This study tested the hypothesis that subsets of human T helper cells can orchestrate leukocyte adhesion to synovial fibroblasts (SFbs), thus regulating the retention of leukocytes in the joints of juvenile idiopathic arthritis (JIA) patients. Several cell types, such as monocytes/macrophages, granulocytes, T and B lymphocytes, SFbs and osteoclasts participate in joint tissue damage JIA. Among T cells, an enrichment of classic and non-classic Th1 subsets, has been found in JIA synovial fluid (SF), compared to peripheral blood (PB). Moreover, it has been shown that IL-12 in the SF of inflamed joints mediates the shift of Th17 lymphocytes towards the non-classic Th1 subset. Culture supernatants of Th17, classic and non-classic Th1 clones, have been tested for their ability to stimulate proliferation, and to induce expression of adhesion molecules on SFbs, obtained from healthy donors. Culture supernatants of both classic and non-classic Th1, but not of Th17, clones, were able to induce CD106 (VCAM-1) up-regulation on SFbs. This effect, mediated by tumor necrosis factor (TNF)-α, was crucial for the adhesion of circulating leukocytes on SFbs. Finally, we found that SFbs derived from SF of JIA patients expressed higher levels of CD106 than those from healthy donors, resembling the phenotype of SFbs activated in vitro with Th1-clones supernatants. On the basis of these findings, we conclude that classic and non-classic Th1 cells induce CD106 expression on SFbs through TNF-α, an effect that could play a role in leukocytes retention in inflamed joints.

Major research efforts have focused on defining cell surface marker profiles for characterization and selection of brain tumor stem/progenitor cells. Medulloblastoma is the most common primary malignant pediatric brain cancer and consists of 4 molecular subgroups: WNT, SHH, Group 3 and Group 4. Given the heterogeneity within and between medulloblastoma variants, surface marker profiles may be subtype-specific. Here, we employed a high throughput flow cytometry screen to identify differentially expressed cell surface markers in self-renewing vs. non-self-renewing SHH medulloblastoma cells. The top 25 markers were reduced to 4, CD271/p75NTR/NGFR, CD106/VCAM1, EGFR and CD171/NCAM-L1, by evaluating transcript levels in SHH tumors relative to samples representing the other variants. However, only CD271/p75NTR/NGFR and CD171/NCAM-L1 maintain differential expression between variants at the protein level. Functional characterization of CD271, a low affinity neurotrophin receptor, in cell lines and primary cultures suggested that CD271 selects for lower self-renewing progenitors or stem cells. Moreover, CD271 levels were negatively correlated with expression of SHH pathway genes. Our study reveals a novel role for CD271 in SHH medulloblastoma and suggests that targeting CD271 pathways could lead to the design of more selective therapies that lessen the broad impact of current treatments on developing nervous systems.

OBJECTIVE

To assess whether endothelial microparticles (EMPs), novel surrogate markers of endothelial injury and dysfunction, are differentially produced in response to acute insulin-induced hypoglycaemia in adults with and without type 2 diabetes.

METHODS

A prospective, parallel study was conducted in individuals with type 2 diabetes (n = 23) and controls (n = 22). Hypoglycaemia (<2.2 mmoL/L: <40 mg/dL) was achieved by intravenous infusion of soluble insulin. Blood samples were collected at baseline and at 0, 30, 60, 120, 240 minutes and 24 hours after hypoglycaemia and analysed for CD31+ (platelet endothelial cell adhesion molecule-1), CD54+ (intercellular adhesion molecule 1), CD62-E+ (E-selectin), CD105+ (endoglin), CD106+ (vascular cell adhesion molecule 1) and CD142+ (tissue factor) EMPs by flow cytometry. The peak elevations (% rise from 0 minutes after hypoglycaemia) in EMP within 240 minutes after insulin-induced hypoglycaemia were modelled using a regression model, with adjustment for relevant covariates. All EMPs were expressed as percentage from 0 minutes hypoglycaemia for each time point and total areas under the curve (AUC0min-24h ) were calculated.

RESULTS

Following insulin-induced hypoglycaemia, levels of circulating EMPs were maximal at 240 minutes (P < 0.001) and returned to baseline values within 24 hours for both groups. The peak elevations (% rise from 0 minutes following hypoglycaemia) seen in CD31+ , CD54+ , CD62-E+ , CD105+ and CD142+ EMPs within 240 minutes were associated with diabetes status after adjustments for all relevant covariates. Individuals with type 2 diabetes showed increased CD31+ EMPs AUC0min-24h (P = 0.014) and CD105+ EMPs AUC0min-24h (P = 0.006) compared with controls, but there were no differences for CD54+ (P = 0.91), CD62-E+ (P = 0.14), CD106+ (P = 0.36) or CD142+ (P = 0.77) EMPs AUC0min-24h .

