,t=7.869,P=0.0002).Besides,the metastasis ability of CD106 ⁺MSCs were significantly stronger than that of CD106 ^- subgroup(114.500±4.481 vs.t=6.900,P=0.0005).The CD106 ⁺MSCs had signifcnatly lower proportions of senescent cells.The expression of aging protein p21 in CD106 ⁺MSCs was significantly lower than that in CD106 ^-MSCs [(17.560±1.421)% vs.(45.800±2.569)%,t=9.618,P=0.0000].Furthermore,there were no visible pigmenting cells after β-galactosidase staining in CD106 ⁺MSCs subgroup.However,in CD106 ^-MSCs,some colored green cells were detected.The rate of NF-κB translocation into nucleus after stimulated by TNF-α was significantly higher in CD106 ⁺MSCs than CD106 ^- MSCs [(37.780±3.268)% vs.(7.30±1.25)%,t=8.713,P=0.0001]. Conclusion Bone marrow-derived CD106 ⁺MSCs possess more powerful biological functions than CD106 ^-MSCs.

Human culture-expanded mesenchymal stromal cells (MSC) are being considered for multiple therapeutic applications because of their regenerative and anti-inflammatory properties. Although a large number of MSC can be propagated from a small initial sample, several lines of evidence indicate that MSC lose their immunosuppressive and regenerative potency aftaer multiple passages. In this report, we use the FACSCAP Lyoplate proteomic analysis system to detect changes in cell surface protein expression of CD45- /CD31- /CD34- /CD73+ /CD105+ stromal cells in unpassaged bone marrow (BM) and through 10 serial culture passages. We provide for the first time a detailed characterization of native unpassaged BM MSC (0.08% of BM mononuclear cells) as well as the changes that occur during the initial expansion. Adipogenic and osteogenic differentiative potential was determined though the serial passages and correlated with immunophenotypic changes and senescence. Among the most prominent were striking decreases in Fas ligand, CD98, CD205, and CD106, accompanied by a gain in the expression of CD49c, CD63, CD98, and class 1 and class 2 major histocompatibility complex (MHC) molecules. Other molecules that are down-modulated with later passage include CD24, CD54, CD59, CD243/P-glycoprotein, and CD273/PD-L2. Early senescence, as defined by the loss of replicative capacity occurring with the loss of differentiative capacity, increase in CDKN2A p16, and increased time to confluence, was accompanied by loss of the motility-associated metalloproteinase CD10 and the proliferation-associated transferrin receptor CD71. Among the strongest statistical associations were loss of MAC-inhibitory protein/CD59, loss of ICAM-1/CD54, and increase in CDKN2A as a function of increasing passage, as well as increased CD10 expression with adipogenic and osteogenic capacities. The data provide a clear set of markers that can be used to assess MSC quality. We suggest that clinically relevant numbers of highly functional low passage MSC can be manufactured starting with large quantities of BM, which are readily available from cadaveric organ donors.

Bleomycin-induced lung disease is a serious complication of therapy characterized by alveolar injury, cytokine release, inflammatory cell recruitment, and eventually pulmonary fibrosis. The mechanisms underlying bleomycin-induced pulmonary fibrosis may be relevant to other progressive scarring diseases of the lungs. Pulmonary vascular endothelial cells are critically involved in immune cell extravasation at sites of injury through adhesion molecule expression and cytokine release. We sought to determine the effects of bleomycin on adhesion molecule expression and cytokine release by pulmonary vascular endothelial cells, and their functional relevance to inflammatory cell recruitment.

The effects of pharmacologically relevant concentrations of bleomycin on adhesion molecule expression and cytokine release by human vascular endothelial cells in vitro were studied by flow cytometry, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. A flow chamber model was used to assess the functional consequences on adhesion of flowing human neutrophils to endothelial cell monolayers.

Bleomycin increased intercellular adhesion molecule 1 (ICAM-1; CD54), vascular cell adhesion molecule (VCAM-1; CD106), and E-selectin (CD62E) expression, and increased monocyte chemoattractant protein (MCP-1) and interleukin (IL-8) release by endothelial cells. Increases in protein expression were accompanied by increased mRNA transcription. In contrast, there was no direct effect of bleomycin on the profibrotic cytokines transforming growth factor-beta (TGF-β), platelet-derived growth factor-BB (PDGF-BB), or endothelin-1. Under flow conditions, endothelial cells exposed to bleomycin supported increased neutrophil adhesion which was independent of ICAM-1 or E-selectin.

Our findings demonstrate that bleomycin promotes endothelial-mediated inflammation and neutrophil adhesion. These mechanisms may contribute to the development of pulmonary fibrosis by supporting immune cell recruitment in the lungs.