CONCLUSIONS

The associations between peak elevations within 240 minutes after insulin-induced hypoglycaemia for CD31+ , CD54+ , CD62-E+ , CD105+ and CD142+ and diabetes status indicate that the assessment of a panel of EMPs within this timeframe would identify a hypoglycaemic event in this population. The greater overall responses over time (AUCs) for apoptosis-induced CD31+ and CD105+ EMPs suggest that hypoglycaemia exerts greater endothelial stress in type 2 diabetes.

The present study investigated the functional maturity of oligodendrocyte derived from rat bone marrow stromal cells (BMSC).

The BMSC were isolated from female Sprague-Dawley rats and evaluated for different markers, such as fibronectin, CD106, CD90, Oct-4 and CD45. Transdifferentiation of OLC from BMSC was obtained by exposing the BMSC to DMSO and 1 µM all-trans-retinoic acid during the pre-induction stage and then induced by heregulin (HRG), platelet-derived growth factor AA (PDGFR-alpha), fibroblast growth factor and T3. The neuroprogenitor cells (NPC) were evaluated for nestin, neurofilament 68, neurofilament 160 and glial fibrillary acidic protein gene expression using immunocytochemistry. The OLC were assessed by immunocytochemistry for O4, oligo2, O1 and MBP marker and gene expression of PDGFR-alpha was examined by RT-PCR.

Our results showed that the fibronectin, CD106, CD90, CD45 and Oct-4 were expressed after the fourth passage. Also, the yield of OLC differentiation was about 71% when using the O1, O4 and oligo2 markers. Likewise, the expression of PDGFR-alpha in pre-oligodendrocytes was noticed, while MBP expression was detected in oligodendrocyte after 6 days of the induction.

The conclusion of the study showed that BMSC can be induced to transdifferentiate into mature OLC.

Bone marrow mesenchymal stromal cells (BM-MSCs) have been demonstrated to contribute to tissue regeneration. However, chronic pathological conditions, such as diabetes and aging, can result in a decreased number and/or quality of BM-MSCs. We therefore investigated the maintenance mechanism of BM-MSCs by studying signaling through the receptor for advanced glycation end products (RAGE), which is thought to be activated under various pathological conditions. The abundance of endogenous BM-MSCs decreased in a type 2 diabetes mellitus (DM2) model, as determined by performing colony-forming unit (CFU) assays. Flow cytometric analysis revealed that the prevalence of the Lin-/ckit-/CD106+/CD44- BM population, which was previously identified as a slow-cycling BM-MSC population, also decreased. Furthermore, in a streptozotocin-induced type 1 DM model (DM1), the CFUs of fibroblasts and the prevalence of the Lin-/ckit-/CD106+/CD44- BM population also significantly decreased. BM-MSCs in RAGE knockout (KO) mice were resistant to such reduction induced by streptozotocin treatment, suggesting that chronic RAGE signaling worsened the maintenance mechanism of BM-MSCs. Using an in vitro culture condition, BM-MSCs from RAGE-KO mice showed less proliferation and expressed significantly more Nanog and Oct-4, which are key factors in multipotency, than did wild-type BM-MSCs. Furthermore, RAGE-KO BM-MSCs showed a greater capacity for differentiation into mesenchymal lineages, such as adipocytes and osteocytes. These data suggested that RAGE signaling inhibition is useful for maintaining BM-MSCs in vitro. Together, our findings indicated that perturbation of BM-MSCs in DM could be partially explained by chronic RAGE signaling and that targeting the RAGE signaling pathway is a viable approach for maintaining BM-MSCs under chronic pathological conditions.

BACKGROUND

To compare the roles of adipose and bone marrow derived mesenchymal stem cells (AMSCs and BMSCs) in multiple differentiation capacity to provide a theoretical basis for stem cell transplantation.

METHODS

We isolated bone marrow and adipose derived mesenchymal stem cells and compared their phenotype, cell doubling time, the secretion of factors, and the ability of multi-differentiation.

RESULTS

BMSCs and AMSCs were similar in cell phenotype and the differences existed only between the expression of CD106. The proliferation rate of AMSCs was faster than of BMSCs (doubling time 28h vs. 39h) and the capacity to suppress T cell proliferation and activation was weakened in AMSCs. In addition, both sources of cells were able to differentiate into bone, fat, and cartilage which proved their stem cell properties.

CONCLUSIONS

Cell origin and abundance were decisive factors in stem cell applications and with the same premise as for AMSCs and BMSCs, adipose tissue is a more promising source of stem cells.

Bone marrow stromal cells (BMSCs) were reported to form floating aggregation of cells with expression of nestin, a marker for neural stem cells (NSCs). The purpose of this investigation is to evaluate the morphology and the molecular markers expressed by NSCs derived from these neurospheres. The BMSCs were isolated from Sprague Dawley rats and evaluated for osteogenesis, lipogenesis, and expression of fibronectin, CD90, CD106, CD31, and Oct4. The BMSCs were cultured with Dulbecco's modified Eagle's medium (DMEM)/F12 containing 15% fetal bovine serum, then with DMEM/F12 containing 2% B27, basic fibroblast growth factor, and epidermal growth factor. The cell aggregates or spheres were stained with acridine orange, which showed that the neurospheres comprised aggregated cells at either premitotic/postsynthetic (PS), postmitotic/presynthetic (PM) phases of cell cycle, or a mixture of both. The NSCs harvested from the neurospheres were polar with eccentric nucleus, and at either a PS or a PM cell cycle phases, some cells at the latter phase tended to form rosette-like structures. The cells were immunostained for molecular markers such as nestin, neurofilament 68 (NF68), NF160, and NF200 and glial fibrillary acidic protein (GFAP). Myelin basic protein (MBP), the pluripotency (Oct4, Nanog, and SOX2), and the differentiation genes (NeuroD1, Tubb4, and Musashi I) were also evaluated using reverse transcription polymerase chain reaction (RT-PCR). Nestin, NF68, NF160, NF200, GFAP, O4, and N-cadherin were expressed in the NSCs. The percentage of immunoreactive cells to nestin was significantly higher than that of the other neuronal markers. MBP was not expressed in BMSCs, neurospheres, and NSCs. The neurospheres were immunoreactive to GFAP. RT-PCR showed the expression of NeuroD1 and Musashi I. The pluripotency gene (SOX2) was expressed in NSCs. Oct4 and Nanog were expressed in BMSCs, while Oct4 and SOX2 were expressed in the neurosphere. This indicates that a pluripotency regularity network existed during the transdifferentiation of BMSCs into NSCs. Image processing of the neurospheres showed that the cells tended to form radial patterns. The conclusion of this study is that the NSCs generated from the BMSC-derived neurospheres have the morphology and the characteristics of neuroepithelial cells with tendency to forming rosette-like structures.

Objective To assess the phenotype of human articular chondrocytes cultured in normoxia (21% O2) or continuous hypoxia (2% O2). Design Chondrocytes were extracted from patients undergoing total knee replacement ( n = 5) and cultured in ~21% (normoxic chondrocytes, NC) and 2% (hypoxic chondrocytes, HC) oxygen in both monolayer and 3-dimensional (3D) pellet culture and compared with freshly isolated chondrocytes (FC). Cells were assessed by flow cytometry for markers indicative of mesenchymal stromal cells (MSCs), chondrogenic-potency and dedifferentiation. Chondrogenic potency and immunomodulatory gene expression was assessed in NC and HC by reverse transcription quantitative polymerase chain reaction. Immunohistochemistry was used to assess collagen II production following 3D pellet culture. Results NC were positive (>97%, n = 5) for MSC markers, CD73, CD90, and CD105, while HC demonstrated <90% positivity ( n = 4) and FC ( n = 5) less again (CD73 and CD90 <20%; CD105 <40%). The markers CD166 and CD151, indicative of chondrogenic de-differentiation, were significantly higher on NC compared with HC and lowest on FC. NC also produced the highest levels of CD106 and showed the greatest levels of IDO expression, following interferon-γ stimulation, indicating immunomodulatory potential. NC produced the highest levels of CD49c (>60%) compared with HC and FC in which production was <2%. Hypoxic conditions upregulated expression of SOX9, frizzled-related protein ( FRZB), fibroblast growth factor receptor 3 ( FGFR3), and collagen type II ( COL2A1) and downregulated activin receptor-like kinase 1 ( ALK1) in 3 out of 4 patients compared with normoxic conditions for monolayer cells. Conclusions Hypoxic conditions encourage retention of a chondrogenic phenotype with some immunomodulatory potential, whereas normoxia promotes dedifferentiation of chondrocytes toward an MSC phenotype with loss of chondrogenic potency but enhanced immunomodulatory capacity.

This study aimed to induce the differentiation of isolated and purified adipose-derived stromal cells (ADSCs) into myoblasts, which may provide a new strategy for tissue engineering in patients with stress urinary incontinence (SUI). ADSCs, isolated and cultured ex vivo, were identified by flow cytometry and induced to differentiate into myoblasts in the presence of an induction solution consisting of DMEM supplemented with 5-azacytidine (5-aza), 5% FBS, and 5% horse serum. Cellular morphology was observed under an inverted microscope. Ultrastructural changes occurring during the differentiation were observed by transmission electron microscopy and confocal laser scanning microscopy. Cellular immunohistochemical staining was applied to determine the expression of desmin protein in cells with and without induced differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were used to detect mRNA and protein expression, respectively, of sarcomeric and desmin smooth muscle proteins. The results showed that ADSCs were mainly of a spindle or polygon shape. Flow cytometry analysis revealed that ADSCs did not express CD34, CD45, and CD106 but high levels of CD44 and CD90, which confirmed that the cultured cells were indeed ADSCs. After induction with a 5-aza-containing solution, morphological changes in ADSCs, including irregular cell size, were observed. Cells gradually changed from long spindles to polygons and star-shaped cells with microvilli on the cell surface. Many organelles were observed and the cytoplasm was found to contain many mitochondria, rough endoplasmic reticulum (rER), and myofilament-like structures. Cell immunohistochemical staining revealed different levels of desmin expression in each phase of the induction process, with the highest expression level found on day 28 of induction. RT-PCR and Western blot results confirmed significantly higher desmin gene expression in induced cells compared with control cells, but no significant difference between the two groups of cells in sarcomeric protein expression. It was concluded that under specific induction setting, ADSCs can be induced to differentiate into myoblasts, providing a potential new option in stem cell transplantation therapy for SUI.

This study was purposed to compare the biological characteristics of umbilical cord-derived mesenchymal stem cells (UC-MSCs) and bone marrow-derived mesenchymal stem cells (BM-MSCs). The frequency of successful isolation, cell yield, colony-forming units-fibroblastics (CFU-F), proliferation capacity, immunophenotype and multi-differentiation potentials of UC-MSCs and BM-MSCs were determined by limiting dilution assay, flow cytometry, invert microscopy, RT-PCR and so on, the determined results were compared. The results showed that MSCs were successfully isolated from all the 36 portion of UC tissue and 8 portion of BM. Although the mean number of nucleated cells isolated from UC tissue was significantly lower than that from BMs (1 x 10(6)/cm vs 5.5 x 10(7)/ml) (p=0.0002), no significant differences of the yield of adherent cells were observed (8.6 x 10(5)/cm vs 8.4 x 10(5)/ml) (p>0.05). UC-MSCs shared the most of the characteristic of BM-MSC, including fibroblastic-like morphology, typical immunophenotype, cell cycle status, adipogenic and osteogenic differentiation potentials. However, the CFU-F frequency was higher in UC (1:1609+/-0.18) than that in BM (1:35700+/-0.01) (p<0.05). Furthermore, the proliferation capacity of UC-MSCs was higher than that of BM-MSCs; the expressions of CD106 and HLA-class I in UC-MSCs were lower than those in BM-MSCs (p<0.05). It is concluded that the cell yield and most biological characteristics of UC-MSCs are similar to BM-MSCs, but UC-MSCs possess the higher proliferation capacity, and the lower expression of HLA-class I and HLA-DR as compared with BM-MSCs, therefore the human umbilical cord tissue may be considered as a promising alternative to bone marrow as a source of MSCs.

T cells have roles in the pathogenesis of native coronary atherosclerosis (CA) and transplant-associated coronary artery disease (TCAD). The mechanisms by which T cells interact with other cells in these lesions are not fully known. CD154 is an activation-induced CD4+ T cell surface molecule that interacts with CD40+ target cells, including macrophages and endothelial cells, and induces the production of pro-inflammatory molecules, including CD54 (ICAM-1) and CD106 (VCAM-1). To investigate whether CD154-CD40 interactions might be involved in the pathogenesis of CA or TCAD we performed immunohistochemical studies of CD154 and CD40 expression on frozen sections of coronary arteries obtained from cardiac allograft recipients with CA (n=10) or TCAD (n=9). Utilizing four different anti-CD154 mAb we found that CD154 expression was restricted to infiltrating lymphocytes in CA and TCAD. CD40 expression was markedly up-regulated on intimal endothelial cells, foam cells, macrophages and smooth muscle cells in both diseases. Dual immunolabeling demonstrated many CD40+ cells co-expressed CD54 and CD106. The extent of CD40, CD54 and CD106 expression showed statistical significant correlation with the severity of disease and the amount of intimal lymphocytes. Together these studies demonstrate the presence of activated CD154+ and CD40+ cells in both CA and TCAD lesions and suggest that CD154-mediated interactions with CD40+ macrophages, foam cells, smooth muscle cells and/or endothelial cells may contribute to the pathogenesis of these diseases.

BACKGROUND

Xenotransplantation of pig organs may provide an approach to alleviate the severe shortage of human organs. Natural antibodies against Galalpha(1,3)-Gal (alphaGal) epitopes cause hyperacute rejection of pig organs in primates. However, evidence for the role of alphaGal in the natural killer (NK) cell-mediated xenoresponse has been contradictory.

METHODS

We investigated the recognition of alphaGal by human NK cells using endo-beta-galactosidase C, an enzyme that cleaves alphaGal, and endothelial cells (EC) from alpha1,3-galactosyltransferase null pigs that do not synthesize alphaGal. Endo-beta-galactosidase C treatment variably reduced the susceptibility of porcine EC to lysis by fresh human NK cells.

RESULTS

Removal of alphaGal from porcine EC using endo-beta-galactosidase C, produced variable results, i.e. cytotoxicity was decreased in half of the human NK cell donors tested. The two EC strains from alphaGal-/- pigs were marginally, and not significantly, less susceptible to lysis by naïve human NK cells compared with alphaGal-expressing cells obtained from animals from the same herd, but these differences were not statistically significant (P>> 0.10). Treatment of porcine EC with recombinant human tumor necrosis factor (TNF)-alpha, which is known to activate porcine EC, enhanced the susceptibility of all target cells to lysis by fresh human NK cells. Surface expression of MHC or adhesion molecules on alphaGal-/- cells, compared with wild type cells, showed no consistent difference in either MHC or adhesion molecules CD106 (VCAM-1), CD31 (PECAM) or CD62E (E-selectin), either with or without TNF-alpha stimulation, that could explain the differential susceptibility to lysis. Strikingly, all alphaGal-/- and wild type EC exhibited similar susceptibility to human NK cells that had been cultured for 5 days with or without interleukin-2.

CONCLUSIONS

These findings demonstrate that human NK cells can kill porcine targets in the absence of alphaGal, and donor variability plays a major role in whether alphaGal has a role in determining susceptibility of porcine EC to lysis. Moreover, susceptibility to lysis of alphaGal null EC is enhanced to the level of wild type EC by activation of either effector or target cells. Elimination of alphaGal alone from source pigs will be insufficient to circumvent the NK cell mediated destruction of porcine EC.

Recruitment and extravasation of T cells through the blood-brain barrier are favored by adhesion molecule-mediated interactions of circulating T cells with endothelial cells. Since a common pathological finding in human T-cell leukemia virus type 1 (HTLV-1)-associated diseases is the infiltration of HTLV-1-infected T lymphocytes into various organs, we have looked for the profile of adhesion molecules expressed by HTLV-1-transformed T cells. Flow cytometry analysis indicated that these cells were expressing high levels of vascular cell adhesion molecule 1 (VCAM-1 [CD106]), a 110-kDa member of the immunoglobulin gene superfamily, first identified on endothelial cells stimulated with inflammatory cytokines. This adhesion molecule was also expressed by T cells obtained from one patient with HTLV-1-associated myelopathy/tropical spastic paraparesis but not by activated T cells isolated from one normal blood donor. The role of the viral trans-activator Tax protein in the induction of VCAM-1 was first indicated by the detection of this adhesion molecule on Jurkat T-cell clones stably expressing the tax gene. The effect of Tax on VCAM-1 gene transcription was next confirmed in JPX-9 cells, a subclone of Jurkat cells, carrying the tax sequences under the control of an inducible promoter. Furthermore, deletion and mutation analyses of the VCAM-1 promoter performed with chloramphenicol acetyltransferase constructs revealed that Tax was trans activating the VCAM-1 promoter via two NF-kappaB sites present at bp -72 and -57 in the VCAM-1 gene promoter, with both of them being required for the Tax-induced expression of this adhesion molecule. Finally, gel mobility shift assays demonstrated the nuclear translocation of proteins specifically bound to these two NF-kappaB motifs, confirming that VCAM-1 was induced on Tax-expressing cells in a kappaB-dependent manner. Collectively, these results therefore suggest that the exclusive Tax-induced expression of VCAM-1 on T cells may represent a pivotal event in the progression of HTLV-1-associated diseases.

Adipose-derived stem cells (ADSC) are adult stem cells which can be induced into motor neuron-like cells (MNLC) with a preinduction-induction protocol. The purpose of this study is to generate MNLC from neural stem cells (NSC) derived from ADSC. The latter were isolated from the perinephric regions of Sprague-Dawley rats, transdifferentiated into neurospheres (NS) using B27, EGF, and bFGF. After generating NSC from the NS, they induced into MNLC by treating them with Shh and RA, then with GDNF, CNTF, BDNF, and NT-3. The ADSC lineage was evaluated by its mesodermal differentiation and was characterized by immunostaining with CD90, CD105, CD49d, CD106, CD31, CD45, and stemness genes (Oct4, Nanog, and Sox2). The NS and the NSC were evaluated by immunostaining with nestin, NF68, and Neurod1, while the MNLC were evaluated by ISLET1, Olig2, and HB9 genes. The efficiency of MNLC generation was more than 95 ± 1.4 % (mean ± SEM). The in vitro generated myotubes were innervated by the MNLC. The induced ADSC adopted multipolar motor neuron morphology, and they expressed ISLET1, Olig2, and HB9. We conclude that ADSC can be induced into motor neuron phenotype with high efficiency, associated with differential expression of the motor neuron gene. The release of MNLC synaptic vesicles was demonstrated by FM1-43, and they were immunostained with synaptophysin. This activity was correlated with the intracellular calcium ion shift and membrane depolarization upon stimulation as was demonstrated by the calcium indicator and the voltage-sensitive dye, respectively.

The alpha 4 beta 1-integrin (CD49d, CD29) constitutively expressed on leukocytes regulates cell migration to inflammatory sites, cell activation, and development through its interactions with two alternate ligands, vascular cell adhesion molecule-1 (VCAM-1; CD106) expressed on cytokine-activated endothelium, dendritic and stromal cells, and the extracellular matrix protein fibronectin. Another alpha 4-integrin receptor, alpha 4 beta 7, expressed on leukocytes also binds VCAM-1 and fibronectin (FN), and controls homing to mucosal tissues through its interactions with mucosal vascular addressin MAdCAM-1. In vitro studies have shown that alpha 4-dependent cell adhesion is regulated by the activation state of the cell and by divalent cations. However, the existence and role of cells with different alpha 4 activation states in vivo have not been defined. Herein we show that a soluble ligand with the two N-terminal domains of human VCAM-1 fused to a human IgG1 constant region, VCAM-Ig, binds selectively to activated alpha 4-receptors on murine cells, such as those induced by Mn2+ in vitro. To determine whether the cells identified by VCAM-Ig were required under physiologic conditions, we assessed its anti-inflammatory effect. We show that VCAM-Ig is not bound to the majority of murine alpha 4+ cells after in vivo administration, yet it significantly delays the onset of adoptively transferred autoimmune diabetes. Thus, soluble VCAM-Ig can modify alpha 4-dependent disease progression, apparently by its selective action on cells with activated alpha 4-integrin receptors, thereby providing evidence for distinct alpha 4 activation states in vivo.

Vascular cell adhesion molecule-1 (VCAM-1; CD106), the receptor for VLA-4, is an important mediator of adhesive and co-stimulatory interactions that govern cutaneous immune responses. Initial studies designed to elucidate temporal aspects of endothelial adhesion molecule induction in murine acute graft-versus-host disease (aGVHD) revealed unexpected and novel VCAM-1 expression by cutaneous and mucosal epithelial cells. Immunohistochemical techniques confirmed VCAM-1 staining as early as 7 days after transplantation in a distinctive subpopulation of squamous epithelial cells that normally occupy focal domains within the epidermal basal cell layer, the follicular infundibulum, and the dorsal lingual epithelium. Specifically, VCAM-1 expression was intimately associated with rete ridge-like prominences in footpad epidermis and in dorsal lingual epithelium. VCAM-1, as evaluated by serial section-labeling techniques, was preferentially expressed at sites of early epithelial infiltration by CD4(+) T cells. Western blot analysis confirmed expression of the 110-kd isoform of VCAM-1 in epithelium isolated from aGVHD animals, and immunoelectron microscopy demonstrated VCAM-1 reactivity restricted exclusively to epithelial cell plasma membranes. It is concluded that VCAM-1 is selectively expressed by discrete squamous epithelial subpopulations in murine aGVHD. As such, VCAM-1 may play a previously unrecognized role in mediating interactions between donor effector T lymphocytes and host epithelial cell targets.

Cytotoxic drugs or irradiation are generally administered before bone marrow (BM) transplantation because of the idea that host bone marrow 'niches' become available to the donor cells for engraftment. How BM stromal cells respond to the radiation, which ultimately modulates grafting of donor cells, is poorly understood. In this study, we examined homing and marrow retention of PKH26+ donor cells in BM of age-matched C57BL/6J mice conditioned at different doses of irradiation. When we injected donor cells into mice that received 900 cGy, the percent homing was highest (15.8 +/- 1.5%) as compared to the lower doses of radiation. Despite the highest levels of homing of donor cells in these mice, about 70% (p < 0.005) homed cells were detached from the marrow within 72 h of transplantation. In contrast, a 2- to 2.5-fold (p < 0.03) multiplication of homed PKH-26+ Sca-1+ cells was observed in sublethally irradiated mice. While determining that CD45- CD106+ cells in BM of the mice received 900 cGy, we found that more than 80% of cells were depleted. It was also revealed from this investigation that grafted cells conferred partial protection to the endogenous myeloid colony-forming cells from radiation injury. Collectively, the present study implicates radiation-induced degeneration of stroma as a cause of poor retention of donor cells in BM of lethally irradiated mice. These results may have important clinical implications in designing conditioning regimens for BM transplantation.

This paper presents a description of the patterns of distribution of accessory cells in helicobacter gastritis and low-grade gastric MALT lymphomas. The use of gastric resection specimens afforded abundant, well-orientated lymphoid tissue. Fifteen cases were selected from patients with MALT lymphomas, three with gastritis alone, and six histologically normal controls. A panel of antibodies working in paraffin-embedded tissue, identifying differing accessory cells, was used. These comprised antibodies to HLA class II (WR18), acid cysteine proteinase inhibitor (ACPI), CD68 (PGM1), Factor XIIIa, S100 protein, CD23 (BU38), CD106 (V-CAM1), CD55 (BRIC 128), and CD21 (1F8). CD68-positive macrophages in the gastritis cases were abundant in the superficial mucosa. Factor XIIIa also identified dendritic cells at deeper sites but these were absent from both the acquired and the neoplastic lymphoid tissue. Antibodies to both S100 protein and ACPI stained dendritic cells localized to areas within and adjacent to the lymphoid tissue only. S100 protein-positive cells were concentrated in close contact with glandular epithelium immediately above the germinal centres, while ACPI-positive dendritic cells were identified, especially around the more blastic reactive follicles, in the intervening space between the germinal centres and the overlying epithelium. Similar patterns of organization were also seen in the areas of mucosal lymphoma. The follicular dendritic markers revealed overlapping but distinct sub-populations within the germinal centres which appeared to alter depending on the activity of the germinal centres. While both ACPI and CD55 stained the germinal centre dendritic reticulum cell networks only, CD21 and CD106 also stained the mantle dendritic cells. The proportion staining with CD23, which stains dendritic reticulum cells within the centrocyte-rich areas of the germinal centres only, was greatest in the more quiescent germinal centres. Similar patterns of staining were also seen in germinal centres within the gastric MALT lymphomas. It is proposed that the sub-populations of dendritic cells staining with S100 protein and ACPI may facilitate helicobacter antigen delivery to the germinal centres. The follicular dendritic cells then promote a sustained B-cell response to the luminal pathogen.

The leukocyte adhesion deficiency syndrome type II (LAD-II) is caused by a general defect in fucose metabolism, which leads to the absence of fucosylated sugar determinants such as the selectin ligand SLe(x). In view of the important role of selectins in lymphocyte migration and homing, we have explored the in vivo immune responsiveness and lymphocyte recruitment to the skin, in response to the neo-antigen keyhole limpet hemocyanin (KLH) in a LAD-II patient. We observed a normal priming of KLH-specific T cells as well as a strong in vivo anti-KLH antibody response, both indicative of a normal T-B cell function and collaboration. Skin biopsies from the patient's skin taken prior to antigenic challenge showed the presence of normal numbers and subsets of T cells. Upon KLH injection, a large number of T cells were found to be recruited to the site of challenge, which was paralleled by up-regulation of the endothelial adhesion molecules ICAM-1 (CD54), VCAM-1 (CD106) and E-selectin (CD62E). The recruited T cell showed a normal subset distribution but lacked cutaneous lymphocyte antigen (CLA), the cutaneous homing receptor. However, the clinical symptoms of delayed-type hypersensitivity in the patient (redness and swelling) were severely depressed compared to that in the controls. In conclusion, the LAD-II patient showed a normal T cell priming and T cell-dependent antibody response, a normal baseline skin homing, and a significant T cell recruitment to the site of KLH challenge, indicating that fucosylated sugar determinants such as SLe(x) and CLA are not strictly required for adequate immune responsiveness and (skin) homing.

OBJECTIVE

Circulating levels of endothelial cell adhesion molecules are elevated in women with preeclampsia. The aim of the present study was to determine levels of these molecules in the fetal circulation of normal pregnancies and pregnancies complicated by preeclampsia.

METHODS

Fetal plasma samples from the umbilical vein and peripheral maternal plasma and serum sample were collected at delivery from women with preeclampsia and women with normal pregnancy. Women with non-proteinuric pregnancy-induced hypertension (PIH) were excluded from the study. A sandwich ELISA technique was employed to quantitate concentrations of soluble ICAM-1 (CD54), VCAM-1 (CD106), and E-selectin (CD62E).

RESULTS

The normal values of soluble endothelial cell adhesion molecules in the fetal circulation were determined as 162+/-45 ng/ml for ICAM-1, 1612+/-582 ng/ml for VCAM-1, and 154+/-58 ng/ml for E-selectin. They were found to markedly differ from the corresponding normal values in the maternal circulation (sICAM-1: 247+/-65 ng/ml; sVCAM-1: 715+/-170 ng/ml; sE-selectin: 34+/-14 ng/ml). The concentrations of sICAM-1, sVCAM-1, and sE-selectin were significantly elevated in women with preeclampsia compared to healthy control pregnant women. In contrast, there was no difference in the circulating fetal concentrations of these molecules between normal pregnancies and pregnancies complicated by preeclampsia.

CONCLUSIONS

Normal values of sICAM-1, sVCAM-1, and sE-selectin in fetal circulation are markedly different from the values obtained for healthy adults. Plasma concentrations of these molecules are elevated in women with preeclampsia but not in the fetal circulation of preeclamptic pregnancies suggesting that based on the analysis of soluble adhesion molecules the fetal circulation may not be affected by the factor(s) that lead to disturbed endothelial cell function in women with preeclampsia.

BACKGROUND

Acute vascular rejection in pig-to-primate xenotransplantation involves recognition and damage of porcine (po) endothelial cells (EC) by human (hu) leukocytes, probably including natural killer (NK) cells. To study such interactions we analyzed rolling and static adhesion of hu NK cells to po EC.

METHODS

The effects of blocking hu and po adhesion molecules on the adhesion hu NK cells to po EC monolayers was analyzed under shear stress (10 min, 37 degrees C, 0.7 dynes/cm2) or under static conditions (10 min, 37 degrees C). All used cell populations were phenotypically characterized by flow cytometry.

RESULTS

Blocking of CD106 on po EC or its ligand CD49d on hu NK cells decreased rolling adhesion of both fresh and activated hu NK cells by more than 75%. Masking of CD62L on fresh but not activated hu NK resulted in a 44% decrease in rolling adhesion, in line with the diminished cell surface expression of CD62L upon activation. Antibodies to CD31, CD54, CD62E, and CD62P on EC or CD11a, CD18, and CD162 on NK cells had only minor effects on rolling adhesion. The adhesion of the FcgammaRIII- hu NK cell line NK92 to po EC was inhibited by 95% after masking po CD106 whereas antibodies to po CD31, CD54, CD62E, or CD62P had no effect, thereby excluding effects of Fc-receptor-dependent binding of hu NK cells to po EC. Static adhesion of activated NK cells was reduced by approximately 60% by blocking either CD49d or CD106, by 47% by blocking CD11a, and by 82% upon simultaneous blocking of CD11a and CD49d.

CONCLUSIONS

Interactions between hu CD49d and po CD106 are crucial for both rolling and firm adhesion of hu NK cells to po EC and thus represent attractive targets for specific therapeutic interventions to prevent NK cell-mediated responses against po xenografts.

In many inflammatory diseases, mononuclear leukocytes (MNLs) accumulate as focal infiltrates in perivascular spaces. We postulated that MNLs migrating through endothelium modify the microenvironment to promote the subsequent migration of additional MNLs into the same area. We found that as monocytes adhere to and migrate spontaneously through an endothelial monolayer, they secrete tumor necrosis factor-alpha (TNF-alpha) and interleukin-1. These cytokines stimulate endothelial cell expression of CD54 (intercellular adhesion molecule-1) and CD106 (vascular cell adhesion molecule-1). Consequently, when freshly isolated MNLs are added to that endothelial monolayer four or more hours later, significantly greater numbers of lymphocytes bind to and migrate through these endothelial monolayers. In addition to its ability to activate endothelial cell adhesion molecules, TNF-alpha induced directed migration of lymphocytes through collagen pads. These results illustrate a potential amplification mechanism by which MNLs moving through a vessel wall may secrete TNF-alpha, leading to the recruitment of additional leukocytes into the same perivascular locus.

We report the characterization of a novel series of human endothelial cell lines (designated SGHEC) regarding the expression and release of adhesion molecules and their binding of lymphocytes. SGHEC expressed significant levels of intercellular adhesion molecule-1 (ICAM-1; CD54) which increased after stimulation with tumor necrosis factor-alpha (TNF alpha), interleukin-1 beta (IL-1 beta), or interferon-gamma (IFN-gamma). Vascular cell adhesion molecule-1 (VCAM-1; CD106) and E-selectin (CD62E) were not detectable on unstimulated SGHEC but substantial levels were expressed after stimulation with either TNF alpha or IL-1 beta but not with IFN-gamma. The increased expression of ICAM-1 and VCAM-1 was evident after 4 h stimulation and was even higher after 24 h; E-selectin was maximal after 4 h and returned almost to basal levels by 24 h. Substantial quantities of immunoreactive ICAM-1 and VCAM-1 also accumulated as soluble material in the supernatants of TNF alpha-stimulated SGHEC (VCAM-1 was substantially higher than ICAM-1), but E-selectin remained below the limits of detection. Various quantitative data suggest that this is a controlled release regulated by cytokine and provide support for a physiological function for these soluble molecules. Primary human lymphocytes and lymphoblastoid cell lines expressing lymphocyte function-associated antigen-1 (LFA-1) bound to SGHEC; this binding increased substantially after activation of either cell type. The binding was inhibited by monoclonal antibodies against LFA-1 and, to a lesser extent, ICAM-1, thus demonstrating the importance of these molecules in the observed binding; neither anti-VCAM-1 nor anti-E-selectin antibodies affected the binding. From these various data, we conclude that LFA-1/ICAM-1 interactions are partially responsible for the binding of lymphocytes to endothelial cells. The SGHEC lines should prove useful in investigating leukocyte-endothelial interactions and the mechanism of release of soluble adhesion molecules.

Upregulation of tissue factor (TF) expression on activated donor endothelial cells (ECs) triggered by the immune response (IR) has been considered the main initiator of consumptive coagulopathy (CC). In this study, we aimed to identify potential factors in the development of thrombocytopenia and CC after genetically engineered pig liver transplantation in baboons. Baboons received a liver from either an α1,3-galactosyltransferase gene-knockout (GTKO) pig (n = 1) or a GTKO pig transgenic for CD46 (n = 5) with immunosuppressive therapy. TF exposure on recipient platelets and peripheral blood mononuclear cell (PBMCs), activation of donor ECs, platelet and EC microparticles, and the IR were monitored. Profound thrombocytopenia and thrombin formation occurred within minutes of liver reperfusion. Within 2 h, circulating platelets and PBMCs expressed functional TF, with evidence of aggregation in the graft. Porcine ECs were negative for expression of P- and E-selectin, CD106, and TF. The measurable IR was minimal, and the severity and rapidity of thrombocytopenia were not alleviated by prior manipulation of the IR. We suggest that the development of thrombocytopenia/CC may be associated with TF exposure on recipient platelets and PBMCs (but possibly not with activation of donor ECs). Recipient TF appears to initiate thrombocytopenia/CC by a mechanism that may be independent of the IR.